Manipulation of gibberellin biosynthesis for the control of plant Eragrostis tef

Manipulation of gibberellin biosynthesis for the control of plant Eragrostis tef
Manipulation of gibberellin biosynthesis for the control of plant
height in Eragrostis tef for lodging resistance
By
ENDALE GEBRE KEDISSO
Thesis submitted in partial fulfilment of the requirements for the degree
PHILOSOPHIAE DOCTOR
Forestry and Agricultural Biotechnology Institute (FABI)
Department of Plant Science
In the
Faculty of Natural and Agricultural Sciences
University of Pretoria
SUPERVISORS:
PROF. KARL KUNERT
DR. URTE SCHLÜTER
May 2012
© University of Pretoria
DECLARATION
This thesis is my original work and has not been presented for a degree in any
other University
Date: May 2012
Signed__________________________________
Endale Gebre Kedisso
Forestry and Agricultural Biotechnology Institute (FABI)
Department of Plant Science, University of Pretoria
This thesis has been submitted for examination with my approval as the
University Supervisor.
Date ___________________________________
Signed___________________________________
PROF. KARL KUNERT
ii
TABLE OF CONTENTS
PAGES
ABSTRACT
vii
THESIS COMPOSITION
ix
ACKNOWLEDGEMENT
xi
ABBREVIATIONS AND SYMBOLS
xv
LIST OF FIGURES
xvii
LIST OF TABLES
xxiii
CHAPTER ONE: INTRODUCTION
1.1.
The problem of lodging
2
1.2.
Plant architecture and lodging
4
1.3.
Genetic control of lodging resistance
6
1.3.1. GA genes and lodging
6
1.3.2. Manipulation of plant height using GA genes
10
1.4.
Brassinosteroid (BRs) genes and lodging
13
1.5.
Induced mutation
14
1.6.
Plant growth regulators for plant height control
17
1.7.
Lodging in E. tef
21
1.7.1.
E. tef growth
21
1.7.2.
Pheno-morphic features related to lodging
22
1.7.3.
E. tef breeding for lodging resistance
24
1.8.
Working hypothesis and aim of study
iii
26
CHAPTER CHAPTER TWO: CONTROLLING PLANT HEIGHT AND LODGING IN
TEF (Eragrostis tef) USING GIBBERELLIN BIOSYNTHESIS INHIBITORS
2.1.
Abstract
28
2.2.
Introduction
29
2.3.
Materials and methods
31
2.3.1. Plant material
31
2.3.2. Plant growth
31
2.3.3. Plant growth regulators (PGRs) treatment
32
2.3.4. Growth measurement
33
2.3.5. Analysis of endogenous GA content
33
2.3.6. Data analysis
34
Results
35
2.4.1. Experiment I
35
2.4.
2.4.1.1.
Analysis of endogenous GA content
2.4.2. Experiment II
2.5.
37
39
2.4.2.1.
Culm and panicle growth
39
2.4.2.2.
Internode growth
40
2.4.2.3.
Tillering and above ground biomass yield
43
Discussion
51
CHAPTER THREE: TRANSFORMATION OF TEF (Eragrostis tef)
3.1
Abstract
58
3.2
Introduction
60
3.3
Materials and methods
61
3.3.1
Preparation of planting material and culture
61
3.3.2
Immature embryo isolation, callus induction and culture growth
62
3.3.3
GA20x and nptII marker gene plasmids
63
3.3.4
Agrobacterium culture, inoculation and co-cultivation
65
3.3.5
Plant regeneration
66
iv
3.4
3.3.6
Preparation of plant material and culture
66
3.3.7
DNA isolation and PCR Screening of E. tef regenerants
67
3.3.8
Phenotypic measurement and characterization of T1 generation
68
3.3.9
Analysis of endogenous content
68
Results
72
3.4.1
Plant transformation
72
3.4.2
Transgene detection
73
3.4.3
Phenotypic characterization
77
3.4.3.1
3.4.4
3.5
Culm, internode and panicle length
Analysis of endogenous GA content
Discussion
77
82
84
CHAPTER FOUR: ISOLATION, CHARACTERIZATION AND EXPRESSION
OF GA GENES WITH PARTICULAR EMPHASIS ON GA20ox IN TEF
(Eragrostis tef)
4.1
Abstract
89
4.2
Introduction
90
4.3
Materials and methods
92
4.3.1 Plant material and plant growth
92
4.3.2 Genomic DNA isolation
92
4.3.3 Gene identification and isolation
93
4.3.4 Isolation of complete E. tef GA20ox coding region
95
4.3.5 Cloning and sequencing of PCR DNA products
97
4.3.6 DNA sequence analysis and phylogentic analysis
98
4.3.7 RNA isolation and cDNA synthesis
99
v
4.4
4.5
4.3.8
Isolation of the Reduced height (Rht) and other E. tef genes
99
4.3.9
GA20-oxidase expression in E. tef
99
4.3.10 Expression of E. tef GA20-oxidase1 (EtGA20ox1) in E. coli
102
4.3.11 HPLC analysis
102
4.3.12 Southern blot analysis
103
Results
104
4.4.1
Isolation of GA genes from E. tef
104
4.4.2
Putative E. tef GA20ox isolation and cloning
105
4.4.3
EtGA20ox copy number
115
4.4.4
GA20ox expression in E. tef
116
4.4.5
In vitro enzymatic activity of GA20ox in a heterologous system
118
4.4.6
Isolation of the Rht and other E. tef genes
119
Discussion
123
CHAPTER FIVE: EVALAUTION AND ANALYSIS OF MUTANT TEF
(Eragrostis tef) LINES FOR DWARFISM FOR LODGING RESISTANCE
5.1
Abstract
129
5.2
Introduction
130
5.3
Materials and methods
131
5.3.1 Plant material
131
5.3.2 Plant growth and GA treatment
131
5.4
5.5
5.3.2.1
Growth measurement
132
5.3.2.2
Data analysis
133
Results
134
5.4.1
Culm height, internode length and diameter
134
5.4.2
Panicle length, tillering, biomass and yield
138
Discussion
145
CHAPTER SIX: GENERAL DISCUSSION AND FUTURE
151
PERSPECTIVE
REFERENCES
154
APPENDIX
176
vi
ABSTRACT
Manipulation of gibberellin biosynthesis for the control of plant
height in Eragrostis tef for lodging resistance
Endale Gebre Kedisso
Plant Science Department, Forestry and Agricultural Biotechnology Institute (FABI), 74
Lunnon Road, Hillcrest 0002, University of Pretoria, South Africa
Supervisor: Prof. Karl Kunert
Plant Science Department, Forestry and Agricultural Biotechnology Institute (FABI), 74
Lunnon Road, Hillcrest 0002, University of Pretoria, South Africa
Co-Supervisor: Dr. Urte Schlüter
Plant Science Department, Forestry and Agricultural Biotechnology Institute (FABI), 74
Lunnon Road, Hillcrest 0002, University of Pretoria, South Africa
Lodging is a key agronomic problem in E. tef. due to morpho-physiological features, such tall
and slender phenotype of the plant. Gibberellins metabolic genes are key targets in the
control of plant height. Plant growth regulators (PGRs) that inhibit GA biosynthesis are used
to shorten stem length thereby increasing lodging resistance. E. tef responded to treatment
with PGRs such as GA, chlormequat chloride (CCC) and paclobutrazol (PBZ). Both PGRs
reduced E. tef plant height but CCC treatment did not affect grain yield. Stem diameter was
not affected by PGR treatment and also not the poor tapering (acropitally increasing
diameter).
Putatively transformed E. tef plants carrying a bean GA 2-oxidase (PcGA2ox) coding
sequence were further produced via embryogenic callus after Agrobacterium-mediated
transformation and plants were successfully grown into mature fertile plants. Eight putative
transformed plants were finally generated carrying the insert (PcGA20 ox or nptII gene
vii
sequence) at the T0 generation. Constitutive expression of the GA 2-oxidase (PcGA2ox)
coding sequence in E. tef resulted in phenotypic changes such as reduction in culm height,
change in biomass, reduction in amount of GA in putative transformant semi-dwarf plants.
The challenges found in the transgene dectection in the T1 generation has been highlighted.
Pheno-morphic changes occurred with little or no effect on yield.
Genes involved in height control (orthologs to the rice sd-1 gene) and signaling (Rht) in E. tef
were also identified and characterized. Activity of the protein for the putative rice sd-1
orthologs was further confirmed by heterologous expression. The three putative sequences in
E. tef were named EtGA20ox1a, EtGA20ox1b and EtGA20ox2. Expression analysis showed
that EtGA20ox2 were much less transcribed compared to the others and EtGA20ox1b could
be the functional equivalent to the rice sd-1 (OsGA20ox2) gene in E. tef.
Further, E. tef mutants with a semi-dwarf phenotype could be developed through mutagenesis
and TILLING. However, regardless of height, grain yield was severely reduced in all mutants
except in the semi-dwarf mutant GA-10. This line also had significantly higher diameter in
most internodes which might contribute to the stiffness of stem. G-10 is therefore a promising
line for further investigations.
viii
Thesis composition
Chapter 1 of this thesis provides a summary of the lodging problem in cereals and
alternative methods (chemical and genetic approaches) used to control lodging as well as the
traits involved. An up-to-date review of the lodging problem in E. tef including phenomorphic features relating to lodging and experiences in other crops as well as in the “green
revolution” are outlined. Approaches solving the lodging problem and genes that play a key
role in plant architecture modification in cereal crops for improving lodging resistance are
discussed. The rationale, aim, and objectives for carrying out this study are further outlined at
the end of the introduction. In Chapter 2, results obtained from treatment of E. tef plants
with GA biosynthesis inhibitors in controlling plant height are presented. This includes
treatment with GA3, CCC and Paclobutrazol and changes in plant height and other phenomorphic and agronomical features due to PGR treatment are reported. Chapter 3 reports
about transforming E. tef plants using immature somatic embryos via embryogenic callus for
Agrobacterium-mediated transformation. Successful regeneration of putative transformed
plants after a transformation procedure using combinations of different media is outlined.
Moreover, characterization of plants over-expressing GA2 oxidase from Phaseolus coccineus
(PcGA2ox1) for inducing dwarfism is presented and results of characterizing putatively
transformed T0 generation plants regarding their morpho-physiological features and
expression of a semi-dwarf phenotype with reduced height are reported. The inconsistent
PCR results at T1 and the possibility that any found differences could also be due to
somaclonal variations owing to the relatively higher rate of auxin applied is indicated.
Chapter 4 outlines the identification, and characterization of height-controlling genes. This
includes the rice homologous SD-1 in E. tef, the wheat Rht orthologue and two Cytochrome
P450 monooxygenase genes (Eui and Brassinosteroid deactivation genes). Also an activity
ix
assay through heterologus expression of EtGA20ox1 in E. coli and specific tissue expression
of the three EtGA20ox homologs as well as copy number of these genes in the E. tef genome
are presented. In Chapter 5, data on phenotype (plant stature) characterization is outlined for
selected mutant E. tef lines developed through mutagenesis and TILLING to generate
sufficient variability for semi-dwarfism in E. tef for lodging resistance. Morphological and
physiological attributes and agronomic relevance of these mutant lines are described in terms
of plant height reduction, tillering, biomass and yield. Chapter 6 finally summarizes the
findings and relevant information developed in this PhD study. It also outlines the salient
features that need to be considered further in a lodging-resistant E. tef ideotype. This is
followed by the list of citations (References) used in this dissertation. The Appendix
provides further sequence results (nucleotides and translated amino acid) from the gene
cloning and characterization study, alignment and phylogentic analysis of E. tef sequences
with different species.
x
ACKNOWLEDGEMENT
First of all, I thank The Lord Almighty for His abundant love and grace, his inspiration
providing me with the wisdom needed to pursue science with enthusiasm. PRAISE YOUR
NAME O LORD!
My deepest gratitude goes to my mother, Askale Nida and my father Gebre Kedisso for their
love and caring implanting in me so much good things. I feel proud of my elder and younger
brothers and sisters for their constant prayers and the pure love we share.
I express my deep gratitude to my supervisor Prof. Karl Kunert for his follow up, guidance
and critical help and inputs encouraging me all throughout my study period. Thank you also
for creating opportunities to attending conferences and research visit to Rothamsted Research
in UK and for creating linkages to research groups in different countries. All this have
considerably facilitated my study and contributed to acquiring skills. Thank you for your
great concern, patience and kindness.
My earnest gratitude also goes to my co-supervisor Dr. Urte Schlüter for her very valuable
inputs, advices and encouragement during the course of my study. Thank you also for the
help creating opportunity linking my research to groups at Rothamsted, UK which has been
very valuable to my study and for successfully finishing my experiments.
I am also very grateful to Prof. Peter Hedden for the supervision and guidance during my six
month stay at Rothamsted Research working in his lab. It was a very valuable time I spent. I
am also indebted to Dr. Simon Volghur for his kind assistance in molecular analysis and
xi
technical help during and after my visit. I also would like to thank colleagues at Molecular
biology lab and Experimental Greenhouse at Rothamsted Research for their kind support.
I also am very grateful to Prof. Maryon J Mayer, Head of the Plant Science Department, and
Rene Stewart Executive Secretary of the department for the positive and kind help and
encouragement in times of need during my study. I also thank the Forestry and Agricultural
Biotechnology Institute (FABI) management and team for the very good working
environment I enjoyed to be part during my study period.
I would like to thank Dr. Solomon Assefa, Director EIAR, for his support and positive
consideration of my extension request and Dr. Adefris T/Wold who shared with me the idea
of working on tef as well as for his kind help and encouragement during the study period. I
would also like to thank Dr. Likyelesh Gugssa for the very valuable help providing me with
technical information on tef transformation and other materials and Dr. Kebebew Assefa
providing me with valuable publications on tef at the beginning of my study. I am also very
grateful to Dr. Zerihun Tadelle for his cooperation to work on some of his mutagenized lines
for my experimentation.
An earnest thank you to my entire lab mates in SA, Berhanu, Rosita, Priyen, Ryhanrd,
Stephan, Magdeleen, Celia, Tsholofelo, Kutzai and Abigel for the nice environment of
interactions we kept alive in the lab, for the concern and help during the long hours of work
together in those years. I am grateful to my FABI friends for the kindness during my study
period. I am particularly very thankful to the unreserved help I received from Tuan, Magriet
and Markus.
xii
I want to sincerely thank my Ethiopian friends studying at UP for the warm friendship and
good social environment we enjoyed together. I thank Ethiopian students who lived in Tuks
Dorp, UP namely Berhanu, Dr. Wubetu, Dawit, Dr. Legesse, Hiywot, Dr. Temesgen, Ato
Habtamu, Dr. Yemaneh, Meheretu, Yebeltal and others for their friendship and support. After
moving to Sunnyside, I missed a lot the football game we established and the small evening
running we used to do. I am also very thankful particularly to Yohannes (John), Dr. Abayneh,
Tedlaye and their families for the unforgettable time my wife and I had together with them. I
also thank Kebron Church leaders and Christian friends in the Ethiopian Christian Fellowship
for the great time we had together that made our stay in Pretoria such a blessing.
This PhD research work was sponsored by the Ethiopian Institute of Agricultural Research
(EIAR), Rural Capacity Building Project (RCBP) and partly by the Rothamsted International
(RI) and was carried out at the Food and Agricultural Biotechnology Institute (FABI),
Department of Plant Sciences, University of Pretoria and at Rothamsted Research, UK. I am
very grateful to the above institutions that constituted the financial backbone for my study.
xiii
DEDICATION
I dedicate this thesis to my wife Meseret Worku, whose understanding, love, kindness and
help enabled me to pursue the work to the end; and to my parents, brothers and sisters who
occupy such a special place in my heart for their love and inspiration uplifting my spirit
through their prayers.
xiv
ABBREVIATIONS AND SYMBOLS
%
µg
µL
2-ODD
bp
BSA
CCC
CCM
CaMV
cDNA
cDNA
CPS
Ct
CTAB
DMSO
DNA
DNase
dNTP
DZ
E. coli
EDTA
EMS
ER
EUI
g
GAn
GA-ox
GA 2-ox
GAI
gDNA
GGPP
GID
GUS
h
H2O
IE
IPTG
KAO
KO
KS
L
LB
LCM
Percentage
Microgram
Microlitre
2-Oxoglutarate dependent dioxygenase
Base pair
Bovine serum albumin
Chlormequate chloride
Co-cultivation medium
Cauliflower mosaic virus
complementary deoxyribonucleic acid
Complimentary DNA
ent-copalyl diphosphate synthase
Cycle threshold
hexadecyl-trimethyl-azanium bromide
dimethyl sulphoxide
Deoxyribonucleic acid
Deoxyribonuclease
deoxy nucleotide triphosphate
Debre Zeit
Escherichia coli
Ethylenediamine tetra acetic acid
ethylmethanesulphonate
Endoplasmic reticulum
elongated uppermost internode
Grams
Gibberellin An
Gibberellin -oxidase
Gibberellin 2-oxidase
Gibberellin insensitive
genomic deoxyribonucleic acid
geranyl-geranyl diphosphate
Gibberellin insensitive dwarf
β-Glucuronidase
hours
Water
Immature embryo
Isopropyl-β -d-thiogalactopyranoside
ent-kaurenoic acid oxidase
ent-kaurene oxidase
ent-kaurene synthase
Litre
Luria broth
Laser capture microdissection
xv
M
min
mL
mM
NaAC
NaCl
NaOH
ng
NTC
o
C
ORF
PBZ
PcGA2ox
PCR
PPFR
qRT-PCR
RACE
RGA
RHT
RNA
RNAase
rpm
s
sd H2O
SD1
SDS
SLN
SNP
TILLING
UV
v/v
w/v
wk
Molar
minute
Millilitres
Millimolar
Sodium acetate
Sodium chloride
Sodium hydroxide
Nanogram
No-target control
Degree Celcius
Open reading frame
Paclobutrazol
Phaseolous cuccinous GA 2-oxidase
Polymerase chain reaction
Photosynthetic photonflux rate
quantitative reverse transcriptase polymerase
chain reaction
Random Amplified cDNA Ends
repressor of ga1-3
Reduced height
Ribonucleic acid
ribonuclease
Revolutions per minutes
Second
Sterile distilled water
semi-dwarf 1
Sodium dodecyl sulphate
Slender
Single Nucleotide Polymorphism
Targeting induced local lesions in genomes
ultra violet
volume per unit volume
weight per unit volume
week (s)
xx
LIST OF FIGURES
Figure 1.1
Phenotypic variations for allelic diversity for semi-dwarfing traits in the green
revolution genes from wheat (Rht) and rice (sd-1)
8
Figure 1.2
Partial protein sequence encoding Rht-B1a and Rht-B1b loci from wheat
showing the few amino acid internal deletion that contributed to wheat semidwarfism
8
Figure 1.3
Simplified scheme of the GA biosynthesis steps and points of inhibition by
growth retardants
12
Figure 1.4
Simplified scheme of the gibberellins (GA) biosynthesis pathways and
deactivation by GA 2-oxidase in plants
19
Figure 1.5
E. tef plant stand in the field at (A) grain filling and (B) at maturity when
almost all plants lodged
23
Figure 2.1
Growth (plant height) response to exogenous application of GA and CCC after
six weeks of Gea Lammie and DZ-01-196 plants grown in greenhouse.
35
Figure 2.2
Tillering, fresh and dry weight responses of six weeks old DZ- 01-196
seedlings to CCC treatment
36
xvii
Figure 2.3
Effect of foliar applied CCC on fresh and dry weight of six weeks old
seedlings
37
Figure 2.4
Effect of CCC (100mM) and PBZ (100µM) on plant height near plant maturity
42
Figure 2.5
Comparison of plant height and panicle growth at plant maturity as affected by
PGRs
43
Figure 2.6
Effect of different concentrations of CCC and PBZ on biomass: fresh weight
(FW) or dry weight (DW) per plant in comparison to biomass of untreated
control plants
46
Figure 2.7
Effect of different concentrations of CCC and PBZ on primary (1o) and
secondary (2o) tiller grain yield per plant in comparison to the untreated
control
47
Figure 2.8
Comparison of panicle elongation with PBZ treatments in proportion to culm
reduction in DZ-01-196
48
xviii
Figure 3.1
Construction of plasmid pGPTV-Kan harbouring Pc2ox1 and the triple 35S
CaMV promoter sequence
64
Figure 3.2
E. tef shoot regeneration using immature embryo from young emerging panicle
as explants
74
Figure 3.3
PCR amplification of PcGA2 ox and nptII sequences from putative transformed
plants of T0 and T1 generation
76
Figure 3.4
Culm and panicle height (cm) and number of tillers per plant of putatively
transformed dwarf E. tef plants
78
Figure 3.5
Selected E. tef dwarf (18) and semi-dwarf T1 generation plants (19(3), 19(2),
28 and 10)
79
Figure 3.6
Seed weight of primary and secondary panicles of dwarf E. tef lines and a
control
80
Figure 3.7
Comparison of endogenous GA levels in the GA biosynthetic pathway between
dwarf and controls plants
83
Figure 4.1
Nucleotide sequence alignment of three putative tef GA20ox sequences
xix
108
Figure 4.2
Derived nucleotide sequence alignment of the two tef GA20oxs (GA20ox1
and GA20ox1b) sequences with sequences from other cereals
110
Figure 4.3
Derived nucleotide sequence alignment of tef GA20ox2 sequence with
orthologous GA20ox gene sequences from other cereals and Arabidopsis
112
Figure 4.4
Molecular
phylogenetic
analysis
of
the
homologous
of
E.
tef
GA20oxsequences (GA20ox1, GA20ox1b and GA20ox2)
114
Figure 4.5
Detection of EtGA20 ox gene copies in the E. tef genome after restriction
enzyme digest using Southern Blot
115
Figure 4.6
Relative expression of GA20ox1 in two E. tef genotypes DZ-01-196 and Gea
Lammie in different plant tissues and growing stages
116
Figure 4.7
Semi quantitative RT-PCR expression analysis of three EtGA20ox genes in
various plant tissues in variety DZ-01-196
117
Figure 4.8
Radiochromatograms after HPLC of E. tef GA20ox1 activity products after
incubation with a 14C-labeled GA12 as a substrate
xx
118
Figure 4.9
Amino acid sequence alignment of the putative tef Rht sequences with
orthologous amino acid sequences from other species
120
Figure 4.10
Molecular Phylogenetic analysis of E. tef Rht peptide sequences by Maximum
Likelihood method
122
Figure 5.1
Culm and panicle length and seed weight (gm) in plants of different mutant
lines and the control
136
Figure 5.2
Culm length of plants of different mutant lines and wild type plants with and
without GA treatment
137
Figure 5.3 Effect of GA application on peduncle and panicle length of mutant
lines and the control
141
Figure A.1
E. tef GA20ox1 full coding region and deduced amino acid residues of E. tef
GA20ox1 sequence with conserved domains.
177
Figure A.2
Homologous sequences of E. tef GA20ox1a and GA20ox2 partial coding
region, and deduced amino acid residues
178
Figure A.3
Partial coding region of the Elongated Uppermost Intenode (EUI) gene in E.tef
Figure A.4
xxi
180
Derived nucleotide sequence alignment of the putative tef Uppermost
Elongated Internode (EUI) with ortholog sequences from other cereals
181
Figure A.5
Molecular phylogenetic analysis of the rice EUI ortholog gene in E. tef
182
Figure A.6
Full coding region of the E. tef RHT gene near full coding region (1397 bp) and
deduced amino acid residues with characteristic DELLA, VHYNP and
VHVVD domains
183
Figure A.7
Putative E. tef brassinosteroid deactivation related Cytochrome P450
monooxygenase gene partial nucleotide sequence
184
Figure A.8
Amino acid sequence alignment of the putative brassinosteroid deactivating
gene sequences from E. tef with amino acid sequences from other species
185
Figure A.9
Molecular Phylogenetic analysis of putative E. tef brassinosteroid deactivation
gene sequence
187
xxii
LIST OF TABLES
Table 1.1
Semi-dwarf sources in wheat induced by chemical or physical mutagens
16
Table 2.1
Quantification of GA intermediates and bioactive forms available in two E. tef
plant varieties at stem elongation
39
Table 2.2
Effect of CCC and PBZ on culm and panicle length, number of tillers and seed
weight per plant of E. tef cv. DZ-01-196
41
Table 2.3
Effects of CCC and PBZ on length of different internodes of tef var. DZ-01196
44
Table 2.4
Effect of CCC and PBZ on diameter of different internodes of E. tef var. DZ01-196
45
Table 2.5
Effect of CCC and PBZ on dry weight of culm and panicle tillers of E. tef var.
DZ 01-196
49
Table 2.6
Correlation coefficients for morphological and yield components of E. tef var.
DZ-01-196
50
Table 3.1
xxiii
Media used for induction of embryogenic callus, co-cultivation, selection and
regeneration of transformed E. tef shoots
69
Table 3.2
Composition of the K99EM medium used for embryogenic callus induction
from E. tef immature embryos
70
Table 3.3
Ionic composition of the K4NB regeneration media used for tef immature
embryo cultures
71
Table 3.4
Survival of non-transformed E. tef seedlings derived from mature embryo on
antibiotic (G418)-containing selection medium
75
Table 3.5
Above ground biomass of putatively transformed dwarf tDZ-01-196 E. tef
plants
81
Table 4.1
Primers used in PCR to amplify GA20ox gene fragments using E. tef genomic
DNA
94
Table 4.2
Primers used or GA20ox sequence RACE- PCR amplification
97
Table 4.3
Primers used in quantitative and semi-quantitative PCR amplification of
GA20ox sequences
101
Table 5.1
xxiv
Peduncle length, internode number, number of tillers, culm and panicle dry
weight per plant of mutant E. tef lines
135
Table 5.2
Response of mutant lines in terms of internode length to exogenously applied
GA3
139
Table 5.3
Peduncle length, internode number, number of tillers, culm and panicle dry
weight per plant of mutagenized E. tef lines
142
Table 5.4
Internode diameter of mutant lines in response to exogenous GA treatment
143
Table A.1
Primers used for PCR amplification of gene fragments of Rht, Eui and BR gene
sequences using E. tef genomic DNA
179
Table A.2
Rht primers used in RACE- PCR
180
xxv
CHAPTER 1
INTRODUCTION
1
1.1
The problem of lodging
Lodging, as a mechanical plant stress, is a complex interaction of the plant with the
environment including factors such as light, wind, temperature, rain, topography, soil type,
nutrition, plant density and diseases (Berry et al., 2004) and is frequently associated with
plant pheno-morphic characteristics (Pinthus, 1973). It is a process by which the shoot of a
small grained cereal is displaced from their vertical stance. The standing strength of the stem,
or the root, to hold up the shoot leverage is threatened by adverse weather conditions.
Lodging is also variety (genotype)-dependent and a tall, weak-stemmed variety is more prone
to lodge than a semi-dwarf variety with stiffer stem. It occurs in the form of stem lodging or
failure of the anchorage system (root lodging) (Pinthus, 1973; Thomas, 1982; Crook and
Ennos, 1993; Graham, 1983 and Delden et al., 2010).
Lodging, causes a direct yield loss due to falling-over of the plant and also an indirect loss
due to limiting optimum use of nitrogen fertilizer. Problem of reduced lignifications of the
stem also reduces stem strength following high nitrogen fertilizer use, which is counterproductive, and any increase in grain biomass is offset by increased lodging (Crook & Ennos
1994). Lodging also restricts mechanical seed harvesting and reduces seed quality.
Furthermore lodging can also limit efficient light interception; reduce translocation and
assimilation, increase respiration and chlorosis thus affecting growth and development
processes in the plant system (Berry et al., 2004). Because of the significance in the global
agricultural economy, wheat and rice were the two main field crops in which it was desirable
to substantially reduce significant yield losses due to lodging of tall, wild-type genotypes.
Applying nitrogen fertilizer was not successful, since it aggravated the lodging problem.
Shorter, sturdier, semi-dwarf varieties were developed that have been far more resistant to
2
lodging and also exhibited an unexpected benefit of improved assimilation into reproductive
organs due to pleiotropic effects (Hanson et al., 1982). Thus an essential trait of the higheryielding varieties was reduced (dwarf) stature that enabled large increases in yield to be
obtained.
Therefore, in many cereal crops, plant height has been the main target for improvement of
lodging resistance incorporating the trait in modern cultivars. For instance the semi-dwarf
wheat variety from Japan (Norin 10) contains the most important dwarfing genes that confer
reduced height trait used today in wheat. This variety originated from a cross between the
native Japanese dwarf variety Daruma with two American wheat varieties, Fultz and Turkey
Red. The genes conferring semi-dwarfing traits are for culm shortening with a semi-dominant
gain-of-function mutation in the REDUCED HEIGHT (RHT) homologous genes in wheat
(Gale and Marhsall, 1976). These genes were introduced into many other varieties grown in
many parts of the world (Silverstone et al., 2001) and 70% of modern wheat varieties carry at
least one of the dwarfing genes (Hedden, 2003). In rice, the recessive semi-dwarf (sd-1) gene
has been used to reduce plant height and considerably improved lodging resistance. The
mutated sd-1 in rice existed in semi-dwarf native varieties. In the 1960s the dwarfing trait
was incorporated into improved rice lines to develop a semi-dwarf phenotype in rice
(Ashikari et al., 2002). In barley, the value of short variety was understood earlier and shorter
barley varieties such as ‘Valticky’ that originated from local landraces was widely grown
replacing taller varieties in Moravia at the beginning of the twentieth century (Bouma and
Ohnoutka 1991).
Generally, taking in to account wheat and rice, mutations in the Rht and sd-1 genes have been
among the main factors responsible for higher yields obtained in the “green revolution”
3
(Yamaguchi, 2008; Kashiwagi and Ishimaru, 2004; Hedden, 2003). As a result the average
yield in wheat has increased from 2.2 t ha-1 to 6.0 t ha-1, whereas in rice yield has increased
due to introduction of semi-dwarf varieties (Berry et al., 2004) from 1.5 t ha-1 to 4.2 t ha-1.
Thus the identification and subsequent introgression of stem height controlling genes was
considered the principal factors for reduced lodging allowing higher amount of fertilizer use
in wheat and rice during the 70s of the last century (Tong, 2007; Kashiwagi and Ishimaru,
2004; Berry et al., 2004; Hedden, 2003).
1.2
Plant architecture and lodging
Plants architectures refers to the degree of branching, internodal elongation, and shoot
determinacy (Wang and Li, 2008).The architecture of plants is linked to plant functions
including efficient water transport, light interception, soil resource acquisition, and the
maintenance of a mechanically stable structure. To optimize their functions plants keep
building their architecture gradually in response to changes in environmental conditions
(Sterck, 2005). When plants lose its optimum architecture orientation or vertical stance
required for optimal function in a given set of environment, due to mechanical failure,
conditions that promote plant growth is interrupted. A favourable environment is created for
diseases increasing harvesting cost and yield loss. The culm structure, such as length,
diameter, shape, composition, degree of branching, leaf arrangement and orientation,
internodal elongation, and shoot (determinate or not), define the type of architecture of a
crop. These factors determine the specific pheno-morphic and physio-morphic features and
define the specific interaction the plant may have with environmental elements such as wind,
rain, soil, nutrient and light (Berry et al., 2004).
4
Lodging occurs when the stem strength, which depends on stem diameter and the
composition and width of the stem wall, is insufficient to hold the shoot up against leverage.
It also occurs due to failure of the anchorage system, which depends on the spread and depth
of the root plate and the strength of surrounding soil, causing the root to have insufficient
strength to hold the shoot up. Stem lodging takes place in the form of “buckling” of the
lower internodes or the middle internodes called “brackling” (Thomas, 1982; Neenan and
Spencer-Smith, 1975), or the breaking of the peduncle known as “necking” found in wheat,
barley, oats (White, 1991). It can also occur in the form of root lodging (failure of anchorage
system) which results in a permanent displacement of the stem found in wheat, barley, and
oats and also in E. tef (Pinthus, 1973; Crook and Ennos, 1993; Graham, 1983 and Delden et
al., 2010). Berry et al. (2004) have further shown that root lodging could be predominant.
Reports, however, have often been in favour of stem (straw) lodging indicating that
misconception can occur about the reason of failure in lodged crops. In E. tef grown in sandy
soils, root anchorage failure and insufficient stem strength have been found (Delden et al.,
2010). However, earlier studies with E. tef have shown that stem length thickness or diameter
of basal internodes, panicle length and weight, and earliness to be important traits for lodging
resistance (Berhe, 1981; Ketema, 1983; Mengesha et al. 1965; Hundera et al., 1999; Asefa et
al., 2000 and Yu et al., 2007).
Further, optimum fertilizer application for increasing grain yield promotes lodging due to
increasing plant height. However, under conditions of high fertilizer (N) and moisture,
varieties having a semi-dwarf stature are less prone to lodging. If semi-dwarf varieties are
further have thick-straw, resistance to lodging is greatly improved.
5
1.3
1.3.1
Genetic control of lodging resistance
GA genes and lodging
The first indication that GAs were endogenous growth regulators in plants was reported in the
1950s, after study showed the height of dwarf pea and maize mutants were restored to normal
by applying gibberellic acid (GA3) (Hedden and Phillip, 2000). Among the many influences
of GA genes on the plant growth and development, their ability to promote internodal
elongation in a wide range of species that belong to the grass family has been of considerable
agronomic importance (Taiz and Zeiger, 2006). Basically the importance of GA was
demonstrated by the discovery of GA-deficient (reduced bioactive GA amounts) or GAinsensitive mutants in several species, including rice (Murakami, 1970), wheat (Maluszynski
and Szarejko, 2005), maize (Phinney, 1956), and Arabidopsis (Koornneef and Van der Veen,
1980). These mutants had typically a dwarf or a semi-dwarf phenotype with a reduced
bioactive GA amount in case of GA-deficient mutants or high bioactive GA concentrations in
certain GA-insensitive dwarf mutants, such as Rht3 wheat and Dwarf-8 maize (Hedden and
Kamiya, 1997) due to a negative feed-back regulation (Alvey and Harberd, 2005). The use of
highly sensitive methods of physio-chemical analysis, such as gas chromatography and mass
spectrometry (GC-MS), has shown that the GAs are large group of natural products, up to
126 different compounds currently known. However, based on the analysis of the GAdeficient mutants only few GAs have intrinsic biological activity and a hormonal actions
(Hedden and Phillip, 2000).
6
Identification and isolation of the homologous sd-1 (GA-deficient ) and Rht (GA-insensitive )
genes in Arabidopsis has further helped to understand the role of GA genes in plant height
control. The sd-1 gene encodes a GA biosynthetic enzyme, GA20 oxidase, and the rice
genome carries four GA20- oxidase genes, GA20ox1-4. The sd-1 corresponds to GA20ox-2,
which is highly expressed in leaves and flowers (Yamaguchi, 2008). The enzymatic action of
the two other oxidases is not well-known (Hedden et al., 2002). GA 20-oxidase is a
regulatory enzyme with multifunctional catalytic activity acting at several stages in the
biosynthesis process and the oxidase is further a prime target in the genetic manipulation of
the GA biosynthetic pathway (Hedden et al., 1998). The function of RHT was only identified
as a result of molecular genetic studies on the analogous gibberellic acid-insensitive (gainsensitive) gai mutant of Arabidopsis. GAI encodes a GA response repressor gene in the GA
response pathway which functions in the absence of GA (Peng et al., 1997). The Arabidopsis
GAI dwarf mutants have further been found to be orthologs of the maize dwarf (D8) genes
(Fu et al., 2001). Unlike sd-1, the phenotype of these dwarf mutants could not be restored to
the wild-type by exogenous GA application due to a mutation in the GA response pathway
(Taiz and Zeiger, 2006). Further studies revealed that RHT has multiple allelic variants
resulting in variations of the RHT mutant (Figure 1.1). Three amino acid deletions and
introduction of a stop codon at the N-terminus of its coding region further resulted in a semidwarf phenotype (Figure 1.2).
7
A
Rht-D1c
Rht-B1c
Br2-Dwarf
Rht-D1d
Rht-D1b
Rht-B1b
Rht-B1d
Mercia
B
Deegeo-woo-gen
(sd1)
Woo-gen
Figure 1.1 A) Phenotypic variations for allelic diversity for semi-dwarfing traits for the
wheat Rht gene (except Br2-dwarf which is a brassinosteroid insensitive dwarf) (Peng et al.
1999 and Pearce et al. (unpublished)) and B) for the semi-dominant (sd-1) semi-dwarfing
gene in rice (Monna et al. 2002, Sasaki et al. 2002 and Spielmeyer et al. 2002).
STOP codon introduced
Re- initiation of
translation?
Figure 1.2 Partial amino acid sequence of the wheat protein encoded by the Rht-B1a and
Rht-B1b loci with amino acid internal deletions of the allele causing the semi-dwarf
phenotype.
8
Dwarfing genes are grouped on their response to applied GA. Mutants of the biosynthetic
pathway, such as rice sd-1, are GA sensitive and the phenotype of the wild-type can be
restored by exogenous application of GA. Dwarf mutants of the GA response, such as wheat
Rht-B1b and Rht-D1b, the maize dwarf 8 (D8) or its ortholog in sorghum dwarf3 (dw3) and
the Arabidopsis GAI, are insensitive to applied GA. In recent years more mutants related to
either sd-1, Rht or other genes in GA metabolism have been identified causing dwarfism in
wheat and rice (Milach et al., 2002; Xu et al., 1995; Carrera et al., 2000; Hedden et al.,
1998).
In gibberellin response mutants, three main classes of mutations have been identified
affecting plant height. These are (a) gibberellin-insensitive dwarfs, (b) gibberellin-deficient
mutants in which the plants can be reversed closer to normal by co-expression of a second
“suppressor” mutation, and (c) mutants with a constitutive gibberellin response also called
“slender” mutants (Taiz and Zeiger, 2006). Examples for gibberellin-insensitive dwarfs are
the wheat “green revolution” mutants with a mutation in the Rht-1 and Rht-2 genes and their
orthologs in maize, Dwarf8 (D8), and in Arabidopsis, Gibberellic acid Insensitive – GAI,
were also found to confer a semi-dwarf phenotype. The Rht locus encodes a repressor protein
(GAI) in Arabidopsis inhibiting stem elongation in the absence of GA. The Arabidopsis
dwarf mutant (gai) protein has a 17 amino acids deletion rendering it insensitive to foliar
application of GA (Fu et al., 2001 and Peng et al., 1999).
A mutation in the rice sd-1 (GA20ox-2) coding region (280 bp deletion) is a loss of function
mutation causing a semi-dwarf phenotype when the mutated gene was expressed in other
crops such as Arabidopsis and potato (Spielmeyer et al., 2002). Rice plants with the mutation
also had a greater harvest index allowing for increased use of nitrogen fertilizers. However,
9
the presence of multiple sd-1 alleles prevented severe dwarfing due to a partial inhibition of
GA production (Yamaguchi, 2008). In Arabidopsis, which carries three GA20 oxidases, such
functional redundancy after mutation, and with GA still produced in other plant parts, caused
a semi-dwarf phenotype (Spielmeyer et al. 2002).
1.3.2
Manipulation of plant height using GA genes
The key role shown above that GA metabolic genes play in plant architecture has made them
prime targets for genetic manipulation. Characterization of these genes has paved the ground
for geneticists and physiologist to target specific metabolic pathways in the production of
higher yielding and hardier plants. However, from the agronomic point of view, not all the
genes involved are of interest (Yamaguchi, 2008; Hedden and Phillips, 2000). Changing or
manipulating the endogenous bioactive GA amount allowed the design of crops with a better
morphological architecture. This approach offers an alternative strategy to introduce
beneficial traits, such as dwarfism, into cereal varieties to improve grain yield.
Modifying the endogenous amount of bioactive GA might occur either through genes
contributing to the production of the bioactive GA or through genes diverting GA forms to
inactive molecules. This includes catabolic inactivation of bioactive GA forms or some of its
precursors (Hedden and Phillips, 2000). Studies by Sun and Kamiya (1994) and Fleet et
al.(2003) have shown that over-expression of the genes encoding enzymes that catalyse the
early stages of GA biosynthesis, e.g. ent-copalyl pyrophosphate synthase (AtCPS) and entkaurene synthase (AtKS) in Arabidopsis, do not significantly increase amounts of bioactive
GA with no effect on plant growth and development. Over-expression of genes downstream
pathway, such as the GA 20-oxidases that are multifunctional and highly regulatory enzymes
10
(Figure 1.4), increased stem elongation, early flowering and decreased seed dormancy
indicating that their activity limits GA biosynthesis (Lang, 1998). Suppression of the different
GA 20-oxidase homologous genes in Arabidopsis through RNAi expression produced
changes in the different parts of the plant showing their tissue specific role (Coles et al.,
1999). Over-expression of its own GA 20-oxidase in Arabidopsis resulted in the elongation of
seedling hypocotyls, increased shoot growth, induced early flowering, and increased GA4
level (Huang et al.1998; Coles et al. 1999). However, over-expression of the same gene from
citrus or Arabidopsis caused an increased amount of bioactive GA and elongated phenotypes
in hybrid aspen and tobacco plants (Eriksson et al., 2000; Vidal et al., 2001; Biemelt et al.,
2004). In potato, Carrera et al. (2000) showed that antisense mRNA expression of a GA20ox
gene reduced stem elongation and increased both tuberization and tuber yield. Studies by
Israelsson et al., (2004) and Phillips (2004) further indicated no difference in the morphology
of transgenic plants following GA 3-oxidase over-expression in hybrid aspen and
Arabidopsis. In rice, antisense copies of GA3ox2 (D18) reduced the final GA amount and
caused semi-dwarf phenotypes in some of the transformants (Itoh et al., 2002). This
phenotype, however, was not stably transferred to the progeny possibly due to gene silencing.
Lowering of the endogenous GA amount is also possible through increasing the expression of
GA 2-oxidase, a positive feed forward regulation enzyme, which catabolises bioactive GAs
and some precursors. After its first isolation using cDNA from runner bean (Phaseolus
coccineus) by a functional screening method (Thomas et al., 1999), an ectopic expression of
OsGA2ox1 gene in rice resulted in reduced stem growth with small, dark green leaves with
reproduction organs severely defective (Sakamoto et al., 2001). However, expression of the
same gene under the control of the shoot-specific OsGA3ox2 promoter induced only a semidwarf phenotype with normal flower and grain development (Sakamoto et al., 2003).
11
Figure 1.3 Simplified pathway of the gibberellin (GA) biosynthesis and deactivation in
plants. Most targeted genes in inducing dwarfism are the 2-ODD family multifunctional
genes catalyzing several steps at the intermediate ( ) and late ( ) steps of the
biosynthesis pathway catalyzed respectively by GA20ox and GA2ox. In addition to
inactivating the bioactive GAs (GA1 and GA4), the GA 2-oxidase also deactivates
intermediates GA9 and GA29. (Adapted from Oikawa et al., 2004)
12
Severe dwarf phenotypes were obtained in Arabidopsis, tobacco and poplar through overexpression of the rice GA 2-oxidase gene (Schomburg et al., 2003; Biemelt et al., 2004;
Busov et al., 2003). Plants with a dwarf phenotype were also produced by over-expression of
a runner bean GA 2-oxidase in Arabidopsis and wheat and Hedden and Phillips (2000)
suggested the superiority of this approach for the breeding of dwarf plants.
1.4
Brassinosteroid genes and lodging
Mutated genes of the brassinosteriod metabolism also causes a dwarf phenotype. The
brachytic2 (br2) mutant in maize and its ortholog in sorghum, dwarf3 (dw3), induce short
internodes (Multani et al., 2003). In barley, uzu dwarfism caused by the missense mutation in
the HvBRI1 gene is a mutation in the brassinosteroid receptor protein resulting in a semidwarf phenotype (Chono et al., 2003). Several other BR deficient and BR-insensitive mutants
have been identified with phenotypic changes including dwarfism, small dark-green leaves, a
compact rosette structure, delayed flowering and senescence and reduced fertility (Sasse,
2002). The practical use of the BR has been limited due to BR deficiency leading to severe
and defected dwarfism and reduced fertility (Divi and Krishna, 2009). However, controlled
changes i.e. slight decrease in BR levels or in BR signaling was found causing significant
increase in yield as a result of change in plant architecture (Divi and Krishna, 2009).
In monocots, only BR-insensitive mutants have been identified (Yamamuro et al., 2000.). A
rice mutation in the C-22 hydroxylase, a BR enzyme involved in leaf inclination, resulted in a
semi-dwarf phenotype that increased above ground biomass by 40% (Sakamoto, 2006). In
tomato, a BR-responsive dwarf (d) mutant was found caused by inactivation of a cytochrome
P450 enzyme (CYP85A1) (Bishop et al., 1996). In Arabidopsis, overexpression of DWF4, a
13
gene that encodes a cytochrome P450 monooxygenase (CYP90B1) (Choe et al., 1998)
resulted in a dramatic promotion of vegetative growth and enhanced seed yields (Fujioka and
Yokota, 2003). In barley, an ortholog of BRI1, a BR-insensitive mutant due to single
nucleotide change in the BR-receptor gene produced a semi-dwarf phenotype with increase in
yield and lodging resistance. Generally, the current knowledge on BR regulation of growth
and development through altered BR activity is growing rapidly through characterization of a
wide variety of BR-deficient and BR-insensitive mutants (Fujioka and Yokota, 2003).
1.5
Induced mutations
Inducing mutation in crops has been long exercised to create variability in germplasm for
species and traits where there is little known variation for a trait of interest. In E. tef the
genetic diversity for lodging resistant traits has not been found to exploit through genetic
introgression into modern cultivars. In other cereal crops, however, inducing mutation to
improve genetic diversity have brought about renewed interest because it mimics natural
variation with relatively high frequency in the selected germplasm with desirable genetic
backgrounds (Maluszynski and Szarejko, 2003). The technique provides novel genes or
alleles (Table 1.1) of known phenotypes (Maluszynski and Szarejko, 2003) in better adapted
or a more desirable background (Konzak, et al., 1984). Through induced mutations, many
genes involved in metabolic pathways responsible for plant development and growth,
response to growth regulators and various biotic and abiotic stresses have been identified
(Barkley and Wang, 2008; Maluszynski and Szarejko, 2003). The most obvious and attractive
feature in inducing mutants is ease of inducing the genetic variation with relatively high
frequency, and very often the mutations mimic natural variations. The genetic changes are
made in advanced genotypes or in genotypes adapted to local environmental conditions or
14
having desirable background traits. Among economic traits largely targeted for improvement
using this technique include semi-dwarfness for conferring lodging resistance in cereal crops
(Barkley and Wang, 2008; Hu, 1973; Ullrich and Aydin, 1985; Maluszynski et al. 2003).
Semi-dwarfness is one of the most desirable traits targeted by induced mutations due to lack
of diversity in desirable phenotypic traits in the genetic pool of many important crops. In rice,
several semi-dwarf mutants, including stiff and lodging resistant, have been selected from
mutated populations and led to the release of important new varieties (Maluszynski and
Szarejko, 2003). Developing and selecting useful mutations involve random mutations for
qualitative traits coupled with large screens of the mutated plants. This requires sufficient
time (1- 2 years) and development of a high quality population. Such technical challenges in
mutation technologies render the method less attractive to many scientists (Barkley and
Wang, 2008; Baenzinger, 1988). In barley, a new semi-dwarf lodging resistance mutant
variety has been selected with an average mature height reduced to 87 cm from a tall, 120130 cm, phenotype. This increased yield by about 15% and with high input about 25%
(Maluszynski and Sigurbjörnsson, 1988; Rutger, 1981). Inducing dwarfism or reduced plant
height without losing the potential yield has been reported for wheat and other cereals (Table
1.1) (Barabäs and Kertész, 1988; Narahari, 1988).
Spontaneous mutation occur with an extremely low frequency, often unnoticed being difficult
to detect in species like the tetraploid E. tef. Thus genetic manipulation such as inducing
mutation in target genes using various mutagens can provide rapid generation and
enhancement of genetic variability. Inducing short stature mutants without changing the
background character of important traits will be extremely beneficial for developing lodging
resistance in E. tef.
15
Table 1.1 Semi-dwarf sources in Rht wheat induced by chemical or physical mutagens
(Maluszynski et al. 2001).
EMS= ethylmethane sulfonate; DES = ; MNH =
Source: Maluszynski and Szarejko, 2003
16
1.6
Plant growth regulators for plant height control
Plant height, particularly culm length, is considered to be among the major factors associated
with lodging sensitivity (Pinthus, 1973; Crook and Ennos, 1994; Berry et al., 2000). Control
of plant growth, such as plant or culm height, can be achieved chemically by using plant
growth regulators (PGRs). Many chemical growth promoters or retardants have been used to
treat crops and plants for controlling growth and development of vegetative or reproductive
parts. PGRs that inhibit gibberellins (GA) biosynthesis are used in high input cereal
management to shorten straw and thereby increasing lodging resistance. They have been
extensively used in many crops to reduce lodging through shortening of the stem and to
maintain a steady improvement in grain yield (Berry et al., 2004; Rajala, 2003). Among the
GA inhibitors that are used to control plant growth are the onium-type compounds, such as
chlormequate chloride (2-chloroethyl-N,N,N-trimethyl-ammonium chloride, CCC) and
mepiquat-Cl, interfering with ent-kaurene synthesis at the early stages of gibberellin
biosynthesis (Rademacher, 2000) (Figure 1.1). Inhibition of the cyclization of geranylgeranyl
diphosphate synthase (GGPP) into copalyl diphosphate synthase (CPP) due to CCC binding
to the enzyme CPP-synthase reduces the availability of bioactive GA (Hedden and Philips,
2000; Graebe et al., 1992; Rademacher, 2000).
A further group are nitrogen containing heterocycles such as triazoles and imidazoles. This
includes paclobutrazol (PBZ) and the closely related uniconazole-P. Both compounds
interfere in the oxidation of ent-kaurene to ent-kaurenoic acid (Rademacher, 2000). Inhibition
of oxidation of mono-oxygenases occurs by sharing lone pair electrons to displace oxygen
from the enzyme binding site at the proto-heme iron, this renders the oxygenase nonfunctional (Rademacher, 2000) (Figure 1.1). Commercially available PBZ in the (2S,3S)-
17
enantiomer is structurally similar to ent-kaurene. More recently developed GA inhibitors
include cimectacarps (trinexapac-ethyl), interfering with the late stages of GA metabolic
reactions mainly by inhibiting 3ß-hydroxylation of GA20 to produce the bioactive GA1
(Rajala, 2003). In general, the stem growth inhibition due to PGRs can be variable, depending
on species and genotypes (Rajala, 2003), and is further based on GA inhibitor mediated stem
shortening by interfering with synthesis of an intermediate precursors, ent-kaurene or entkauronic acid, or by inhibiting 3ß- hydroxylation of GA20 to bioactive GA1 (Graebe et al.,
1992; Rajala, 2003; Hedden et al., 2010).
18
MVA
PGA
Onium-type compounds
FPP
Nitrogen containing
heterocycles
Paclobutrazol
Ancymidol
Uniconazol- P
Florprimidol
Chlormeqat - Cl (CCC)
Mepiquat – Cl
AMO - 1618
GGPP
CPP
Ent-Kaurene
ent Kaurenic Acid
GA 12 Aldehyde
cyclohexanetriones
Prohexadione – Ca
Trinexapac - ethyl
Daminozide
GA 53
GA 19
GA 20
Active GA
Figure 1.4 Simplified scheme of GA biosynthesis steps and points of inhibition by
plant growth regulators. Broken line represents minor inhibitor activities
(Rademacher, 2000).
Reduction in plant height following application of PGRs is associated with reduced
endogenous bioactive GA amounts and reduced elongation of internodes particularly of the
uppermost internodes and peduncle (Sanvicente et al., 1999; Rajala, 2003). CCC inhibits
stem elongation in wheat by reducing up to 40% plant height (Humbries et al., 1965). In
oilseed rape, foliar treatment with a combination of CCC, ethephon and imazaquin reduced
main stem length by 7% in the field and 16% under greenhouse conditions. The uppermost
three internodes contributed significantly to the reduction (Sanvicente et al., 1999). In barley,
19
responses to CCC were genotype-dependent (Rajala, 2003), but when wheat or barley plants
were treated with CCC, the PGR had no effect when plants already contained dwarfing genes
such as Rht1, Rht2, or Dw6 (Abbo et al., 1987; Peltonen-Sainio and Rajala, 2001). PBZ
application reduced stem length in some rice cultivars by 90%, lodging from 60% (in
controls) to 0% and increased yields up to 15% when compared to controls (French et al.,
1990). PBZ and the closely related uniconazole-P are highly active PGRs with practical uses
in rice, fruit trees and ornamentals (Rademacher, 2000) and about 84% of the winter wheat in
UK is treated with PGRs (Berry et al., 2004).
PGR application (CCC treatment) has been found to increase lodging resistance due to an
increase in stem diameter (Tolbert, 1960). However, other research groups showed no change
in the content of structural compounds (cellulose, lignin and hemicelluloses) in the plant stem
following CCC and ethephon treatment (Clark and Fedak, 1977; Knapp et al., 1987). In
barley, no clear relationship was found between stem diameter and cell wall thickness of the
two basal internodes with lodging susceptibility (Stanca et al., 1979). Shortening of stem
after PGR treatment may also not necessarily result in reduced lodging as reported for wheat
and barley (Knapp et al., 1987; Ma and Smith, 1992).
Information about the use of PGRs in E. tef is very limited but increased yield following CCC
application at 0.7 - 2.0 l a.i. ha-1 has been reported but lodging was not prevented (Alkamper,
1970). This early E. tef result has been further supported by Berry et al (1998) applying CCC
to a lodging-prone crop and reduced lodging area only from 88 to 83% at harvest.
20
1.7 Lodging in E. tef
1.7.1
E. tef growth
Tef, Eragrostis tef (Zucc.) Trotter, is a small-seeded full grain cereal with high economic
importance in Ethiopia. It is the most resilient crop with low risk of failure (Tefera and
Ketema, 2001) grown under very diverse environments and exhibits high diversity in most
pheno-morphic and agronomic traits (Assefa, 2003). In Ethiopia, E. tef is grown on over 2.56
million ha1, accounting for about 28% of the total acreage and 19% of the gross grain
production of the major cereals (CSA, 2008). The lives of estimated over 50 million people
depend directly on E. tef as a staple food. E. tef grows on water-logged vertisol in the
highlands as well as water-stressed areas in the semi-arid regions (Takele et al., 2000).
Suitable growing rain-fed areas are reported to be those with a growing period of 100 - 150
days, rainfall of 375 - 700 mm and a mean temperature of 12 - 22oC (Takele et al., 2000).
However, Kebede et al. (1989) reported that higher dry matter accumulation occurs at 35°C
than at 25°C with the highest leaf carbon exchange rate, 31.8 µ-mol m-2 s−1, occurs at this
temperature.
Grain yield varies from 1-2.5 t ha-1 with a national average yield of about 1.0 t ha-1 and grain
yield potential which might be elevated to 4.5 t ha-1 (Tefera and Belay, 2008; Teklu and
Tefera, 2005). Yield ranges between 2.5- 4.5 t ha-1 have been reported for research plots using
improved varieties and with support of a net to prevent lodging (Tefera et al., 2001; Mamo
and Parsons, 1987; Delden et al., 2010). Early local varieties maturing in less than 85 days,
such as Gea-Lamie, Dabi, Shewa-Gimira, Beten and Bunign, are widely used under short
growing conditions experiencing low moisture stress in the mid and low altitude or low
21
temperature at high altitudes. Under a suitable growing environment, local cultivars, such as
Alba, Ada and Enatit, are used. Modem varieties, such as DZ-01-354, DZ-01-196, DZ-01787, are widely grown by farmers in areas with optimal rainfall and DZ-Cr-37 is grown in
low-moisture stress areas. These varieties give mean grain yields ranging from 1.4 to 2.7 t ha1
(Assefa, 2010; Ketema, 1997; CSA 2008).
1.7.2
Pheno-morphic features related to lodging
Lodging is a key agronomic problem in E. tef production (Yu et al., 2007) and up to 23%
yield loss is accountable to lodging under natural conditions (Ketema, 1983) i.e. with
minimal or no fertilizer condition. Even with good crop management practices, lodging is a
major limitation to sustainable improvement of the crop. E. tef generally has a tall culm
height up to 155 cm and a fine or slender stem with first and second basal culm internode
diameter range from 1.2-4.5 mm (Aseffa et al., 2010; Teklu and Teferea, 2005; Ketema,
1983). Thus E. tef is characterized by a low root-collar diameter to plant-height ratio. Nearly
all improved varieties have a tall phenotype with culm height reaching up to 150 cm and with
a basal internode diameter of <4.5 mm (Figure 1.5). The root system is fibrous and shallow
emerging from nodes above the base, and growing 4 - 8 cm deep under field conditions. The
panicle forms about a third of the culm length (Ketema, 1997; Kebede et al., 1989). Most of
the above characteristics appear to be typical making the crop very susceptible to lodging
(Figure 1.5) due to weak stem-base having insufficient strength to hold the shoot up against
leverage.
22
A
B
Figure 1.5 E. tef plant stand in the field at (A) grain filling and (B) at maturity when
almost all plants lodged ( Source: Dr. Likyelesh Gugssa (Holetta Agricultural Research
|Center).
23
Most studies showed stem lodging due to bending at the basal internodes to be the major
problem in E. tef (Ketema, 1983; Asefa et al., 2000)). In a modelling work for the lodging
character in E. tef Mark (1985) took into account node diameter, 1st and 2nd internode length,
biomass and wind among external forces acting on the plant. According to him the lodging
score (S) is computed as:
S=
bo + b1h (W + Q) , where:
D3 (1-t4)
S = lodging score; bo, bi = empirical constant; W = tiller weight, Q = drag force due to wind,
t = node diameter ratio (inner: outer); D = mean internode length (1st & 2nd). Because of a
high correlation of panicle length with yield and hence tiller weight (Mengesha, 1965),
panicle length has been substituted for (W + Q) and mean diameter of the 1st and 2nd for
internode length. However, this model could predict only about 33% of the variance recorded
for lodging implicating possible involvement of other factors not accounted for. Further
development of models to predict and improve the lodging character in E. tef has not been
made except in a recent study to examine applicability of other crops’ models developed for
wheat and barley (Delden, 2010).
1.7.3
E. tef breeding for lodging resistance
E. tef breeding has mainly resulted in tall phenotypes with low root collar diameter to plant
height ratio (Ketema, 1983) and most varieties so far developed for high yield have this tall
phenotype (Teklu and Teferea, 2005). No genotype has been found so far to be lodging
resistant and there is no clear agreement on the important trait to look for. Berhe (1981)
regarded short, stiff-strawed genotypes as important, others suggest short plants (possibly
24
straw + panicle) (Ketema, 1983; Mengesha et al. 1965) and they reported a high correlation
for lodging with stem diameter, plant height, panicle length and yield. Hundera et al. (1999)
reported days to heading and maturity to be negatively associated with lodging while plant
height, culm length, panicle length, culm diameter, panicle weight, and shoot biomass were
highly significant and positively associated with lodging resistance. Asefa et al., (2000)
recommended stem morphology related characters, such as total height, number of nodes,
thickness and length of basal internodes, to be important. Teferra et al. (2003) reported that
high yielding lines tend to lodge more severely because of failure to bear the heavy panicles,
indicating that lodging also imposes limitation on genetic improvement in E. tef for further
yield increase. Recent studies in E. tef have shown strong correlations between lodging,
panicle type, culm thickness, and grain yield (Yu et al., 2007). Lodging index showed
positive and highly significant correlations with primary shoot weight, 100 seed weight, grain
yield, shoot biomass and negative correlations with peduncle length thus, high yielding lines
tended to lodge. The positive and strong relationship of lodging with plant height and plant
height with yield and other important yield component traits indicates lodging resistance
improvement will remain challenging in E. tef until it is possible to uncouple plant height and
yield traits. Overall, lack of knowledge of exact traits to look for is still the most critical
drawback in modern E. tef cultivation.
Van Delden et al., (2010) also reported that E. tef has the lowest value for plant base
diameter, the diameter of tillers at the soil surface, and the average root plate diameter
compared to other cereals like wheat and rice and emphasized the significance of root failure
as yet another serious factor in E. tef lodging. However, it is not yet clear if root lodging
could well be associated with E. tef root morphological attributes such as root strength and
rigidity, root number and length, or stem characters like ticker stem base. Moreover, how
25
these factors interact with different soil characteristics to cause the lodging problem needs to
be investigated.
1.8
Working hypothesis and aim of study
In this PhD study the problem of reducing plant height in E. tef was addressed to improve
lodging resistance in the crop. Since the GA metabolism plays a significant role in plant
height control, it was hypothesized that regulation of the GA amount in E. tef will change
pheno-morphic and also agronomic characteristics that will affect lodging and also
decoupling plant height from yield. This study had therefore the aim to reduce plant height by
either chemical PGR treatment or manipulation of gene expression to reduce plant height and
also to study in more detail the expression of height regulating GA genes in E. tef. The
objectives of the study were (i) to study the in vivo response of two E. tef genotypes (short
variety: Gea Lammie and long variety: DZ-01-196) to treatment with PGRs to confirm a role
of GA in E. tef plant height control, (ii) to optimize E. tef plant transformation and
regeneration for the production of transformed E. tef plants with reduced GA content (iii) to
characterize transformed E. tef plants over-expressing GA2-oxidase (PcGA2ox1) under the
control of a CaMv3x35S promoter to decrease bioactive GA amounts (iv) to identify and
characterize the genes involved in plant height control (rice sd-1 and wheat Rht orthologous
genes) in E. tef and (v) to characterize morphologically and physiologically existing semidwarf E. tef mutants derived from a TILLING process.
26
CHAPTER 2
CONTROLLING PLANT HEIGHT AND LODGING IN TEF
(Eragrostis tef, Zucc.) USING GIBBERELLIN BIOSYNTHESIS
INHIBITORS
27
2.1 Abstract
Tef (E. tef) is a small seeded nutritious cereal and a primary food source in Ethiopia grown on
over 2.56 million ha in Ethiopia. Tef productivity is low, 1.0 t per ha, due to several factors,
among which lodging is the most critical causing direct losses of about 23% under natural
condition. High yielding cultivars are usually tall and more susceptible to lodging and
breeding effort has not yet succeeded to decouple height from yield. Inhibitors of gibberellin
(GA) biosynthesis such as chlormequat chloride (CCC) are used extensively to restrict
growth and improve lodging resistance in cereals. First, responsiveness of tef plants to GA3
and CCC application was determined using two tef varieties Gea Lammie (short) and DZ-01196 (tall). At 10-2M CCC plant height was reduced by 43% and 21% in the tall and short
variety, respectively, within six weeks after plant emergence. CCC at 10-1M reduced tiller
number in both varieties. More detailed analysis of growth regulator application by including
Paclobutrazol (PBZ) on the tall tef variety DZ-01-196 revealed that, both CCC and PBZ
reduced culm length, with a much stronger reduction from paclobutrazol. Grain yield on the
other hand was not affected by CCC treatment. CCC-treatment reduced culm length by
affecting all internodes, with the 1st- 3rd internodes, followed by the 6th and 7th most severely
affected, whereas paclobutrazol treatment strongly affected all internodes, with greatest effect
on the uppermost 4 internodes. Internode diameter was unaffected by both CCC- and
paclobutrazol-treatments. A steady increase in mean internode diameter until the 6th internode
was found for CCC-treated and also control plants revealing a poor tapering in tef plants.
Reduction of GA amount in tef might be a target for improving lodging resistance allowing
uncoupling of plant height and yield.
28
2.2 Introduction
Tef (Eragrostis tef (Zuccagni) Trotter) is a panicle bearing, small-seeded nutritious cereal
grown extensively in Ethiopia in diverse climatic and soil conditions with low risk of failure
(Assefa et al., 2010). Tef is grown on about 2.6 million ha and accounts annually for about
28% of the total acreage of cereal production in Ethiopia. However, tef suffers from low
productivity with average yields of only 1.0 t ha-1. Among the factors contributing to low
yield, lodging is the most important (Assefa et al., 2010; Tefera et al., 2003; Yu et al., 2007).
In general, lodging interferes with water and nutrient transport, reduces light interception,
provides a favourable environment for disease, increases harvesting cost and losses and
decreases grain yield and quality (Tripathi et al., 2003). It occurs either by buckling / bending
at the basal culm internodes, or due to root lodging or failure of the anchorage system of the
plant (Assefa et al., 2000; Ketema, 1983; Pinthus, 1973). Culm length and the strength of the
basal part of the culm are considered major factors associated with lodging sensitivity
(Rajala, 2003; Tripathi et al., 2003).
In cereals, improvement of lodging resistance has been predominantly achieved by reducing
plant height, in particular by chemical inhibition of gibberellin (GA) production
(Rademacher, 2000) or by the use of semi-dwarf varieties with reduced GA biosynthesis or
signal transduction (Hedden, 2003). Chlormequat chloride (CCC), the most commonly used
plant growth retardant (PGR), blocks GA biosynthesis by inhibition of the cyclization of
geranylgeranyl diphosphate (GGPP) to ent-copalyl diphosphate (CPP) by CPP synthase
(Rademacher, 2000). Triazole PGRs, such as paclobutrazol (PBZ), inhibit the conversion of
the GA precursor ent-kaurene to ent-kaurenoic acid (Rajala, 2003; Hedden and Graebe,
29
1985). In general, PGRs have been extensively used in many crops to reduce lodging through
shortening of the stem and to maintain a steady improvement in grain yield (Berry et al.,
2004; Rajala, 2003).
Reduction in plant height due to PGR treatment, is associated with reduced elongation of
internodes particularly of the uppermost internodes and peduncle (Sanvicente et al., 1999;
Rajala, 2003). CCC inhibits stem elongation in wheat (Humbries et al., 1965) and in oilseed
rape. Foliar treatment with a combination of CCC, ethephon and imazaquin reduced main
stem length in barley where shortening of the uppermost three internodes contributed
significantly to the reduction (Sanvicente et al., 1999). PBZ application was found to reduce
stem length and lodging in rice and increase yield by up to 15% compared to controls (French
et al., 1990).
In tef, cultivars bred for improved grain yield possess a tall phenotype and are highly
susceptible to lodging (Assefa et al., 2010; Yu et al., 2007). Thus, lodging susceptibility has
prevented the introduction of higher yielding varieties with good grain quality, and also
hampered the use of input-intensive husbandry. Currently, there is no detailed information
available for tef on the effect of PGR treatment on lodging and yield responses. The objective
of this study was therefore to investigate morphological and yield changes in tef following
GA biosynthesis inhibitor treatment under controlled environmental conditions. Results
obtained demonstrate that CCC treatment of tef plants significantly reduces plant height
without affecting yield.
30
2.3 Materials and Methods
Two experiments one with preliminary observation (also referred in this chapter as
Experiment I) involving two growth regulators and two varieties and a second one (also
referred to as Experiment II) involving one variety and two growth regulators have been
carried out.
2.3.1
Plant material
Seed material of the tef (Eragrostis tef) varieties DZ-01-196 and Gea Lammie used for the
experiments was obtained from the Ethiopian Institute of Agricultural Research, Holetta
Agricultural Research Center, Ethiopia. Plants of variety DZ-01-196 have a tall phenotype,
derived from a conventional breeding program and is widely used for cultivation while Gea
Lammie is a landrace grown by farmers for its earliness.
2.3.2
Plant growth
Experiment I was done using varieties DZ-01-196 and Gea Lammie grown in a greenhouse at
University of Pretoria. Plants were grown on a germination soil mix in a pot supplemented
with a Hoagland fertilizer solution (Kebede et al., 2008). With the second set of experiments,
plants were grown at Rothamsted Research, UK from May to August using pre-germinated
seeds in moist soil in pots. In both experiments a total of 12-16 plants were maintained in 3 to
4 per treatment. About 7-10 days after emergence thinning was done or transplanting of
uniform seedlings was carried out to new pots [15 cm diameter (top) x 12.5 cm (height) and
10 cm diameter (bottom)]. Seedlings were grown on either a commercial germination mix
31
soil supplemented with half strength Hoagland solution in the observation trial or in a
compost mix consisting of peat (75%), sterilized loam (12%), vermiculite (3%) and grit
(10%), which was supplemented with a slow release fertilizer. Plants were well-watered
every other day and the temperature was maintained at 23-27°C (day) and 15-18°C (night).
Seedlings were grown for 14 weeks until plant maturity in an environmentally controlled
greenhouse using a 16-h photoperiod provided by natural light supplemented with light from
sodium lamps to maintain a minimum PAR of 350 µmol m-2 s-1.
2.3.3
PGRs treatment
In the first experiment, the above two genotypes were considered for the investigation.
Gibberellic acid (GA3) and a GA-biosynthesis inhibitor, chlormequate chloride (2chloroethyl-N,N,N-trimethyl-ammonium chloride, CCC) were applied by rubbing the
underside of the leaf lamina. Application began after plant thinning and continued for six
weeks every week. Both compounds were applied (10µl) to the lower surface of the
uppermost expanding young leaf per week, at concentrations ranging from 10-1M to 10-6M. In
the second experiment, only one variety, DZ-01-196 was considered for a more detailed
study. Plants were treated with CCC and a potent GA- biosynthesis inhibitor, paclobutrazol
(PBZ), (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl) pentan-3-ol).
CCC was applied at 10 mM and 100 mM and PBZ at 10 µM and 100 µM. For both
inhibitors, individual solutions were prepared in dsH2O and 100 ml of individual solution
were applied every two weeks to the base of the pot when watering of plants was carried out.
Treatment with inhibitors started 3 wks after seed germination.
32
2.3.4
Growth measurements
Culm, individual internodes and panicle lengths of tillers were measured from the subtending
nodes using a ruler. The internode and tiller number were also recorded for each plant.
Primary tiller refers to the main tiller that emerges first as the seed germinates. Secondary
tillers refer to shoots that emerge at a later stage during seedling growth. The internode
diameter was measured at 3 mm above the node using a Standard Digital Caliper. Dry weight
was determined from above-ground plant material by drying fresh material at 80oC for 2 days
in an oven. Grain yield was determined by measuring the weight of seeds from all tillers.
2.3.5
Analysis of endogenous GA content
The upper most two internodes including their nodes at shoot elongation stages and just
before panicle initiation were harvested and stored at -800C until analysis. Endogenous GA
levels were monitored using stored internodal tissue after samples were freeze-dried and
grinded using a ball mill for extraction, purification, and analysis of GAs. Powdered replicate
samples of about 0.5g were re-suspended in 80% aqueous MeOH with addition of mixture of
2H- and 3H-labeled GA internal standards. The aqueous extract was then subjected to a
rotation vacuum evaporator at 40-450C to remove methanol. The pH of the aqueous extract
was adjusted to 3.0 using 1 mol/l HCl before further partitioning three-times with watersaturated ethyl acetate. The combined organic phases were reduced to dryness under vacuum
at 420C to remove ethyl acetate. After column purification and full methylation with ethereal
diazomethane, samples dissolved in methanol were injected onto an analytical C18 reversed
phase HPLC column for fractionation. Recovery of fractions was monitored using triturated
33
(3H) internal standards and GAs were quantified using gas chromatography- mass
spectrometry (GC-MS) system using selective ion monitoring.
2.3.6
Data analysis
Growth and yield data were collected after six weeks and at plant maturity for the first and
second sets of the experiments from 12 individual plants and their tillers per treatment.
Analysis of variance (ANOVA) and Pearson Correlation Coefficients were performed for
data analysis using the SAS statistical package (SAS Institute Inc., Cary, NC, USA).
Statistical significance of difference between treatment means was determined using the
Tukey's Studentized Range (HSD) Test. A P-value of <0.05 was considered as significant.
34
2.4 Results
2.4.1
Experiment I
The effect of exogenous application of gibberellic acid (GA3) and the GA inhibitor (2chloroethyl-N, N, N-trimethyl-ammonium chloride (CCC) on two tef varieties, Gea Lammie
and DZ-01-196, was investigated. In this preliminary observation, the two genotypes showed,
to a considerable degree, contrasting response to the application of exogenous GA3 and CCC.
Different GA3 or CCC amounts affected plant height beginning in the first week of its
application in both varieties when compared to untreated control plants. In general, GA3
application increased plant height whereas CCC reduced the plant height in both varieties
(Fig. 2.1A and B).
B
A
Plant height (cm)
40.0
Gea Lammie
GA3
CCC
35.0
30.0
*
*
*
25.0
*
20.0
15.0
10.0
CCC
35.0
*
30.0
25.0
20.0
*
15.0
10.0
5.0
5.0
0.0
GA3
45.0 DZ-01-196
40.0
Plant height (cm)
45.0
Control
1.0
10
0.0
100
Control
1.0
10
100
GA3 (µM) /CCC(mM)
GA3 (µM) /CCC(mM)
Figure. 2.1A and B. Growth (plant ht) response after six weeks of Gea Lammie and
DZ-01-196 grown in greenhouse to exogenous application of GA and CCC.
Treatments:
C=Control;
GA3/CCC:
100=100µM/100mM
35
1=
1µM/1mM,
10=10µM/10mM
and
Application of GA3 (10-2M) significantly increased (p<0.01) plant height by 41% in plants of
the short genotype and 13% of the tall genotype under greenhouse conditions. In contrast,
applying CCC (10-2M) significantly reduced (p<0.01) plant height by 46% in the tall
genotype compared to a 27% reduction in the short genotype. GA3 treated plants were tall
and had slender stems compared with plants treated with CCC (data not shown). The above
results led to further study in more detailed (second experiment) only one variety, DZ-01196, using CCC and PBZ. Results show that both fresh and dry weight of DZ-01-196 plants
were affected by CCC treatment. At a CCC concentration of (10-1M) fresh weight of plants
was significantly reduced (p<0.001) in the taller genotype when compared to the untreated
control (Fig. 2.3). Number of emerging tillers was also significantly (p<0.05) reduced by the
highest CCC level (10-1M) in the taller genotype. There was at most one tiller per plant at
elongation stage (in addition to the main tiller) in those treated with CCC (≥10-1M) when
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
A
6.0
B
5.0
*
Weight (g)
No. of tillers per plant
compared to the untreated control (Fig. 2.2).
4.0
*
3.0
2.0
*
1.0
Control
10
1.0
CCC (mM)
0
100
Fresh wt
Dry wt
Control
*
*
10
1.0
CCC (mM)
100
Figure 2.2. Tillering (A), fresh and dry weight (B) responses of six weeks old DZ- 01-196
seedlings to CCC treatment.
36
Tiller
20
CCC concentration (mM)
10 Tiller
*
20
20 Tiller
*
10
0
0.001
0.01
0.1
0
Con
10
B
0.001
*
30
0.01
20
0.1
10 Tiller
Con
30
A
Length (cm)
Length (cm)
40
CCC concentration (mM)
Figure 2.3 Effect of foliar applied CCC at different concentrations of foliar applied CCC on
the length of the 7th internode (A) and 8th internode (B) of primary (10) and secondary (20)
tillers in comparison to the untreated control. Data represent the mean ± SE of tiller length of
12 individual plants.
Most of the reduction in the culm length was due to a significant reduction in internode
elongation particularly in the upper-most two internodes following CCC treatment (Fig. 2.3 A
and B). However, in the uppermost (8th) inernode, it has been observed that the primary tiller
has been more sensitive to CCC and the secondary tiller. Length of the secondary tillers was
reduced at lower CCC levels than at higher level compared to the main tiller. Such
contrasting response was not observed between the main and secondary tillers for the 7th
internode. The reason for this is not clear.
2.4.1.1 Analysis of endogenous GA content
The two genotypes of tef, Gea Lammie and DZ-01-196 were characterized to determine the
state of GA concentrations in them and reveal the existence and nature of the relationship
between GA and plant height.
37
The predominant GA pathway and the primary biologically active product in vegetative shoot
of tef was previously unknown. On the other hand, endogenous GA content determination in
plant material is a challenging task because of GA’s extremely low concentration in plants
and its complex biological matrices (Ge et al., 2007). Other characteristics such factors as
low ultraviolet (UV) absorption, absence of fluorescence, and distinguishing chemical
characteristics as well as the need for specific chemical assay makes GA analysis
complicated (Ge et al., 2007). Therefore, the determination of GA levels in plants had to be
carried out at Rothamsted Research/UK where expertise and facility were found.
Results from the endogenous GA analyses of plant tissues taken from the upper most two
internodes including their nodes using analytical HPLC column fractionation and GC-MS
monitoring shows that the shorter genotype Gea Lammie has generally lower level of
bioactive GA and most of the precursors than the tall genotype (Table 2.1). The content of the
most abundant bioactive GA3 was about three fold less in the shorter genotype Gea Lammie
compared to DZ-196-01. The bioactive GA level corresponds with plant heights, and
associated other growth characteristics of the two genotypes. Moreover, the concentration of
most of the immediate precursors in Gea Lammie were about half the amount in DZ-01-196.
The amount of endogenous GA1 concentration was moderately related with plant height than
biomass or number of tillers. Concentrations of precursors for the Non- 13-Hydroxylation
pathway such as GA15, GA9, and GA34 were extremely low or nil in most cases. The amount
of and the bioactive final product, GA4, of this pathway was nil in both genotypes (Table
2.1). Therefore, the analysis showed that the early 13-hydroxylation is the major or preferred
pathway in E. tef and the most abundant bioactive form was GA1 (Table 2.1).
38
Table 2.1 Quantification of GA intermediates and bioactive forms from internode sample
analysis at stem elongation stages of two tef varieties, DZ-01-196 (tall) and Gea Lammie
(short).
Genotype
GA1§
GA4§
GA8
5.71** 2.25 3.17 0.0
0.0
9.36 6.65 19.22 0.18 ND
10.68
0.0
5.35 4.63 10.97 0.3
4.28
GA29
GA3§
GA15
GA20
GA19
GA34
GA9
GA53
DZ-01-196
Gea Lammie
4.22
1.12 1.07 0.0
SE
0.25
0.71 1.39
0.53 0.31 0.53
0
0.28 1.06 0.25
ND =not determined; §Bioactive forms of GA; *E. tef has the 13β-hydroxylation as a
major pathway for GA biosynthesis hence GA1 is a major bioactive product.
**
Values are in ng per g dry weight and a data point represents average of three samples
2.4.2
Experiment II
2.4.2.1 Culm and panicle length
In the second experiment treatment of tef plants with PBZ using soil application of the
growth regulator significantly reduced culm and panicle length of plants by internode
shortening when compared to CCC treatment or to the untreated control (Fig. 2.4; Table 2.2).
PBZ also showed significant effects on growth parameters at a much lower concentration
than CCC (Table 2.1). When plants were treated with PBZ at 10 µM and 100 µM, culm
length was reduced by 92% and 98%, respectively. However, CCC treatment only reduced
39
culm length by 9.3% (10 mM) and 22.3% (100 mM). Further, PBZ treatment significantly
reduced also panicle length (Table 2.2). In contrast to PBZ treatment, CCC treatment
increased panicle length at both concentrations applied (10 mM and 100 mM) with a
significant increase at 100 mM when compared to the untreated control (Table 2.2). In
addition, the panicle length to culm length ratio of 1.61 (10 µM PBZ) and 1.51 (100 µM
PBZ) decreased to 0.37 and 0.49 when plants were treated with either 10 mM CCC or 100
mM CCC, respectively. This indicates that both inhibitor treatments had a stronger effect on
culm length than on panicle length (Fig. 2.5), with CCC treatment resulting in an increased
panicle length at both concentrations. Furthermore, after PBZ and CCC treatment panicle
length was strongly correlated with culm length, culm dry weight and total above ground
shoot dry weight and also negatively correlated with tiller number per plant and dry weight
per height ratio (Table 2.5).
2.4.2.2 Internode growth
Both internode length and diameter were significantly reduced by PBZ treatment as a soil
application when compared to CCC treatment or to the untreated control (Tables 2.2 and 2.3).
In PBZ-treated plants, internode length was reduced in all internodes. However, the two
upper most internodes of plants treated with 10 µM PBZ and the four uppermost internodes
of plants treated with 100 µM PBZ completely failed to elongate (Table 2.3). In CCC-treated
plants, reduction in internode length also varied between the different internodes. The first
three internodes (internodes 1-3) contributed 48%, whereas the last three internodes
(internodes 5-7) contributed 46% to the total internode length reduction. In the preliminary
study when CCC was used as a foliar application and not as a soil application, the upper-most
two internodes contributed most of reduction in internode length (See Fig. 2.3: A and B).
40
However, for the 8th internode, length of the secondary tillers was less at lower CCC levels
than at higher level.
The lowermost two internodes (I1+I2) of CCC-treated plants were further more positively
correlated (r = 0.70, P < 0.05) to culm length when compared to the uppermost two
internodes (I7+I8) (r = -0.04, P < 0.05) (data not shown).
Table 2.2 Effect of CCC and PBZ on culm and panicle length, number of
tillers and seed weight per plant of tef cv. DZ-01-196.
Treatment
Culm
length (cm)
Panicle
length (cm)
No. of
Tillers
Seed wt (g)
159.9± 3.7a
50.26±1.9c
5.0±0.6b
3.79±0.5a
10mM
145.0±2.2b
53.93±0.9b
6.0±0.6b
3.63±0.7a
100mM
124.2±3.3c
61.50±0.7a
7.3±1.1b
3.78±0.3a
10µM
11.5±1.1d
18.57±0.8d
17.3±2.8a
1.11±0.3b
100µM
3.9±0.3e
5.87±0.3e
15.0±2.1a
0.05±0.03b
Control
CCC
PBZ
Significance
***
***
***
***
Letters within the column denote significance as determined using the Student's
t-test. Data shown represent mean values ±SE of 12 individual plants.
Significance level was determined using ANOVA (*** P < 0.001) and difference
between treatment means was determined using the Tukey's Studentized Range
(HSD) Test. Means followed by the same letter are not significantly different.
41
50cm
Figure 2.4 Effect of CCC (100mM) and PBZ (100µM) on plant height
near plant maturity in comparison to the untreated control.
The internode diameter was, however, unaffected by either PBZ or CCC treatment except for
the first internode after 10 mM CCC treatment, internodes 5 and 6 after 10 µM PBZ
treatment and internode 4 after 100 µM PBZ treatment. In CCC-treated and control plants,
the internode diameter steadily increased from the base up to internode 6. This indicated a
poor tapering characteristic of tef plants under the selected growth conditions (Table 2.4).
42
Figure 2.5 Comparison of plant height and panicle growth at plant maturity
as affected by PGRs (CCC: 100mM and PBZ: 10µM) application.
2.4.2.3 Tillering, above ground biomass and yield
PBZ-treated plants had a three-fold increase in the number of tillers per plant whereas CCCtreated plants had no significant increase in the number of tillers when compared to the
untreated control (Table 2.2). PBZ treatment also significantly reduced culm and panicle dry
43
Table 2.3 Effects of CCC and PBZ on length of different internodes of tef var. DZ-01-196.
Internode length (cm)
Treatment
I-1
I-2
I-3
I-4
I-5
I-6
I-7
I-8
8.6±0.9a
14.3±0.85a
18.7±0.5a
18.3±1.1a
21.9±0.9a
23.0±2.0a
27.2±1.1a
28.4±3.3a
10mM
8.1±0.9a
12.3±0.5b
15.1±0.9b
17.3±0.8a
20.4±0.1b
22.7±0.9a
24.1±0.9b
25.0±2.7a
100mM
3.3±0.6b
8.9±0.6c
12.8±0.7c
15.8±0.5b
17.0±0.6c
18.3±0.8b
20.5±0.7c
27.6±1.3a
10µM
1.3±0.5c
2.0±0.5d
3.0±0.5d
4.0±0.7c
1.7±0.8d
0.8±0.1c
100µM
0.45±0.1d
0.9±0.3e
1.0±0.3e
0.8±0.2d
***
***
***
**
**
NS
Control
CCC
PBZ
Significance
***
***
Letters within the column denote significance as determined using the Student's t-test. Data
shown represent mean values ±SE from 12 individual plants. Significance level was determined
using ANOVA (** P < 0.01; *** P<0.001; NS = not significant) and difference between
treatment means was determined using the Tukey's Studentized Range (HSD) Test. Means
followed by the same letter are not significantly different.
44
Table 2.4 Effect of CCC and PBZ on diameter of different internodes of tef var. DZ-01-196.
Internode diameter (mm)
Treatment
I-1
I-2
I-3
I-5
I-6
I-7
3.2±0.1a
3.7±0.1a
4.2±0.2a 4.34±0.2a
4.6±0.3a
4.8±0.3a
4.7±0.3a 4.0±0.3a
10mM
2.8±0.1b
3.5±0.1a
3.9±0.1a 4.25±0.1a
4.6±0.1a
4.9±0.1a
4.9±0.1a 4.2±0.4a
100mM
3.2±0.1a
3.9±0.12 4.1±0.1a 4.22±0.1a
4.4±0.1a
4.4±0.1b 4.4±0.2a 3.8±0.2a
10µM
3.7±0.5a
3.9±0.4a
3.7±0.6a 3.66±0.6a
2.0±0.8b 0.8±0.6c
100µM
3.8±0.5a
4.1±0.4a
3.7±0.6a 0.56±0.4b
Signif.
***
NS
Control
I-4
I-8
CCC
PBZ
NS
**
*
**
NS
NS
Letters within the column denote significance as determined using the Student's t-test. Data
shown represent mean values ±SE from 12 individual plants. Significance level was
determined using ANOVA (* P <0.05; ** P < 0.01; *** P<0.001; NS = not significant) and
difference between treatment means was determined using the Tukey's Studentized Range
(HSD) Test. Means followed by the same letter are not significantly different.
45
weight in both the primary and secondary tillers when compared to CCC treated plants or
to the untreated control (Table 2.6). PBZ also significantly suppressed above-ground
shoot dry weight by 43.2% and 75.9% at 10 µM and 100 µM PBZ, respectively (Table
2.5; Fig. 2.6). In plants treated with 10 mM CCC secondary tiller culm and panicle dry
weights were increased significantly as was total above-ground shoot dry weight when
compared to untreated plants and those treated with PBZ l. In CCC-treated plants, the
change in the above-ground shoot dry weight was all due either to the increase (at 10
mM) or the decrease (at 100 mM) in secondary tiller growth.
*
*
*
*
*
Figure 2.6 Effect of different concentrations of CCC and PBZ on biomass: fresh weight
(FW) or dry weight (DW) per plant in comparison to biomass of untreated control plants.
Data represent the mean ± SE of 12 individual plants. (CCC1 = 10 mM CCC, CCC2
=100 mM CCC, PBZ1 =10 mM PBZ and PBZ2 = 100 mM PBZ).
46
*
*
* *
Figure 2.7 Effect of different concentrations of CCC and PBZ on primary (1o) and
secondary (2o) tiller grain yield per plant in comparison to the untreated control. Data
represent the mean ± SE of tiller grain yield of 12 individual plants. (CCC1 = 10 mM
CCC, CCC2 =100 mM CCC, PBZ1= 10 mM PBZ and PBZ2 = 100 mM PBZ).
Further, in GA inhibitor-treated plants, panicle dry weight contributed less (by 8.4 14.5%) to above ground shoot dry weight while for panicle in the untreated control was
higher (12.0%). Also, in all GA inhibitor-treated plants the tiller number per plant was
negatively correlated with above-ground shoot dry weight but positively correlated with
the ratio of dry mater to shoot height (Table 5). PBZ treatment (10 µM and 100 µM)
significantly reduced the seed weight per plant when compared to the untreated control.
Such a significant reduction was not found after CCC treatment. In CCC treated plants
panicle bearing secondary tillers further contributed 63.4% to the total yield per plant
(Fig. 2.7). This indicates that PBZ-induced profuse tillering, resulting mainly in non-
47
panicle bearing tillers, which did not contribute to grain production. Nevertheless PBZ
had a stronger effect on culm than on panicle length and also clearly demonstrated that
the responsiveness of E. tef to GA inhibition has been different between culm and panicle
(Fig. 2.8).
~11cm
~4cm
PBZ 10mM
PBZ 100mM
Figure 2.8 Comparison of panicle elongation with
PBZ treatments in proportion to culm reduction in
DZ-01-196.
48
Table 2.5 Effect of CCC and PBZ on dry weight of culm and panicle tillers of tef var.
DZ-01-196.
Total shoot (g)
Primary tiller (g)
Secondary tiller (g)
Treatment
Culm
Culm
Panicle
Control
CCC
7.11±0.3a 2.01±0.2a
25.67±1.8b
5.52±0.6b
10mM
100mM
6.70±0.3a 2.17±0.2a
31.10±1.3a
6.93±0.6a
4.60±0.3b 1.87±0.1a
19.89±1.6c
6.51±0.6ab
Panicle
40.30±1.9b
46.89±1.7a
34.53±1.9c
PBZ
16.10±1.8d
10µM
100µM
Significance
0.72±0.1c 0.73±0.2b
0.41±0.1d 0.31±0.1c
**
11.81±1.2d
5.63±0.3e
**
2.82±0.6c
0.29±0.1d
**
**
6.41±0.4e
**
Letters within the row denote significance as determined using the Student's t-test. Data
shown represent mean values ±SE from 12 individual plants. Significance level was
determined using ANOVA (** P < 0.01) and difference between treatment means was
determined using the Tukey's Studentized Range (HSD) Test. Means within a column
followed by the same letter are not significantly different.
49
Table 2.6 Correlation coefficients for morphological and yield components of tef var.
DZ-01-196.
CL
PL
PaL
TH
IN
PL
PaL
TH
IN
NT
CDW
PDW
TSDW
DW/Ht
S/P
0.5352
0.9281
0.9872
0.9291
-0.6415
0.8872
0.7779
0.8966
-0.8687
0.7191
0.5555
0.5663
0.5579
-0.2571
0.5012
0.4383
0.5065
-0.4561
0.4557
0.9511
0.9276
-0.6028
0.8356
0.8251
0.8748
-0.8585
0.7587
0.9321
-0.6145
0.8609
0.7859
0.8833
-0.8848
0.7538
-0.6252
0.8486
0.7447
0.8634
-0.8532
0.6522
-0.4258
-0.4248
-0.4307
0.8029
-0.4401
0.7904
0.9803
-0.7199
0.6682
0.8809
-0.6543
0.8923
-0.7372
0.7511
NT
CDW
PDW
TSDW
DW/Ht
0.9869
CL = Culm length; PL = Peduncle length; PaL = Panicle length; TH= Total height; IN=
Internode number; NT = No. of tillers; SFW= shoot fresh weight; CDW = Culm dry weight;
PDW = Panicle dry weight; SDW = Shoot dry weight; DW/Ht = shoot dry weight per height
ratio; S/P = seed weight per plant. Significance level for the Pearson Correlation Coefficients
was determined using the SAS statistical package and all values are significant at P<0.001.
50
2.5 Discussion
Results have shown a GA-dependent control of plant height in tef. Application of GA3
(10-2M) increased plant height by 41% in plants of the short genotype and by 15% in the
tall genotype under greenhouse conditions. Applying CCC at 10-2M reduced plant height
by about 43% in the tall genotype DZ-01-196 when compared to a 21% reduction in the
short genotype Gea Lammie during the six weeks of plant growth. Application of the GA
inhibitor CCC reduced the plant height in plants of DZ-01-196 (tall phenotype). This
effect was more dramatic than the effect on plants height in Gea Lammie. Since GA
increased height in Gea Lammie and CCC reduced height in both, the short phenotype in
Gea Lammie is not considered to be either due to interference in the GA-biosynthesis or
GA- response but could be associated to other factors not relevant to GA metabolic
genes. Studies in wheat has shown that tall and intermediate varieties showed a greater
response to CCC than the short or semi-dwarf varieties containing mutant GA genes like
Rht1/Rht1 (Börner & Meinel, 2006; Abbo et al., 2004).
The content of the bioactive GA1 and GA3 and all the precursors were higher in the tall
genotype, DZ-01-196, than in Gea Lammie showing in most cases about a two fold
increase. Thus the bioactive GA concentration was a good reflection of the difference in
plant height between the two genotypes. This also explains the differential responses to
exogenous GA3 application where DZ-01-196 was less responsive to exogenous GA
treatment compared with Gea Lammie because of higher bioactive GA amount in the
plant tissue.
51
Further study using only the tall variety (Experiment II) has demonstrated that the GA
inhibitors CCC and PBZ reduce stem growth in tef but with a much stronger effect of
PBZ when compared to CCC. Culm length was the most responsive plant part to inhibitor
action and CCC reduced culm length by one quarter but without reducing panicle growth
and grain yield.
Based on our finding that CCC treatment reduces plant height in E. tef, the PGR effect on
DZ-01-196 was investigated in greater detail because this variety has been widely grown
for its high yield and grain quality. It is also used as a parental line in E. tef breeding
program but suffered from lodging losses (Yu et al. 2007; Assefa et al. 2010). Therefore,
effect of GA inhibitors CCC and PBZ on DZ-01-196 was specifically studied further.
Both GA inhibitors CCC and PBZ significantly reduced stem growth in DZ-01-196 but
with a much stronger effect of PBZ at a much lower concentration when compared with
CCC because of its stronger inhibitory effect on GA biosynthesis than CCC (Lurie et al.,
1997)). Culm length was most responsive and CCC reduced culm length almost by a
quarter without reducing panicle growth and grain yield A further reduction of culm
height might be achieved by increasing CCC concentration, but such increased
concentration should not affect panicle growth. Yield increase associated with CCC
application, reported for other crops (Berry et al., 2004), was not found for E. tef. This
could be because of absence of increased head-bearing tillers following CCC application.
PBZ, greatly reduced culm elongation particularly affecting elongation of the uppermost
four internodes which in most cases was completely inhibited. Also, a lower PBZ
52
concentration might be applied so that a less drastic effect is achieved in reducing the
plant height and with minimal effect on panicle growth and yield.
Since GA inhibitor action greatly affected culm length, this indirectly imply that a higher
demand for endogenous GA may exist by the elongating stem than by the panicle. This
may be due to a higher meristematic activity and cell elongation in the intercalary
meristem of the elongating stem rendering it more sensitive to changes in the GA
amount. Shortening was more pronounced in the lowermost internodes with up to a 2.6fold reduction when compared to the control. Shortening of basal internodes, if associated
with stem wall thickening or increase in dry weight per unit of basal internodes, will
further minimize the lodging risk. For example, the basal internode length and plant
height in wheat are the two most important culm traits closely associated with lodging
(Kelbert et al. 2004).
It was also observed that soil-applied CCC shortened all internodes, foliar application
shortened the uppermost two internodes (Gebre, et al., 2011). A similar effect for foliarapplied CCC has been previously also reported for barley (Sanvicente et al., 1999) and
hybrid rye (Froment and McDonald, 1997). The results also indicate a possible
differential response to mode of application which could be related to the translocation of
CCC to the site of the biochemical targets being more localized in leaf application. E. tef
may benefit more from foliar than soil application. Irregularity of responses has been
noted in other crops presumably for differences in the decomposition of the chemical in
the soil, depending on prevailing temperature and humidity conditions (Radmacher,
2000; Pintus, 1973).
53
In our study CCC treatment even increased panicle length, but this increase was not
related to any change in seed weight per plant. Whereas in a previous study with wheat
improved grain set thus increased harvest index was obtained following shortening of
stem (Rajala, 2003). Absence of a CCC effect on E. tef panicles possibly indicates a low
demand for bioactive endogenous GA for panicle growth and therefore inhibition of GA
biosynthesis does not greatly affect yield while reducing plant height. Agronomically,
this would be advantageous allowing high productivity in tef plants while minimizing
lodging.
In this study, we also investigated the effect of the GA-biosynthesis inhibitors on stem
diameter. In PBZ-treated plants tapering stem morphology with a steadily increasing stem
diameter was found. In contrast CCC-treated and control plants only had an increase in
stem diameter but in the upper internodes only. Such steady acropetal increase in stem
diameter might exacerbate lodging susceptibility and this problem might even become
more serious with increasing N-fertilization allowing minimum wind speed or rain to
cause lodging. This also indicates that E. tef plants have a weak transition from shoot to
root with a smaller plant-base diameter causing poor tapering. Absence of an effect on
stem-base diameter by CCC is not unique to E. tef as it has also been reported in wheat
and barley (Gendy & Hofner 1989; Berry et al. 2000).
PBZ-treated plants had a higher tiller number compared to CCC-treated plants. A
continuous application of a high PBZ dose strongly promoted tillering but inhibited tiller
elongation. This limits leaf expansion and photo-assimilation thereby reducing
carbohydrate reserves required for rapid growth (Stavang et al., 2009). Promotion of
54
tillering by GA deficiency and inhibition of stem elongation has been recently also
reported for other cereals (Rajala, 2003; Lo et al., 2008). However, in contrast to wheat,
barley and oats, where increased tillering was found after foliar and seed treatment with
CCC (Naylor et al., 1989; Craufurd and Cartwright, 1989; Peltonen and Peltonen-Sainio,
1997; Peltonen-Sainio et al., 2003), no significant increase in tillers was found in tef
when treated with CCC. It has been observed, in general, that gibberellins tend to cause
less development of auxiliary buds while promoting the elongation of already growing
(initiated) stem as well as tillers (Peltonen and Peltonen-Sainio, 2001). On the other hand
the application of GA-inhibitors promote tiller initiation and stunting of the central stem.
Tillering is generally considered an adaptation to environmental changes. Under a longday condition, as applied in this study, CCC treatment has been found to produce in other
cereals more tillers per main shoot at maturity (Rajala, 2003). Further, in wheat a high
number of tillers reduces plant productivity in terms of grain production and lodging
resistance and it has been further suggested that a low tiller number per m2 is required for
lodging resistance (Tripathi et al., 2003). In tef breeding, however, the main focus has
been on obtaining high tillering with tall plants for improved grain yield. It would be
therefore important for developing a lodging resistance tef ideotype where plant height is
differentially controlled (decoupling of plant height and yield) while maintaining
optimum number of panicle bearing tillers that is yet to be determined from the lodging
stand point.
In E. tef breeding, taller plants with higher number of tillers have been the main focus for
grain yield improvement. In this study, it has been demonstrated that CCC can reduce
plant height without affecting grain yield. It is therefore important to develop a lodging-
55
resistant E. tef ideotype differentially controlling plant height and yield. Generally, this
study has demonstrated that GA biosynthesis is a prime target for plant height regulation.
Since CCC reduces plant height without affecting grain yield, this compound might be
suitable for lodging prevention providing the advantage of high productivity. Treatment
with CCC did not increase stem diameter, the diameter to height ratio however increased
and CCC treatment would therefore also improve plant standability. Although PBZ is
probably useful at lower concentrations, its high cost and persistence in the soil would
restrict its wider application. However, future fine-tuning might be required to optimize
CCC use for commercial application in lodging prevention without compromising seed
yield. However, extensive use of chemicals such as CCC may not be sustainable for
environmental concerns. In which case long term solutions through genetic modification
of GA metabolism and targeting genes, such as the rice sd-1 and the wheat Rht orthologs
in E. tef, could be a strategy for plant height control in E. tef.
56
CHAPTER 3
TRANSFORMATION OF TEF (Eragrostis tef)
57
3.1
Abstract
Successful application of genetic transformation for integration of a transgene is much
dependent upon availability of an efficient in vitro plant regeneration procedure and
detection of transgene insertion and expression. Isolated immature embryos of E. tef
cultivar DZ-01-196 were used for embryogenic callus formation and callus was
transformed with the GA inactivating coding sequence (PcGA2ox) under the control of a
triple CaMV 35S promoter using the Agrobacterium transformation procedure. Media
K99 was applied as basal medium and both the MS-based co-cultivation medium (CCM)
and regeneration medium (K4NM) were used for embryogenic callus induction from
immature embryos, Agrobacterium transformation and regeneration of embryogenic calli.
Transformed E. tef callus was tolerant to treatment with the selectable marker kanamycin
which inhibited growth of non-transformed shoots derived from matured embryos. A
total of 55 plants were regenerated from callus to fully viable plants setting seeds at
maturity. Eight putatively transformed T0 plants were produced carrying the transgene in
their genome which was detected by PCR. Sequence analysis confirmed that the
amplified PCR product had 97.2 and 99.8% sequence similarity to PcGA2ox and nptII,
respectively, but detection of the PcGA2ox or nptII transgene in the T1 generation was
inconsistent although phenotypic characterization of semi-dwarf T1 generation plants
showed changes in agronomic characters such as plant height, number of internodes,
tillering, panicle length, biomass and yield as well as changed GA content. Results
showed a GA-deficient growth characteristic (semi-dwarf phenotype) in putatively
transformed plants associated with a low level of bioactive GA1 and immediate
precursors. Culm reduction was due to absence of elongation of the upper-most
58
internodes. Panicle length in semi-dwarfed plants showed no relation with Culm length.
Up to 3.7 fold increase in grain yield per plant was found in some semi-dwarfed plants.
Lack of detection of transgene insertion in T1 generation is still a major concern and
further studies are necessary to rule out that somaclonal variation has not been the source
of variation in plant height and other plant characteristics.
59
3.2
Introduction
Agrobacterium-based transformation still imposes a considerable challenge in cereal
transformation. Some of the salient features that determine success of the method have
been previously extensively investigated (Shrawat and Lörz, 2006). Further, variations in
transgene expression are influenced by several factors including somaclonal variation
induced by tissue culture process, the copy number of the transgene incorporated into the
host genome, truncation of the transgene, and epigenetic gene silencing (Shrawat, 2007).
Silencing of the transgene is often associated with a high transgene copy number or
transgene promoter activity and occurs either at the transcriptional or post-transcriptional
level.
The successful application of Agrobacterium-based genetic transformation systems and
progress in precision of integration of the transgene is dependent upon availability of an
optimized efficient in vitro plant regeneration procedure. This has been one of the
objectives for this study. In vitro regeneration protocols for E. tef have been reported by
several research groups to regenerate plants from various explants such as roots, leaf
bases and seeds (Bekele et al. 1995; Mekbib et al. 1997; Assefa et al. 1998). However,
obtained regeneration was poor to consider further application (Gugssa, 2006). Moreover,
immature reproductive organs, such as embryos, had not been used until recently (Gugssa
et al., 2006) where regeneration of haploid plants has been achieved using gynogenic
tissue of E. tef and immature zygotic embryos (Gugssa, 2008). However, regeneration of
transformed plants has so far only been obtained by Gugssa (2008) regenerating a single
plant expressing the Green florescent protein (Gfp).
60
The objectives of this study were therefore establishing a transformation and regeneration
procedure using protocols developed for various cereals and detecting integration and
expression of the GA inactivating gene (PcGA2ox) in E. tef. The method of
transformation of immature embryos and production of transformed embryogeneic callus
was applied to regenerate transformed shoots that develop into fertile plants. Putatively
transformed E. tef plants with changed plant stature (dwarf/semi-dwarf) were also
characterized for such traits as plant height, tillering, stem diameter, panicle length,
physiological (biomass and yield) and biochemical characteristics (GA content).
3.3 Materials and Methods
3.3.1
Preparation of plant material and culture
Seeds of improved variety DZ-01-196 were obtained from the Ethiopian Institute of
Agricultural Research (EIAR). The seeds were germinated on germination mix soil and
the seedlings were grown in pots under a 26±2 / 18°C day/night temperature and a 14 hr
day length. Plants were further supplemented with a full-strength Hoagland nutrient
solution until immature zygotic embryos, referred to as immature embryos (IEs) in this
study, could be harvested from the developing panicle 2-3 weeks after panicle
emergence.
61
3.3.2
IE isolation, callus induction and culture growth
Immature embryos (IEs) from E. tef plants of cultivar DZ-01-196 were isolated and callus
was induced according to the protocol reported by Gugssa (2008). Callus inoculation and
co-cultivation with Agrobacterium during E. tef transformation and further regeneration
was carried out following various protocols (Gugssa, 2008; Hensel and Kumlen, 2004;
Rao et al., 2007; Toki, 1997; O’Kennedy et al., 2004). Immature embryos were collected
from flower spikes 7 to14 days post anthesis and IEs were isolated using a binocular
microscope (Gugssa et al., 2008). The middle segment was selected for isolation of
embryos. Freshly detached spikes were used for immediate isolation and culture after
sterilization, or spikelets were pre-treated at 4oC for a day before isolation and
disinfection of the immature embryo. For sterilization, the intact spikelet was cut short to
3 - 5 cm segments before isolation, this allowed better handling and culture of IEs. Intact
spikelets were surface-sterilized with 70% ethanol for 1 min followed by washing in
2.0% chlorox containing 0.1% Tween 20, 2 - 3 drops of savlon for 12 min under shaking
(modified from Gugssa, 2006 and O’Kennedy et al., 2004), which was followed by a 4 5-times rinse in ddH2O (sterile) by working in a laminar air-flow cabinet (LAFC). The
IEs were isolated aseptically with forceps under sterile conditions and were placed,
scutellum side-up, on petri-dishes containing K99EM embryogenic callus induction
medium (Table 3.1; Gugsa et al., 2008). Embryonic calli initiated from IEs were used in
this study as explants source for Agrobacterium-mediated transformation. Viable looking
proliferating embryogenic calli were transferred to fresh CI medium (Table 3.1) every
second week. Infection with Agrobacterium for transformation was done at this stage
using 2-3 weeks old young calli.
62
3.3.3
GA2ox and nptII marker gene plasmids
The hybrid binary plasmid pGPTV-kan containing the coding sequence for neomycin
phosphotransferase (nptII), which confers resistance to kanamycin and its analogue
geneticin (G418), under the control of the nos promoter and terminator sequences was
used in E. tef transformation. The plasmid T-DNA region also contained the coding
region of GA2 oxidase (about 1 kb) isolated from runner bean (Phaseolus coccineus)
(PcGA2ox1) obtained through functional screening (Thomas et al., 1999). The transgene
is under the control of a triple CaMV 35S promoter sequence located next to the right
border of the T-DNA (Fig. 3.1). The full construct containing the transgene, promoter
and Kan resistance was a gift of Dr. Hedden, Rothamsted Research, UK. The E. coli
strain JM109 (Invitrogen, USA) was used to maintain the plasmid before transforming
cells of Agrobacterium tumefaciens strain LBA4404. The presence of the insert in the
plasmid was confirmed using agarose gel electrophoresis for plasmid DNA digested with
restriction enzymes.
Competent Agrobacterium cells were used for transformation with the plasmid.
Agrobacterium cells were transformed with the plasmid DNA of pGPTV-kan by mixing
60 µl competent cells with 10 µl plasmid DNA harbouring the transgene and incubating
on ice for 5 min before transferring the mixture to liquid nitrogen for 5 min. The mixture
was then incubated at 37oC in a water bath for 5 min. LB medium (1 ml) was added to the
tube containing the Agrobacterium-plasmid mixture, the tube was sealed and was shaken
on a rocking table for 2 - 4 h at room temperature. After briefly spinning the tube in
Eppendorf microcentrifuge to collect the cells, 150 µl of the mixture was poured onto
63
solid LB medium containing the two antibiotics kanamycin (50 mg l-1) and rifampicin (25
mg l-1) and plates were incubated for 2 days at 28oC for selection of transformed cells.
Single colonies were randomly selected and cultured on a new antibiotic containing LB
plate for two more days. A liquid culture of the re-streaked colony was established to
verify after plasmid isolation by PCR the presence of the transgene in the plasmid.
Figure 3.1 Construction of plasmid pGPTV-Kan harbouring Phaseolus coccineus
GA2ox1 (PcGA2ox1), the triple 35S CaMV promoter sequence, the nos terminator (Tnos)
sequence and also the nptII selectable marker gene.
64
3.3.4
Agrobacterium culture, inoculation and co-cultivation
For E. tef transformation, transformed Agrobacterium (strain LBA4404) cells (500 µl)
were transferred into a 500 ml Erlenmeyer flask containing 250 ml LB/YEP medium and
kanamycin (50 mg l-1). The culture was shaken at 200 rpm at 28oC until the OD660 was
about 1.0. About 30 ml of Agrobacterium cells were centrifuged (3,500 rpm, 10 min) in a
bench top centrifuge and the cell pellet was re-suspended in 30 ml liquid co-cultivation
medium (CCM; Table 3.2) (Hensel and Kumlehn, 2004). The virulence activator
acetosyringone (Table 3.1) was added immediately before inoculation. The cultures were
stirred at 50 rpm for about 1 h before infection. After infection for 6 – 12 h, calli were
blotted onto sterile tissue paper and briefly rinsed with liquid CCM. Washed calli were
then co-cultivated by growing on K99EM callus induction medium (K99EM-CIM) for 23 days with 2.0 mg l-l of the auxin 2, 4-dichlorophenoxyacetic acid added (2, 4-D) but
without addition of any antibiotic (Table 3.1). The surviving calli were then transferred to
a selection medium. For the control, embryogenic calli were kept uninfected with
Agrobacterium but were subject to all post infection treatments excluding antibiotic
treatment.
After co-cultivation, and further growth for 2-3 days in antibiotic free medium, calli were
then transferred to CI - SL media (Table 3.1) supplemented with 2.17 mg l-1 2,4-D, 250
mg l-1 cefotaxime (or 250 mg l-1 timetin) and 100 mg l-1 kanamycin and then cultured for
2 to 3 weeks. After this time, calli were transferred to a CI - SL medium supplemented
with 2.17 mg l-1 2,4-D, 200 mg l-1 cefotaxime (or 200 mg l-1 timetin) and 100 mg l-1
kanamycin before plant regeneration. All culturing until the regeneration stage was done
65
using culture plates (50 mm x 10 mm) which were kept in dark at 24±20C. Developing
embryos (sometimes turning green) transferred to K4NM regeneration medium were
grown under a 16 h photoperiod maintaining a temperature of 24±20C.
3.3.5
Plant regeneration
After selection, calli that were still creamy-white were transferred to K4NM preregeneration medium (Table 3.1). The medium had no 2, 4-D addition and a reduced
concentration of kanamycin (50mg l-1) and cefotaxime (125 mg l-1). After two rounds of
selection on this medium, calli were transferred to a regeneration medium (Table 3.2) for
6 to 8 weeks which was refreshed after 3 weeks (with no antibiotics added). Developed
regenerated shoots (2 to 4 cm long) were transferred for 1 week to partly ventilated baby
jars containing the regeneration medium. Plantlets were then transferred to an
environmentally controlled phytotron for hardening-off.
3.3.6
Preparation of plant material and culture
Regenerated T0 plants were acclimatized and grown in an environmentally controlled
greenhouse with a 16-h photoperiod provided by natural light supplemented with light
from sodium lamps to maintain a minimum PAR of 350 µmolm-2s-1. The temperature was
maintained at 23-27°C (day) and 15-18°C (night). Seeds from selected T0 plants that
showed positive PCR amplification of GA2ox1 insert were further grown in pots [15 cm
diameter (top) x 12.5 cm (height) and 10 cm (bottom)]. A soil mixture consisting of peat
66
(75%), sterilized loam (12%), vermiculite (3%) and grit (10%) was used supplemented
with a slow release fertilizer.
3.3.7
DNA isolation and PCR screening of E. tef regenerants
All regenerated plants grown in the phytotron were screened for the presence of the
transgene by PCR using gene specific primers for PcGA2ox and nptII. Leaf tissue from
putatively transformed plantlets was used to extract genomic DNA using a modified
CTAB method (Harini et al., 2008). Same procedure was applied to extract DNA from
untransformed plants (control) that however were regenerated through the whole process
except the Agro-infection. DNA amplification was carried out in a 25 µl reaction mixture
with template DNA (ranging between 100-150 ng), 0.5 µl dNTPs (10 mM stock), 1.2µl
MgCl2 (25 mM stock), 0.5 µl primer (10mM), 5 µl of a 5X reaction buffer, and 0.15 µl
Taq polymerase (Fermentas, Canada). Amplifications were carried for 35 cycles (DNA
denaturation: 940C, 30 sec.; primer annealing: 600C, 30 sec.; DNA extension: 720C, 30
sec.). Sequences of the PcGA2ox and nptII gene primers used for PCR amplification
were: one sense primer (PcGA2ox): 5’- TCA TAG TGA ACG CCT GTA GG- 3’ and two
anti-sense primers: 5’-TGT TCT TCA CTG CTG TAA TG - 3’ and 5’- ACC TGC TTA
ACG TAT TCC TCT G – 3’obtained from NCBI database mRNA nucleotide sequence
(Acc. No. AJ132438 for PcGA2ox). Expected fragment size after amplification of GA2ox
gene were 321 and 391 bp, respectively. PCR amplification of the nptII gene was
performed under identical conditions as used for PcGA2 ox. Sequences of the nptII
primers used for PCR amplifications were: primer 1: 5’-AGA CAA TCG GCT GCT
CTG AT-3’ and primer 2: 5’- ATA CTT TCT CGG CAG GAG CA-3’. PCR products
67
with expected size of 365bp were analyzed by gel electrophoresis on a 1.0% agarose gel
(Sigma, St. Louis, MO) to confirm that a correct size product was amplified. The sizes of
the amplified fragments were determined using a molecular weight marker after ethidium
bromide staining to view fragments on the gel (GIBCO BRL, Gaithersburg, MD).
3.3.8
Phenotypic measurements and characterization of T1 generation
T1 generation transformed plants were grown from seeds after selfing putative
transformed plants (T0 generation) that have shown GA2ox1amplification by PCR from
isolated genomic DNA. T1 generation plants that showed a dwarfed phenotype at
seedling stage were further phenotypically characterized for growth and yield. Control
plants that were subjected to transformation and had a wild-type phenotype were used for
comparison. Measurements were taken at plant maturity to determine plant height, length
of culm, length and diameter of individual internodes, above ground biomass, tillering,
yield and yield components. Dry weight for above ground biomass was determined by
drying fresh material at 80oC for 2 days in an oven. Grain yield was determined by
measuring the weight of seeds from main and secondary tillers. All data were collected at
plant maturity and analyzed using GenStat statistical package.
3.3.9
Analysis of endogenous GA content
From selected dwarfed T1 plants, sample of near equal weight were harvested during the
stem elongation stage before panicle initiation from the secondary tillers. The upper-most
68
two internodes including its nodes were cut and weighed and stored at -800C until
analysis. The same procedure was followed as described in Chapter 2 Section 2.3.5.
Table 3.1 Media used for induction of embryogenic callus, co-cultivation, selection and
regeneration of transformed E. tef shoots.
K99EM* based callus induction medium
K99EM
Modified MS salts and Organic I* and Organic II* containing in 1 L
(CIM)
medium: 1.023 mg (7 mM) glutamine; 250 mM casein hydrolysate; 213.2 mg
(1 M) MES; 90 g (250 mM) maltose H2O; 2.17 mg (10 µM) 2,4-D; pH 5.8
MS based co-cultivation medium
CCM
Medium ( 1 L) contains: MS salts and vitamins (4.4 g); 30 g maltose; 800 mg
L-cysteine; 500 mg L-proline; 300 mg casein hydrolysate; 350 mg myoinositol; 98 mg acetosyringone; 2.5 mg DICAMBA, 2.0 mg 2, 4-D; pH 5.8
K99EM based selection medium
CI- SL
Modified MS salts and Organic I and Organic II containing in 1 L: 1.023 mg
(7 mM) glutamine; 250 mM casein hydrolysate; 213.2 mg (1 M) MES; 90 g
(250 mM) maltose H2O; 2.17 mg (10µM) 2,4-D; pH 5.8, and 250 mg
cefotaxime; 100 mg kanamycin
K4NB** based regeneration medium
PRE-RE
Medium ( 1 L) contains: 0.25 M glutamine, 10 mM CuSO4, 36 g (100 mM)
maltose H2O; 1 mM BAP; 50 mg kanamycin; 125 mg cefotaxime; pH 5.8
RE
Medium ( 1 L) contains: 0.25 M glutamine; 10mM CuSO4, 100 mM maltose
H2O; 1 mM BAP; pH 5.8
* (See Table 3.2); ** (See Table 3.3); CIM = callus induction medium is equivalent
medium to K99EM (Gugssa, 2008); CCM = co-cultivation medium + antibiotics
(Hensel and Kulmen, 2004); CI-SL = callus induction and selection medium (K99EM +
antibiotics); PRE-RE = pre-regeneration medium; RE= regeneration medium.
69
Table 3.2 Composition of the K99EM medium (Gugssa, 2008) used for embryogenic
callus induction from E. tef immature embryos.
Media Components
Inorganic salts
Conc. (mg l-1 )
(NH4)2SO4
80 (1 mM)
Retinol
1.01 (0.04 µM )
Malic acid
40 (0.3 µM)
KNO3
2,022 (20 mM)
Thiamine HCl
1.0 µM
Citric acid
40 (0.1 µM)
KH2PO4
340 (2.5 mM)
Riboflavin
0.2 (0.5 µM)
Fumaric acid
40 (0.3 µM)
CaCl2.2H2O
441 (3 mM)
Ca-panthenate
1.0 (4.2 µM)
Na-pyrovate
20 (0.2 µM)
MgSO4.7 H2O
246 (1 mM)
Folic acid
0.4 (0.9 µM)
Glutamine
1.023 (7 mM)
NaFeEDTA
27.5 (75 µM)
Pyridoxine HCl
1.0 ( 4.9 µM)
Casein-
250 µM
Organics I
Conc. (mg l-1 )
Organics II
Conc. (mg l-1 )
hydrolysate
MnSO4.4 H2O
11.2 (50 µM)
Cobalamine
0.02
MES
213.2 (0.1 M)
H3BO3
3.1 (50 µM)
Ascorbic acid
2.0 (11.4 µM)
Maltose H2O
90,000 (250 mM)
ZnSO4.7 H2O
7.2 (25 µM)
Calciferol
0.01 (0.03 µM
2,4-D
2.17 (10 µM)
Na2MoO4.2 H2O
0.125 (5 µM)
Biotin
0.01 (0.04 µM)
CuSo4.5 H2O
0.025 (0.2 µM)
Cholin chloride
1.0 (7.1 µM)
Phytagel
0.3%
CoCl2.6 H2O
0.025 (0.2 µM)
p-aminobenzoic
0.02 (0.1 µM))
pH
5.8
acid
KI
0.17 (1µM)
Myo-inositol
100 (0.6 µM)
Nicotinic acid
1.0 (8.1 µM)
70
Table 3.3 Ionic composition of the K4NB regeneration media used for E. tef immature
embryo cultures.
Components of K4NB regeneration medium
Inorganic salts
Organics
Concentration
(mg l-1)
Macro-nutrients
(NH4)2SO4
KNO3
KH2PO4
CaCl2.2H2O
320 (4 mM)
3640 (36 mM)
340 (2.5 mM)
441 (3 mM)
MgSO4.7 H2O
Na-FeEDTA
246 (1 mM)
27.5 (75 µM)
Concentration (mg l-1)
Organics I
T-vitamins (1000x)
Thiamine HCl
10.0
Pyradoxine HCl
1.0
Organics II
Glutamine
CuSO4
0.25 mM
10.0 mM
MaltoseH2O
100 mM
2,4-D
Phytagel (SigmaPB169)
pH
1µM
0.3%
Micro
MnSO4.4 H2O
H3BO3
ZnSO4.7 H2O
11.2 (50 µM)
3.1 (50 µM)
7.2 (25 µM)
Na2MoO4.2 H2O
0.12 (0.5 µM)
CuSo4.5 H2O
CoCl2.6 H2O
KI
1.25 (5 µM)
0.024 (0.1 µM)
0.17 (1 µM)
71
5.8
3.4 Results
3.4.1
Plant transformation
Agrobacterium (strain LBA4404)-mediated transformation was carried out using the
embryogenic callus from the scutellum region of immature E. tef embryos. The immature
embryo produced embryogenic callus from the scutellum side within 2 weeks of culturing
the embryos on embryogenic callus induction medium (Figs. 3.1 A and B). In some
cases, callus already appeared within a week, this callus was also used for transformation.
Further, not all embryos formed callus and some embryos only formed callus after 2
weeks. This callus was not further used.
The antibiotic-containing selection medium was optimized for selecting kanamycinresistant germinating mature embryos (Table 3.1). At 25 mg l-1 G418, up to 75% of
shoots derived from non-transformed embryos wilted after 12 days exposure to the
antibiotic without completely collapsing. The shoots also showed a yellowing of leaf tips.
At 40 mg l-1 G418, up to 90% of non-transformed shoots collapsed and all shoots turned
brown. Germinating shoots did not survive treatment with 75 mg l-1 G418 after 12 days
of treatment with G418 (Table 3.1).
Immature embryos developed in to embryogenic callus (Figs. 3.2A and B) proliferated
into shoots after 2 months of culturing the embryogenic callus on induction medium
followed by 2 months of culturing on K4NM regeneration medium (Fig. 3.2C). Several
regenerated shoots turned white or failed to survive while growing in vitro (data not
72
shown). Regenerated green plantlets were obtained which were hardened-off and grown
to maturity in an environmentally controlled phytotron (Fig. 3.2E). A total of 55 plants
were regenerated to fully viable plants setting seeds at maturity (Fig 3.1D). The
regenerated putative transformed plants had generally a slower growth when compared to
non-transformed plants. Putative transformed plants were kept under high humidity in the
phytotron with a perforated polyethylene bag covering the pots and growing plants for
about 1 to 2 weeks. E. tef being strictly selfing, no bagging was required to avoid
crossings. At maturity, all transplanted and successfully grown plants produced fertile
panicles setting seeds.
3.4.2
Transgene detection
In 8 of the 55 putative transformed plants (T0 generation), which were regenerated and
grown in the phytotron, the genome-inserted PcGA2ox or nptII sequences were detected
by PCR in isolated genomic DNAs (Fig. 3.3). Sequence analysis of the amplified PCR
product confirmed that amplified products using two sets of PcGA2ox primers with the
sizes of 321 bp and 391 bp (PcGA2ox), and 365 bp (nptII) had a 97.2 to 99.8% sequence
similarity to PcGA2ox and nptII, respectively. However, detection of PcGA2ox or nptII
sequences was inconsistent in the T1 generation where from several plants with a semidwarfed phenotype the two sequences could not be consistently amplified by PCR from
isolated genomic DNA in repeated amplifications.
73
A
C
B
D
Figure 3.2 E. tef shoot regeneration using immature embryo from young emerging
panicle as explants. (A) Embryogenic callus deriving from immature zygotic embryos;
(B) embryogenic callus proliferating into shoots; (C) embryogenic callus tissue
proliferating into shoots after 2 months of culture on embryogenic callus induction
medium followed by two months of culture on K4NM regeneration medium; (D) fertile
regenerating plants.
74
Table 3.4 Survival of non-transformed E. tef seedlings derived from
100 mature embryos on antibiotic (G418)-containing selection
medium.
Remarks
G418 (mg l-1)
Survival (%)
0
100
20
100
<50% yellowing of leaf
25
100
<75% wilting and yellowing
30
100
100% yellowing of leaf with
1/3rd leaf top area burning
40
10
90% collapsed and brown
50
5
95% collapsed and brown
75
0
100
0
125
0
75
A
Event Number (PcGA2ox )
M
M
Control
321bp
1
PC NC
2 3 4 5 6 7 8 9
B
PcGA2 ox
M
nptII
N
391bp
365bp
1 2 3 4 56 7 8
A BCDEFG
C
PcGA2ox detection at T1
M
L11(1)
L12(3)
L13(1)
L13(2)
L13(3)
L14(1)
L14(2)
L7(3)
L8(1)
L8(2)
L8(3)
NC
*L18
L5(1)
L5(2)
L6(1)
PC
*L19(1)
*L19(2)
*L21
L2(8)
L2(3)
L3(1)
*L4(2)
L4(3)
321bp
Figure 3.3 PCR amplification of PcGA2 ox (A and C) and PcGA2ox and nptII (B);
sequences from putative transformed plants of T0 (A & B) and T1 (C) generation.
(M) 1kb (A & B) and 100 bp (C) ladder molecular size markers; (NC) negative
control without template DNA added in a reaction mix; (PC) a positive control with
plasmid pGPTV-kan. Lines with asterisk (*) are among semi-dwarf phenotypes
used for further phenotypic analysis.
76
3.4.3
Phenotypic characterization
3.4.3.1 Culm, internode and panicle length
Despite inconsistent PCR results, significant (P<0.001) variation in mean culm height
was found between T1 plants, with a dwarf/ semi-dwarf phenotype, and wild-type nontransformed plants. Plants with a dwarf/semi-dwarf phenotype had a Culm height ranging
from 65 cm to 117 cm whereas the wild type control plants had a height of 157 cm
(Figures 3.4 and 3.5). E. tef plants of line number 18 had the shortest Culm length (65
cm) followed by plants of line 23 (98 cm). In plants with a dwarf or semi-dwarf
phenotype, major reduction in plant height originated from reduction of the Culm and not
from reduction of the panicle. Culm reduction was mostly due to absence of elongation of
the upper-most internodes (data not shown). In some dwarf plants elongation of the 7th
and/or 8th internodes did not occur when compared to control plants. In most dwarfed
plants reduction in length was found in all internodes (data not shown).
There was no significant variation in internode diameter between dwarf plants and wildtype (control) plants. Generally, internode diameter increased up to the 3rd internode
when overall mean values were compared (data not shown). However, control plants had
a small and steady increase in diameter up to the 6th internode. Plants showed acropetal
increase in diameter upwards in both semi-dwarf and control plants demonstrating a weak
tapering in these plants.
77
Panicle length of the semi-dwarf plants ranged from 60 - 80 cm when compared to
panicle length of the control (67 cm) (Figure 3.4). Panicle elongation was not associated
with any change in plant height but positively correlated with tiller number. Panicle
emergence was delayed by a few days to a few (2 - 3) weeks in more dwarfed plants. In
semi-dwarf plants the number of tillers per plant varied between 18 - 67 and the number
was significantly higher (P<0.001) (1.3 - 4.8 fold) than in control plants (Figure 3.4).
Grain weight per main tiller was not significantly different (P > 0.05) between semidwarf plants and control plants and differences originated from secondary tillers.
Culm
Panicle
Tiller
180
80
*
Length (cm)
60
120
90
40
*
60
*
20
30
0
0
Control 19 (2)
19(1)
4
19 (3)
10
21
25
28
23
18 (1)
Dwarf lines and a control (wild type)
Figure 3.4 Culm and panicle height (cm) and number of tillers per plant of
putatively transformed dwarf E.tef plants. Standard error (SE) values and
significance level was determined by Student's t-test using GenStat Discovery
Edition (VSN International Ltd) (P ≤ 0.001).
78
No. of tiller plant -1
150
Control
19(3)
19(2)
28
10
18
Figure 3.5 Selected E. tef dwarf (18) and semi-dwarf (19(3), 19(2), 28 and 10 T1
generation plants. Control is a plant subjected to the transformation process without
addition of Agrobacterium and antibiotic selection.
79
Up to four-fold increase in grain yield per plant was also found in some semi-dwarf
plants (Figure 3.6). Higher biomass was found in the majority of semi-dwarf plants when
compared to control plants (Table 3.5). Further, most of above-ground shoot weight
increase was due to more tillering (Table 3.5).
25
Main tiller
Secondary tiller
Total
*
*
20
Grain wt.(g) plant -1
*
*
15
*
*
*
*
10
5
0
Control
4
10
18(1)
19(1)
19(2)
19(3)
21
23
25
28
Dwarf lines and a control (wild type)
Figure 3.6 Seed weight of primary and secondary panicles of putatively transformed dwarf
E. tef lines and a control. Standard error (SE) values and significance level was determined
by Student's t-test using GenStat Discovery Edition (VSN International Ltd) (P ≤ 0.001).
80
Table 3.5 Above ground biomass (gm) of putatively transformed dwarf tDZ-01-196 E. tef
plants
Lines
Shoot FW
Culm + Leaf
DW
Panicle
DW
Tillers
Culm +
Leaf DW
Tillers
panicle
DW
Total
Shoot DW
Control
213.7
5.9
2.7
54.9
10.8
74.2
4
429.9
4.7
2.3
120.8
28.9
177.7
10
375.3
3.8
2.2
103.2
12.8
174.0
18 (1)
155.9
4.2
2.6
42.0
10.1
144.2
19(1)
493.3
4.5
2.3
134.4
36.5
156.7
19 (2)
504.1
4.4
2.8
140.4
32.8
122.1
19 (3)
418.8
5.6
4.3
116.4
50.0
180.3
21
465.7
4.1
3.0
125.3
41.6
176.4
23
171.2
4.3
4.5
43.1
14.7
113.9
25
424.4
3.6
2.3
113.3
25.0
66.6
28
346.0
3.2
1.9
89.5
19.3
58.8
Mean
363.5
4.4
2.8
98.5
25.7
131.4
SE
38.35
0.24
0.25
10.90
4.07
14.24
Significance
***
***
***
***
***
***
FW = Fresh weight (gm); DW = Dry weight (gm)
Standard error (SE) values and significance level was determined by student's t-test using
GenStat Discovery Edition (VSN International Ltd). (*** =P<0.001).
81
3.4.4
Analysis of endogenous GA content
Semi-dwarf plants had lower amounts of bioactive GA as well as lower amounts of
precursors than the control plants when the endogenous GA content of plant tissues taken
from the upper-most two internodes at shoot elongation stages were analyzed (Figure 3.7).
The content of the most abundant bioactive GA form, GA1, in dwarf plants such as L18(2),
L19(2), L9(1), L21, L18 and L10, was considerably less when compared to the control.
Amounts of immediate GA1 precursors, such as GA44, GA19, GA20 and in particular GA19,
were also reduced in the dwarf plants (Figure 3.7). An expected increase in GA29 due to
GA2ox over-expression was, however, not found. GA amount was also compared with the
GA amount in the E. tef dwarf landrace variety, Gea Lammie, grown under similar
conditions. Bioactive GA amount (including GA precursors in the 13β-hydroxylation
pathway (See Table 2.1) except in some cases like GA29 and GA51) in putative transformed
plants was much lower than in Gea Lammie.
82
25.0
D1a
G1a
L18(2)
L19(2)
L9(1)
L10
L18
L21
ng per g dry wt.
20.0
15.0
10.0
5.0
0.0
GA53 GA1 GA29 GA8 GA20 GA44 GA4 GA51 GA19 GA34 GA9
Analyzed intermediates and bioactive GAs
Figure 3.7 Comparison of endogenous GA levels in the GA biosynthetic pathway between
dwarf plants and wild-type controls (DZ-01-196 tall phenotype and Gea Lammie short
phenotype). Dwarf lines (L18(2), L19(2), L9(1), L21, L18 and L10) represent semi-dwarf
phenotypes. Standard error (SE) values (bar) was determined by Student's t-test using
GenStat Discovery Edition (VSN International Ltd).
83
3.4 Discussion
This is the first report about E. tef transformation with the aim of modifying plant stature
through over-expression of a GA inactivating gene (GA2ox) from Phaseolus coccineus. In
vitro regenerated plants were successfully grown into seed producing mature fertile plants.
For Agrobacterium-mediated transformation, a combination of different media has been
successfully applied for embryogenic callus induction, Agrobacterium inoculation and cocultivation and plant regeneration. These media have been previously used for plant
regeneration from immature embryos in barley and rice as well as in E. tef (Hensel and
Kumlehn, 2004; Gugssa, 2008; Ramana Rao and Narasimha Rao, 2007). However, in
contrast to previous reports about E. tef transformation either expressing the Gfp protein,
which either resulted in a single transformed E. tef plant (Gugssa, 2008), or integrating the
gus gene into callus tissue (Mengiste, 1991), 8 putative transformed plants carrying the insert
(PcGA20 ox or nptII gene sequence) at the T0 generation were regenerated in this study.
Success in embryogenic callus induction using zygotic immature embryos as explants and
regeneration into shoots was dependant on the age (size) of the embryos. Older immature
embryos developed callus later with limited differentiation, very small embryos died on the
callus-inducing medium. Intermediate-sized immature embryos successfully developed into
embryogenic callus and regenerated ultimately into a fertile seed-setting plant. Some of the in
vitro regenerated shoots lost, however, their green pigment during growth and died during the
selection/regeneration stage. The reason for this is still unclear and requires further
investigation. Also, anti-necrotic compounds, such as ascorbic acid, cysteine, and silver
nitrate in co-cultivation and subsequent culture media, might be applied in future work to
fine-tune the transformation process.
84
High natural kanamycin resistance of E. tef callus was also found in this study. Such natural
antibiotic resistance of callus has been already reported for E. tef (Mengiste, 1991) as well as
for rice (Twyman et al., 2002). To overcome natural resistance against kanamycin, a more
potent kanamycin derivative, geneticin (G418), was used in this study. This antibiotic
completely controlled shoot regeneration from mature E. tef embryos at 75 mg l-1. Gugssa
(2008) previously found no inhibition of E. tef callus induction and somatic embryo
formation from immature embryos with 50 mg l-1 G418. However, the amount required for
complete killing of untransformed callus was not reported. A future study might, therefore,
investigate if selection using G418 and others such as glyphosate or hygromycin resistance
would be a more powerful selection system for transformed E. tef.
Agrobactrium growth during inoculated callus development was suppressed using 250 mg l-1
cefotaxime, but Agrobacterium growth was sometimes not completely blocked. A higher
cefotaxime concentration (500 mg l-1), in addition to better suppression of bacterial growth,
has been reported inducing embryogenesis in rice and sugarcane (Mittal et al., 2009) whereas
Ratnayake et al. (2010) recently found inhibition of rice embryogenesis with amounts higher
than 500 mg l-1. Therefore, a future study might also determine the optimum cefotaxime
amount suitable for E. tef transformation.
Plant height significantly varied among the semi-dwarf plants and maximal reduction in
Culm height was 56% when compared to the height of control plants. Reduction was across
internodes except for the most dwarfed plants. In these plants, there was no elongation of the
upper most three internodes. Results are in agreement with earlier observations modifying
height controlling GA genes (Lo et al., 2008; Hedden 1999). In this study, reduction in plant
85
height in the semi-dwarf E. tef plants was also associated with reduced amounts of bioactive
GA1, a metabolite of 13β-hydroxylation and a dominant GA biosynthesis product in E. tef.
However, accumulation of GA8, a deactivation product of the bioactive GA1, in the semidwarf plants was not proportional to the relative height differences or to deactivation of GA1.
In rice, GA deactivation decreased height (Lo et al., 2008) with increasing yield (Ookawa et
al., 2010). Results in this study also showed that yield increased in some semi-dwarf plants.
This was not entirely due to an increase in the number of tillers per plant because some plants
had a low total tiller number but had still a higher grain yield per plant (e.g. line 23 and 19(2)
vs. 19(3)). Yield increase was also highly and positively related to shoot and panicle dry
weight. Although semi-dwarf plants had generally delayed panicle initiation, shoot growth,
including panicle growth, was extended due to delayed maturity with fertility of panicle and
seed set not affected.
Although putatively transformed plants showed the semi-dwarfed phenotype, detection of
integration of the transgene in T1 plants by PCR was inconsistent and no amplification
product was found when the PCR reaction was repeated. Therefore, the final proof that plants
are indeed transformed has so far failed despite several attempts repeating genomic DNA
isolation and changing the PCR conditions. Currently, the possibility that any found morphophysiological differences are due to somaclonal variations owing to the relatively higher rate
of 2, 4-D applied, which is known to cause such changes, cannot not be ruled out (Banerjee et
al., 1985). Therefore, dwarfed plants have to be further characterized to consistently show
GA2ox transgene integration into the genome, including Southern blot analysis, and if
expression of integrated GA2ox is always associated with reduction in plant height. However,
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if somaclonal variation has caused the phenotype change, this technique might also be
considered as an excellent tool to develop semi-dwarf phenotypes in E. tef.
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CHAPTER 4
ISOLATION, CHARACTERIZATION AND EXPRESSION OF
GA GENES WITH PARTICULAR EMPHASIS ON GA20ox IN
TEF (Eragrostis tef)
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4.1 Abstract
To isolate GA20ox and Rht genes and further characterize and monitor the expression of
GA20ox in E. tef, degenerated primers derived from rice, wheat, maize and sorghum ortholog
sequences were used of full or partial sequences (422 to 1500 bp) by normal, “anchored” and
nested PCR. Three putative GA20ox genes orthologous to the rice sd-1 (semi-dwarf 1) were
identified. Sequences contained the characteristic domains KLPWKET and NYYPXCXXP
and the conserved H and D amino acid residues. Three more sequence orthologous to the
wheat Rht gene, the rice Elongated Uppermost Internode (Eui) and a Cytochrome P450
monooxygenase gene involved in brassinosteroid (BR) deactivation were also isolated. The
E. tef Rht sequence had the conserved motifs DELLA, VHYNP, VHVVD, and a C-terminus
of the GRAS domain. E. coli expressed GA20ox1a catalyzed the conversion of the [14C]labelled gibberelline precursor GA12/ to GA9. The three GA20ox were further differentially
expressed in different plant tissues. EtGA20ox1a and EtGA20ox1b had highest transcription
in the uppermost internodes whereas EtGA20ox2 transcription was low in most tissues.
Further, in E. tef EtGA20ox1b was possibly the functional equivalent to the rice sd-1 gene.
All sequences showed close homology with ortholog genes from sorghum, maize, rice barley.
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4.2 Introduction
Among the genes of GA-biosynthesis, GA 20-oxidase is shown to be an important regulator
in the pathway. It catalyses several late steps converting GA12 and GA53 in parallel pathways
to respective products GA9 and GA12 which then are converted to bioactive forms, GA4 and
GA1 by GA 3β-hydroxylase (see chapter 1 “Introduction”). Availability of genes encoding
GA-biosynthetic enzymes has allowed understanding how these genes function. In many
species the members of the dioxygenases family, GA 20-oxidases, are encoded by several
genes. They show distinct spatial and temporal expression patterns with some overlapping
function in plant development regulated by environmental signals and endogenous factors
(Hedden and Phillips 2000; Lange, 1998; Hedden and Kamiya, 1997). Such functional
redundancy in the GA 20-oxidases has been found for many crops and has been considered as
the reason why null mutations in some of the genes exhibit a semi-dwarf phenotype. The
existence of an overlap between expression patterns of isozymes and mobility of the GA
products within a plant system prohibits severe dwarfing of the stem (Hedden and Phillip,
2000). Due to their regulatory role determining GA concentrations, members of this gene
family have been the target for plant genetic manipulation and introduction of agronomically
useful traits (Appleford et al., 2006; Carrera et al. 2000; Sakamoto et al. 2003).
Genomic DNA amplification by PCR for isolation of gene sequences is a common procedure
and has also been used in this part of the study to isolate GA biosynthesis and signalling
genes. A genomic survey at the NCBI database (http://www.ncbi.nlm.nih.gov), as done for
this study, is also a vital tool to provide information for the design of primers for gene
amplification in PCR application for reverse transcription (e.g. RACE). The Rapid
Amplification of cDNA Ends (RACE) is a method for amplifying DNA sequences from a
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mRNA templates between a defined or identified internal site and unknown sequences at
either to the 3' or the 5' -end of the mRNA. It requires two gene sequence-specific primers
that flank the region of sequence to amplify relatively few target molecules in a complex
mixture (Miao et al., 2010). Therefore, 3' and 5' RACE methodologies offer possible
solutions to the severe limitation impose on the PCR to amplify regions of unknown
sequences. The 5' RACE, also known as “anchored” PCR, is a technique that facilitates the
isolation of unknown 5' ends from low-copy transcriptions. Low level of concentration is
expected for phytohormones and their corresponding related genes such as GA20ox that
involve in the, GA, regulation (Hedden and Phillip, 2000).
No GA gene so far has been isolated from E. tef to allow studying the allelic diversity of GA
genes in the genetic pool, explore their functional roles to identify a useful mutation in GA
genes. Having verified the importance of GA in tef height regulation, therefore, the objectives
of this part of the study was to first identify and clone full-length cDNAs from various GA
genes in particular encoding the multifunctional GA20-oxidase. Secondly, the objective was
to characterize these cloned GA gene sequences on the genomic level and study their
expression in various plant parts during plant development.
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4.3 Materials and Methods
4.3.1
Plant material and plant growth
Seed material of tef (Eragrostis tef) variety DZ-01-196 for this experiment was obtained from
the Ethiopian Institute of Agricultural Research, Holetta Agricultural Research Centre,
Ethiopia. Seeds were germinated on germination mix soil and the seedlings were grown in
pots (about 225 plants per m2) under a 26±2 / 18°C day/night temperature and a 14 hr day
length. Plants were further supplemented with a half-strength Hoagland nutrient solution until
all samples were collected.
4.3.2
Genomic DNA isolation
Young leaves were homogenized in liquid nitrogen and transferred to a reaction tube
containing pre-heated (600C) extraction buffer according to the method of Harini et al. (2008)
with a modification omitting the NaCl addition. DNA spooling was further replaced by
precipitating the DNA. Samples not immediately used were frozen and stored at -800C. The
mixture of fine-powdered plant material was immediately incubated after homogenization in
extraction buffer at 600C for 30 min with intermittent shaking. This was followed by adding
an equal volume of chloroform: isoamyl alcohol (24:1) and subsequent centrifugation at 6000
g for 10 min. The aqueous phase was further extracted with an equal volume of phenol:
chloroform: isoamyl alcohol (25:24:1) and centrifuged at 8000 g for 10 min. After
centrifugation, the supernatant was transferred into 2.5 volumes of chilled ethanol and
genomic DNA was precipitated. Further purification was made using isopropanol which was
followed by centrifugation at 8000 g for 10 min at 4oC to collect precipitated DNA. Washing
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of precipitated DNA was carried out with 70% ethanol twice followed by centrifugation. The
genomic DNA was finally dissolved either in TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH
8.0) or dsH2O and further treated with RNase and precipitated again as outlined above. DNA
concentration of samples was determined using the nanodrop technique (Thermo Scientific
NanoDrop 3300) and then visually by running 2 µl of sample DNA on a 1% agarose gel to
check for purity.
4.3.3
Gene identification and isolation
Identification and cloning of GA biosynthetic genes, the rice SD-1 homologues (GA20oxidases) in E. tef was carried out using degenerated oligonucleotide (sense and antisense)
primers (Table 3.1). Primers for specific amplification of the genes sequences were designed
based on the published sequences of the GA20ox genes of different monocot plants accessed
from the GenBank. Primers were designed based on conserved amino acid domains of rice,
sorghum, maize and wheat orthologous sequences obtained from the GenBank database. The
sequences of these nucleotides were designed using the primer3 program (Rozen and
Skaletsky, 2000). The PCR conditions were optimized using these primers to amplify either
genomic DNA or cDNA.
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Table 4.1 Primers used in PCR to amplify GA20ox gene fragments using E. tef genomic
DNA
Target gene
Primers
Degenerate/specific primers (5’ – 3’)
GA20 ox
F1
GCTGCCGTGGAAGGAGAC
F2
CACCGATGATGATGATGATGATG
F3
CTACGCGAGCAGCTTCACG
R1
CGCCGATGTTGACGACGA
Primers for GA20ox were targeted to the region encoding the amino acid sequence
KLPWKET (sense) and NYYPXCXXP (antisense). PCR amplification of E. tef genomic
DNA (gDNA) and complementary DNA (cDNA) was done using either Dream Taq
(Fermentas, Canada) or FastStart Taq (Roche, UK) DNA-polymerases. A 50 µl reaction
mixture consisted of the following components: 5x PCR buffer, 10 µl MgCl2 (25 mM), 2 µl
dNTPs (10 mM each); 1 µl each for forward and reverse primers (10 µM), 1 µl DNA (100
ng/µl); 1 µl DMSO; 0.25 µl Dream Taq DNA-polymerase (5 U/µl); 33.75 µl H2O. PCR
reactions were prepared on ice and run at the following standard cycling conditions: 94°C (2
min) followed by 35 cycles consisting of 94°C (30 sec); 55°C-65°C (30 sec); 72°C (1
min/kb). This was followed by DNA extension at 72°C for 7 min and then holding the
reaction at 4°C.
When amplification of a high-fidelity PCR component was required, the “Phusion”
polymerase proof reading enzyme was used (Finnzymes). The PCR reaction contained the
following: 10 µl 5x Phusion “GC” buffer; 1 µl dNTPs (10 mM each); 1 µl each for forward
94
primer (10 µM); 1µl DNA (100 ng/µl); 1 µl DMSO; 0.5 µl Phusion polymerase (2 U/µl); 34
µl H2O. Preparation of the PCR reactions were carried out on ice with reactions running at the
following standard cycling conditions: 98°C (30 sec) followed by 35 cycles with 98°C (10
sec); 55°C-70°C (30 sec); 72°C (1 min/1kb) and DNA extension after 35 cycles at 72°C for 7
min followed by holding the reaction at 4°C. Genomic DNA amplifications were done using
GC-rich buffer and addition of DMSO (molecular grade) in reactions to improve
amplification efficiency. PCR products were visualized on a 1% agarose gel. For cloning,
bands were excised from the gel and purified with a commercially available PCR product
purification kit (Qiagen, Germany). The extracted DNA was used as a template for cloning,
or nested PCR using the conditions described above. Cloning was carried out using the
pGEM-T Easy cloning vector (Invitrogen, USA).
4.3.4
Isolation of complete E. tef GA20ox coding regions
For extension of GA20ox homologous (GA20 ox1-ox3) to their unknown 5’ and 3’, a total of
20 primers either as specific primers SPR1, SPR2 or SPR3 (nested primers for upstream
extension in 5’ direction) and SPF1 and SPF2 (nested primers for downstream extension in
the 3’ direction) were designed and used for nested PCR amplification (Table 5.2). cDNA
synthesis was done using anchor primers (supplied with cDNA synthesis kit; Invitrogen,
USA) and manually designed specific primers (SP primers) (Table 5.2). Primers were
allowed to anneal identified polymorphic regions to also enable selective amplifications of
homologous upstream and downstream regions of alleles using reverse PCR (RACE-PCR).
cDNA synthesis. PCR amplification procedures were done using the 2nd Generation 5’/3’
RACE Kit (Roche, Switzerland) following the manufacturers recommendations for fulllength cDNA synthesis of genes of interest.
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Primers for isolation of a full-length sequence of GA20 ox1 (1420 bp), but containing also
the start and stop codon and restriction sites BamHI and HindIII (underlined), were designed
as follows, sense: 5’-AGG GAT CCA GCC AGC TGC CCG TGA TG-3’ and antisense: 5’TGA AGC TTA ACA GAA CAG GCG GTC ATG GAT GAC-3’. A nucleotide (“A”) was
added to this primer to ensure that the start codon (ATG) in the GA20 ox1 is “in-frame”
during after cloning into the plasmid pET-32a(+). In order to avoid error incorporated during
the PCR-amplification, high fidelity “Phusion” polymerase proof reading enzyme
(Finnzymes) was used and DNA sequences were determined from at least 3 -5 independent
clones of the amplified product.
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Table 4.2 Primers used for GA20ox sequence RACE- PCR amplification
Target gene
Primers
Degenerate/specific primers (5’ – 3’)
GA20 ox1
GA20 ox1SPR3
GCGGCAGCGTGAAGAAGGCGTCCAT
GA20 ox1SPR2
CACATCATCATCATCATCATCGGT
GA20 ox1SPR1
CTCGGCGGGTAGTAGTTGAGGCGCAT
GA20 ox1SPF1
CCTTCGTCGTCAACATCGGCG
GA20 ox1SPF2
CGGAGACAACCAAAGGAGGCG
GA20 ox3SPR3
GTCTCCTTCCACGGCAGCAATCG
GA20 ox3SPR2
CGATTGGCAGTAGTCTCGGAACA
GA20 ox3SPR1
CGGGCACGGCGGGTAGTAGTTG
GA20 ox3SPF1
GCAGGGACTTCTTCGCCGACG
GA20 ox3SPF2
CGGGAGCCATCGTCGTCAACATC
GA20 ox3
4.3.5
Cloning and sequencing of PCR products
Cloning of the purified PCR products was done using the pGEM-T Easy vector system
(Promega, UK). Insert and plasmid DNA (pGEM-T Easy) was mixed at a 3:1 ratio in a 10 µl
ligation mixture with 1 µl T4 DNA ligase (5 U/µl) and 1x T4 DNA ligase buffer (Invitrogen,
USA). The mixture was incubated at room temperature overnight. Competent Top10 or
DH5α E. coli cells were transformed with DNA fragments legated into the pGEM T-easy
plasmid by heat shock treatment at 42°C for 45 sec followed by 2 min incubation of
transformed cells on ice. E. coli cells were then incubated for 1-1.5 hr at 37°C with rotation
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(150 rpm) on a shaker after adding 250 µl SOC media (20 g/l bacto-tryptone, 5 g/l bactoyeast extract, 0.5 g/l NaCl, 20 mM glucose, 2.5 mM KCl, 10 mM MgCl2, pH7.0).
Transformed cells (200 µl) were then plated onto 2YT agar plates (16 g/l bacto-tryptone, 10
g/l bacto-yeast extract, 5 g/l NaCl, 1.5% (w/v) agar, pH 7.0) supplemented with 0.5 mM
IPTG, 0.01 mg/ml X-Gal and 0.1 mg/ml ampicillin allowing blue-white selection of
transformed colonies.
Five white colonies were cultured overnight in 5 ml 2YT media supplemented with 0.1
mg/ml ampicillin. Plasmid DNA was purified from cells using the “QIAprep plasmid mini
kit” (Qiagen, Germany) according to the manufacturer’s instructions. Isolated DNA was
eluted in 50 µl buffer EB (Qiagen, Germany). Positive cloned inserts were confirmed by
EcoRІ digestion followed by agarose gel electrophoresis. Confirmed DNA samples were
further run for sequencing in a PCR reaction containing 2 µl BigDye Terminator 3.1, 2 µl
Sequencing Buffer (5X), 1 µl M13 or custom-designed sequencing forward or reverse primers,
2 µl sdH2O and 3 µl template DNA. The PCR reaction product was then purified using
Sephadex G-50 Fine Grade Slurry (Sigma, UK) and Centri-Sep columns before sequencing.
4.3.6
DNA sequence analysis and phylogenetic analysis
DNA sequences were analyzed using the BLAST program (http://www.ncbi.nlm.nih.gov
/blast/) by comparing with sequences in the GenBank database. Candidate nucleotide
sequences were translated using the ExPASY Translation Tool (http://au.expasy. org/
tools/dna. html) before BLAST search for determining homology of deduced amino acid
sequences of closely related plant species. Phylogenetic analysis was computed using all the
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amino acid sequences of related genes from the different species using CLC Bio Mainbench
(Version 5.5)/ MEGA 4.1 (beta).
4.3.7
RNA isolation and cDNA synthesis
The frozen immature leaf sample of DZ-01-196 was homogenized in liquid nitrogen followed
by total RNA extraction using the RNeasy kit (Qiagen, Germany) following the instructions
by the manufacturer. Up to 2 µg of total RNA and oligo dT (18 - mer) was used for cDNA
synthesis following first-strand cDNA which was reverse transcribed by using the
SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, UK) according to the
manufacturer’s instruction.
4.3.8
Isolation of the Rht and other genes
Using an identical PCR approaches stated above (Section 4.2.3 to 4.2.7) were employed for
designing primers, running PCR, cloning and sequencing of PCR products for the isolation of
from E. tef homologous sequences to the wheat Rht gene and other genes (the rice Elongated
Uppermost Internode (EUI) and brassinosteroid deactivation (BR)). Primers used are shown in
Table A1 and A2 (See Appendix).
4.3.9
GA 20-oxidase expression in E. tef
The expression of the three E. tef GA20 oxidases in different E. tef tissues at different
developmental stages was determined by a quantitative real time PCR (qRT-PCR) using
purified total RNA from different tissues. The qRT-PCR analysis was done using the
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LightCycler technique for quantitative reverse transcription (RT)-PCR of the mRNA levels of
the gene of interest using SYBR Green (fluorophore that binds double-stranded DNA) to
produce fluorescence for detection. Various internal control primers were designed (Table
4.1) based on constitutively expressed house-keeping tef Actin and 25S rRNA gene to
optimize qRT-PCR measurements. The E. tef 25S rRNA gene was selected and amplification
was optimized based on the LightCycler 480 (Roche Diagnostic, UK) result. The crossing
point value (CP) was generated representing the fractional cycle number at which the amount
of amplified target reaches a fixed threshold. The following protocol was used to determine
the expression ratio: the Ct value difference between each triplicate was kept below 1.5 and
outliers with SD > 2 were removed from the analysis and the mean Ct and standard deviation
(SD) for each sample were determined. In the first step of analysis (∆Ct), all samples values
were normalized with respect to the least expressed sample. The highest Ct value was
subtracted from the Ct value of each sample where the least expressed sample will have a ∆Ct
value of 0. The difference of samples from the reference value was computed using the
function (∆Ct sample - ∆Ct reference) to obtain a ∆∆Ct value used to calculate 2-∆∆Ct along with
calculating the means for each group, the standard deviation and standard error of the mean
(SEM). This provides a relative expression ratio (in arbitrary units) and variation between
ratios.
Expression of GA20ox sequences from E. tef was further analyzed using total RNA extracted
from different E. tef tissues at the same time i.e. at rapid stem elongation stage (10-15 days
before panicle emergence), except the young panicle (inflorescence), which was sampled
three weeks later. The RNase Plant Mini Prep kit (Qiagen, Germany) was used for RNA
isolation according to the manufacturer’s guide followed by DNAase treatment. RNA (2.0
µg) was converted into cDNA using the Superscript III First-Strand Synthesis System for RT-
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PCR (Invitrogen, UK). The reaction was stopped by heat inactivation at 94 °C for 5 min.
PCR amplification of tef GA20ox cDNA using 0.4 mM of the specific primers (Table 5.1)
that have been designed on the basis of polymorphic regions of the three cloned homologous
sequences of the EtGA 20-oxidase gene. PCR DNA amplification was carried out at 94 °C
for 1 min followed by a 32 cycles for GA20ox1 and GA20ox1b and a 40 cycle for GA20ox2
of 94 °C for 1 min, 67 °C for 30 sec and 72 °C for 30 sec. The length of the expected
amplification fragment ranged between 80-190 bp. Tef SDH gene was used as a control.
Table 4.3 Primers used in quantitative and semi-quantitative PCR amplification of GA20ox
sequences
Target gene
Primer
Primer sequence (5’ – 3’)
GA20 ox1
GA20 ox1 02F
ATGTGGTGGGCTACTACGTCAGCAAG
GA20 ox1 01R
TCATCTCCGAGCAGTAGCGCCCGT
GA20 ox2 02F
CGGCCCACACCCTCTTGCTCCA
GA20 ox2 02R
TTGACGACGATGGCTCCCGGCTT
GA20 ox2
GA20 ox3 01F GA20 ox3 02F
GA20 ox3 01R
SDH
25S
GGACTACCTGGTGGGCCGC
TAGTAGTTGAGCCGCATGATGGAGTCG
SDH 01F
CACAGCTGAGCGCTACGTTCTC
SDH 01R
CCCAATGCAACACCGAAAATACG
25S 01F
ATAGGGGCGAAAGACTAATCGAACC
25S 01R
GAACTCGTAATGGGCTCCAGCTATC
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4.3.10
Expression of EtGA20ox1a in E. coli
The activity of EtGA20 ox1 was monitored using a gel-purified PCR product (1.2 kb) that
was amplified using primers sense: 5’-AGG GAT CCA GCC AGC TGC CCG TGA TG-3’
and antisense: 5’-TGA AGC TTA ACA GAA CAG GCG GTC ATG GAT GAC-3’ from a
cDNA template. The PCR amplification was done using “Phusion” polymerase proof reading
enzyme (Finnzymes) and the blunt-end PCR product was gel-purified and cloned into the
plasmid pET32a vector (Novagen, UK) using the BamHI and HindIII (underlined) restriction
sites. Competent E. coli DH5-α cells were transformed with the E. tef GA20ox-1 containing
plasmid. From transformed cells, recombinant plasmid DNA was isolated and E. coli BL21
cells were transformed with the pET32-a fusion by heat shock treatment. A transformed
colony was selected and cells were cultured overnight in 5ml 2YT containing 0.1 mg/ml
carbenicillin for selection. A cell suspension (500 µl) was then added to 50ml 2YT containing
0.1 mg/ml carbenicillin and incubated at 37oC for 2 hr and then 50 µl of 1M IPTG was added
to the cell suspension. Cells were then cultivated at 25oC for 6 hr under shaking (200 rpm).
The cell suspension was then centrifuged for 5 min and the cell pellet was frozen at -20oC
overnight.
4.3.11
HPLC analysis
Defrosted cells were re-suspended in 1.5 ml lysis buffer containing in 10 ml 1 ml 1 M TrisHCl, pH 7.5, 50 µl of 1M DTT, 200 µl of 50 mg/µl lysozyme, 8.75 ml sdH2O. The cell
suspension (1.5 ml) was incubated for 15 min at room temperature, treated with DNase and
cell debris were removed by centrifugation for 10 min in an Eppendorf centrifuge at 13 000 g
at 4oC and the cell lysate was kept for at -800C. The lysate was thawed and the following
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added to 90 µl supernatant: 5 µl dioxygenase co-factor mix (containing in 5 ml 80 mM 2oxoglutarate, 80 mM ascorbate, 80 mM DTT, 10 mM FeSO4, 40 mg mL21 BSA, and 20 mg
mL21 catalase in 100 mM Tris-HCl, pH 7.5; Williams et al., 1998) and 5 µl substrate (GA1214
C). The mixture was incubated for 2 hr at 300C under shaking at 200rpm. Glacial acetic acid
(10 µl) was added to the mixture and further diluted with 140 µl sdH2O and centrifuged for
10 min at maximal speed in an Eppendorf centrifuge before HPLC analysis to measure the
GA intermediate products catalyzed by this protein (GA20 ox1).
4.3.12
Southern blot analysis
A DNA hybridization probe of 450 bp was synthesized using specific primers for a coding
region of GA20 ox and a DIG labelling probe synthesis kit (Roche, Switzerland). Purified
genomic DNA (15 µg) was digested overnight with the restriction enzymes BamHI, BglII,
EcoRI and HindIII. Digested genomic DNA was fractionated on a 0.7% agarose gel and
transferred by the alkaline transfer technique (Roche, UK) to a nylon membrane (HybondN+, Amersham Pharmacia Biotech, UK). Transferred DNA was hybridized with a DIGlabelled probe in an oven at 420C overnight. Membranes with hybridized DNA were incubated
in a color substrate solution in the dark without shaking for at least 30 min for detection of
hybridized DNA bands.
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4.4
Results
4.4.1
Isolation of GA genes from E. tef
Identification and cloning of a E. tef ortholog of the rice SD-1 (SEMI-DWARF 1) gene was
carried out using a total of 5 degenerate oligonucleotides that were designed based on
orthologous sequences of the conserved amino acid regions from rice, sorghum, maize, wheat
and barley. Sequences were retrieved from the GenBank database using accession numbers of
known gene sequences from rice and wheat using BLAST search (NCBI). A partial sequence
closely related to the 2-oxoglutarate dependent oxygenase gene family was first obtained by
PCR amplification from E. tef DNA, which shares conserved domains of GA biosynthetic
pathway genes. However, analysis revealed that this sequence is lacking the important
domains that are characteristic of the 2-oxoglutarate dependent dioxygenase (2ODD) genes
(GA20ox, GA3ox and GA2ox) involved in the GA biosynthetic pathway (Hedden and
Kamiya, 1997). When primers F1, F2, F3 and R1 were used, three PCR fragments (E. tef1. E.
tef2. E.tef3) were obtained with sizes between 422-523 (Figs. A.1A and B; Fig. A.2C, D, E
and F see Appendix). After cloning of fragments into the plasmid pGEM-T Easy and
sequencing the nucleotide sequence of these fragments showed a >60% homology with
known GA20ox orthologous sequences from closely related cereal species such as sorghum,
maize, rice, wheat and barley.
Using an identical PCR approaches GA signaling gene from E. tef homologous to the wheat
REDUCED HEIGHT (RHT) gene was isolated. Additional gene sequences isolated were the rice
Elongated Uppermost Internode (EUI) and the Cytochrome P450 monooxygenase gene involved
in brassinosteroid (BR) deactivation both involved in plant height development. Primer pairs
RhtF1/ F2, RhtR1/R2 and Rht SPF and RhtSPR for Rht gene; EUIF1 and EUIR1 for Eui gene
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and BRF1 and BRR1 for BR gene (Tables A.1 and A.2, see Appendix) allowed PCR
amplification of the following fragments: (1) 1395 bp (near full coding region of Rht gene)
that shares over 85% homology with the above species (Figs. 5.9, 5.10 and A.6 (see
Appendix)), (2) 344 bp (Eui gene) which shares over 60% homology with EUI orthologous
sequences from sorghum, wheat, maize, brachypodium and rice (Figs. A.3 and A.4 and A.5,
see Appendix); and (3) 749 bp (BR gene), another Cytochrome P450 monooxygenase gene
but with brassinosteroid deactivation activity was partially cloned using semi-degenerate
oligonucleotides (Figs. A.7K and L; A.8 and A.9, see Appendix). The peptide sequence
shared 49 - 52% homology with orthologous peptide sequences of genes involved in BRs
catabolism.
4.4.2
Putative E. tef GA20 ox isolation and cloning
Based on the known sequence information obtained previously for the three putative
sequences of E. tef1-3 sequence specific primers (SP primers) (Table 4.2) were designed and
used for nested PCR amplification. Generation of full-length sequences of coding regions
was done using 5´/3´ RACE (Random Amplified cDNA ends). Application of RACE for E.
tef GA20ox resulted in two 5´ and two 3´ short sequences (contigs) with sizes of 238, 263 and
121 and 305bp. No further sequence extension could be obtained for E. tef2 and E. tef3 and
GA20ox2. The four fragments of E. tef1 were aligned using “contig assembly” of the vector
NTI program (Vector NTI Advance™9.0). The consensus sequence of 1448bp was translated
using the ExPASY protein translation tool. The sequence consisted of an open reading frame
encoding a putative polypeptide of 482 amino acids. Comparison of the deduced amino acid
sequence with other plant species (sorghum, rice, maize, wheat and barley) showed the
presence of all characteristic conserved (consensus) amino acids sequences (domains):
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LPWKET, (144-149 aa) and NYYPXCXXP (228-236 aa of the putative sequence) and three
His residues for binding Fe2+ (245-247 aa counting from the start codon). BLAST results of
the full coding region of putative E. tef GA2ox1 showed high identity scores with orthologous
genes from S. bicolor (68.3%), Z. mays (68.7%), O. sativa (59.3%), L. perenne (64.6), T.
aestivum (66.6%), H. vulgare (67.5%) and Z. japonica (54.5%) (Figs. 4.1 - 4.6).
Similarity among the tef GA20ox homologs and comparison with orthologs of other monocot
species was determined based on amino acid sequences using partial sequences trimmed to
core areas present in all the three sequences. E. tef1 showed 89.6% and 59.8% homology to
E. tef3 and E. tef3 sequences, respectively. GA20ox isolated from different species exhibits a
conserved domain of amino acids with identity ranging from 50 % - 75% (Hedden and
Kamiya, 1997). E. tef1 and E. tef3 sequences were found to be closely related to each other
and E. tef GA20ox2 sequence was found to be closely related to GA20ox2 genes sequences
from various other monocot species (Figs. 4.4 – 4.6). The three homologous sequences were
tentatively named as E. tef GA20ox1a, E. tef GA20ox1b and E. tef GA20ox2.
The E. tef GA20ox2 partial coding sequence has high polymorphism at the N-terminus when
aligned and compared to orthologs from other closely related monocot cereal species.
Generally, alignment and phylogenetic relationship further showed identity with GA20ox
sequences from other plant species such S. bicolor (81%), O. sativa (sd1) (68%), O.
rufipogon (72%), T. aestivum (57%), H. vulgare (58%), Z. mays (56%), A. thaliana (57%)
and L. perenne (58%) when 112-122 aa sequences present in all sequences from those species
were aligned (Figs. 4.3 and 4.4). EtGA20ox2 also has a 120 bp intron between the two
conserved functional domains (Fig. 4.3). Further, EtGA20ox1a, EtGA20ox1b groups closely
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with GA20ox1 sequences from sorghum, maize, zyocia spp. and rice. A similar result was
found for EtGA20ox2 closely grouping with GA20ox2 orthologs from these plant species.
107
E. tef 1
120
240
360
480
600
19
19
E. tef 2
E. tef 3
TGCGGCAGCGTGAAGAAGGCGTCCATTCGAGTGGAGTCTCGGAATCCTCAAACTCACTGCAGCAGCAGCAGCAGTAGTAGTAGTAGTCCTCCTCCTAGTCAATCAGAGAGAGAGAGAGAG
AGAGAGAGAGAGAGAGAGAGCCAAGCCAGCTGCCCGTGATGGTGGTGGTAGCGCAGGCGCAGGCGCAGGCGCAGATAGAAGTAGAAGCAGATGAGCCGCCGCAGCAGCTTGAGGTGGTGG
TGTTCGACGCGGCCCGTCTGAGCGGGCTGAGTGACATCCCGGCCCAGTTTTTGTGGCCGGAGGAGGAGAGCCCGACGCCTGACGCGGCGGAGGAGGAGCTGGACGTTCCTCTGATCGACC
TCTCCGGCGACGCGTCGGAGGTGGTCCGTCAGGTGCGTGAGGCCTGCGAGGCGCACGGCTTCTTCCAGGTGGTGAACCACGGCATCGACGCCGGCCTCGTGGCGGAGGCGCACCGCTGCA
TGGACGCCTTCTTCACGCTGCCGCTGCCGGAGAAGCAGCGCGCCCAGCGCCAGCCCGGCGACTGCTGCGGCTACGCCAGCAGCTTCACGGGCCGCTTCGCCAGCAAGCTGCCATGGAAGG
-----------------------------------------------------------------------------------------------------CGATTGCTGCCGTGGAAGG
-----------------------------------------------------------------------------------------------------CGATTGCTGCCGTGGAAGG
E. tef 1
E. tef 2
E. tef 3
AGACGCTCTCCTTCCGGGCCAAGGCCAAGGCCACCGATGATGATGATGATGATGTGGTGGGCTACTACGTCAGCAAGCTGGGCGAGGCCTACAGGCGGCACGGCGAGGTGTACGGGCGCT 720
AGACGCTCTCCTTC--------GGCCACCGCGAC-------------------GTCGTGGAGTACTTCACATCCACCCTCGGCAGCGACTTCAAACC-CCTAGGGAGGTGTTCCGAGACT 111
AGACGCTCTCCTTCCGGGCCA-GCCCAACGTCGCCGGCCTTG-----------GTGGAGGACTACCTGGTGGGCCGCCTTGGCGACGAGTACAGGCGGCACGGCGAGGTGTACGGGCGCT 127
E. tef 1
E. tef 2
E. tef 3
ACTGCTCGGAGATGAGCCGGCTGTCGCTGGAGATCATGGAGGTGCTGGGCGAGAGCCTGGGCGTGGGCCGCCGCTGCTTCCGCGACTTCTTCCAGGACAACGACTCCATCATGCGCCTCA 840
ACTGCCAATCGATGAAGGAGGTGTCGCTGGCGATCATGGAGGTGCTGGGCGCGAGCCTGGGCGTGGGGAGGCGCTACTGCAGGGACTTCTTCGCCGACGGCTGCTCCATCATGAGGTGCA 231
ACTGCTCGGAGATGAGCCGGCTGTCGCTGGAGATCATGGAGGTGCTGGGCGAGAGCCTGGGCGTGGGCCGGCGCTGCTTCCGCGACTTCTTCCAGGACAACGACTCCATCATGCGGCTCA 247
E. tef 1
E. tef 2
E. tef 3
ACTACTACCCGCCGTGCCAGCGGCCCATGGAGACGCTGGGCACGGGCCCGCATTGCGACCCCACCTCCCTCACCATCCTGCACCAGGACCACGTCGC---CGGCCTCCAGGTCTTCGCCG 957
ACTACTACCCGCCGTGCCCGGAGCCGGACCGGACGCTGGGCACGGGGCCCCACTGCGACCCGGCGGCCCTCACCCTCTTGCTCCAGGACGACGACGTGGACGGGCTCCAGGTGCTCGTCG 351
ACTACTACCCGCCGTGCCAGCGGCCCATGGAGACGCTGGGCACGGGCCCGCATTGCGACCCCACCTCCCTCACCATCCTGCACCAGGACCACGTCGC---CGGCCTCCAGGTCTTCGCCG 364
E. tef 1
E. tef 2
E. tef 3
CCGGCCGGTGGCTCTCCATCCGCCCGCACGCCCAGGCCTTCGTCGTCAACATCGGCGACACCTTCATGGCGCTCTCCAACGGCCGGTACAAAAGTTGCCTGCACAGGGCGGTGGTCAACA 1077
ACGGCGAGTGGCGGCCCGTGCGGCCCAAGCCGGGAGCCATCGTCGTCAACATCGGCGA-------------------------------------------------------------- 409
GCGGCCGGTGGCTCTCCATCCGCCCGCACGCCGCCGCCTTCGTCGTCAACATCGGCGA-------------------------------------------------------------- 422
E. tef 1
GCAGCGTCCCGCGGAGGTCCCTGGCCTTCTTCCTCTGCCCGGAGATGGACAAGCTCGTCACGCTCCCGCCGCAGCTTCTTCCTGATCTGCCCGGAGACAACCAAAGGAGGCGCCCCTACC 1197
CGGACTTCACGTGGCGCACCCTGCTCGAATTCACGCAGAAGCACACAGGGCCGACATGAAGACGCTCGAGGTCTTCTCCAACTGGCTCCGCCATGGCCAGGACAAGGTAGCGCTACCTCC 1317
CCTATGCTAGTTATTCTCTCCATACCAATATGTAATGTAATGTAATGAATGTAGTCATCCATGACCGCCTGTTCTGTTCAAAAAAAAAAGTCGACATCGATACGCGTGGTCCGCCTCCTT 1437
TGGTTGTCTCC 1448
***
***
108
Figure 4.1 Nucleotide sequence alignment of three putative E. tef GA20ox sequences.
Putative Fe2+- binding consensus regions are indicated with highlighted asterisks (*), the 2oxoglutarate-binding motif is indicated over-lined, and the nucleotide sequence for the
conserved LPWKET region, considered to be involved in the binding of GA substrate, is
shown with double over-line. In GA20ox2, the 120 bp intron between LPWKET and
NYYPPCPEP functional domains is not included in the alignment. Dashes (-) have been
inserted to maximize sequence homology. Identical and similar regions are shown by light
and dark shaded areas and dots whereas number indicates the position of the conserved
regions within the predicted nucleotide sequence.
109
MVVVAQAQAQAQIEVEADEPPQQLEVVVFDAARLSGLSDIPAQFLWPEEESPTPDAAEEELDVPLIDLSG-------DASEVVRQVREACEAHGFFQVVNHGIDAGLVAEAHRCMDAFFT 113
------------------------------------------------------------------------------------------------------------------------ 1
MV-------..SLG.PLLLQPPPPSL..............Q.....AD........-...A........-------..A.......R..DL............DA.LQ........... 105
MV-------L.AHD---------PPPL..............Q..I..AD......S.-...A........-------..A.......R..DL.......G.....A.T............ 96
MVVQQ------------------EQE......V...QTE..S..I..A....GSV.V-...E.A...VGAGA-----ER.S.....G....R....L......E.A.LE........... 98
MVQP-----------------------.....L..AQ....S..I..AD.......T-...H.....IG.LLSGDREA.A..T.L.GD...R.............E.LGH.R-.V..... 95
MVRP-----------------------.....V...R....S..I...G........-...H....NIG.MLSGDAAA.A..T.L.G....R.............E.L.D....V.N... 96
MVQP-----------------------.....L...RA...S..I...G........-...H.....IG.MLSGDADA.A..T.L.GQ...R..L..........E.L.D....V..... 96
MVQP-----------------------.....V...RT...S..I...G.......T-..MH.....IG.MLSGDPRA.A..T.L.G....R.............Q.L.D....V..... 96
MAAASTPNG--------------------.PPVR.LATTV.V.AVLFDIDGTLC.SD-PLHH.AFQE.LL----------.IGYNNGVPIG-............QEPL........N... 88
LPLPEKQRAQRQPGDCCGYASSFTGRFASKLPWKETLSFRAKAKATDD-DDDDVVGYYVSKLGEAY-RRHGEVYGRYCSEMSRLSLEIMEVLGESLGVG---RRCFRDFFQDNDSIMRLN
-----------------------------............SPTSPAL-VE.----.L.GR..DE.-................................---..................
..MSD......RQ..S........................YSDDQ-G.G--.V..D.F.D...D..-.H..................L...........---..H..R...G........
....D......RQ..S........................YTDDDDG.KSK.V.AS.F.D....G.-.H..................L...........---..H..R...G........
...G....RSGAR.-RTA......................YSSAGDEEG-EEG.GE.L.R...AEHG..L....S...H........L.........IVGDR.HY..R...R........
MS.QD....L.R..ES........................SCPSE-----P.L..D.I.AT...DH-..L....A........................---.AHY.R..EG.E......
M........L.R..ES........................SCPSD-----PAL..D.I.AT...DH-..L....A........................---.AHY.R..EG........
M........L.R..ES........................SCPSD-----PAL..D.I.AT...GH-..L....A........................---.AHY.R..EG........
M........L.R..ES........................SCPSD-----PAL..D.I.AT...DH-..L....A........................---.AHY.R..EG.E......
...........RQ.ES............C...........YSSNPSS---P.L..D.F.E....D.-.H..A..A............M.GI........---.DH..R...P.E......
* *
YYPPCQRPMETLGTGPHCDPTSLTILHQDHVAGLQVFAAGR------WLSIRPHAQAFVVNIGDTFMALSNGRYKSCLHRAVVNSSVPRRSLAFFLCPEMDKLVTLPPQLLPDLPGDNQR
........-.............................G..------...-....A.......--------------------------------------------------------........YD...................D.G.....D.ATGPGTGR.R.....PG..................R..........R................V.RP.AE.V-----.DAN
........YD...................D.G.....D.AT----LA.R....RPG..................R..........R.A..............V.RP.KE.V-----.DAN
...A....LD.....................G..E.W.E..------.RA...RPG.L...V.........A..R..........TA..............TV.RP.EE.V-----.DHH
........N....................D.G....H.D..------......R.D.............................R...K............V.AP.GT.V-----.EAN
........L....................N.G....HTE..------.R....R.D.............................R...K............V.AP.GT.V-----.ASN
........Y....................D.G....HTE..------.R....R.D.............................R...K............V.AP.GT.V-----.AAN
........L....................D.G....HTD..------.R....R.D.............................R...K............V.AP.GT.V-----.AAN
........L......................G.....TD..------.R......G..........TGALQRPVPKL........L...K............V.CP.EG.V-----EAGM
Identity (%)
RRPYPDFTWRTLLEFTQKHTGPTRRSRSSPTGSAMARTRRYLPYASYSL 391
100
E. tef 1
------------------------------------------------- 138
80
E. tef 3
P.A..........D..MR.YRSDM.TLEAFSNWLN---HGG-HLS.PPP 378
68
S. bicolor GA20ox
P.A..........D..MR.YRSDM.TLEAFSNWLSTSSNGGQHLLEKKK 372
69
Z. mays GA20ox
P.V.......A..D...R.YRADM.TFQAFSDWLN----HHRHLQPTIY 370
59
O. sativa GA20ox
P.A.......A..D.....YRADMKTLEVFSDWIQQGHQP-----AATT 359
67
L. perenne GA20ox
P.A.......S..D.....YRADMKTLEVFSSWIVQQQQGQ.ALQPAMT 365
67
T. aestivum GA20ox1A
P.A.......S..D.....YRADMKTLEVFSSWVVQQQQ------AAMT 359
67
D. villosum GA20ox
110
P.A.......S..D.....YRADMKTLEVFSSWVVQQQP-----..ART 360
68
H. vulgare GA20ox1
P.A..........D...RRYRADM.TLEVFSNWLRHGQDKTP.TSLIHR 359
54
Z. japonica GA20ox
228
83
218
212
216
206
207
207
207
201
342
138
333
323
325
315
316
316
316
310
Figure 4.2 Derived amino acid sequence alignment of two putative E. tef GA20ox (E. tef1
and E. tef3) sequences with orthologous GA20ox gene sequences from sorghum (S.
bicolor; Acc No. XP_002463483.1), maize (Z. mays; Acc No. ACF83905.1), rice (O.
sativa; Acc No. P93771.2), wheat (T. aestivum; Acc No. 004707.1 ), barley (H. vulgare;
Acc No. AAT49058.1), lolium (L. perenne; Acc No. AAG43043.1), Zyocia (Z. japonica:
Acc No. ABG33927.1) and Dasypyrum (D. villosum; Acc No. ACU40946.1). Putative
Fe2+- binding consensus regions are indicated with asterisks (*), the conserved motifs,
NYYPXCXXP and LPWKET are shown with deep dark shades. Identical and similar
regions are shown by light and dark shaded areas and dots whereas number indicates the
position of the amino acid within the predicted peptide.
111
E. tef 2
LPWKETLSFGHRD----------VVEYFTSTLGSDFKPLGEVFRDYCQSMKEVSLAIMEVLGASLGVGRRYCRDFFADGC 70
TTVEIASFATFFFVRTRDRSSESFYNDCTDRAMHGRR
S. bicolor GA20 ox2
O. sativa sd1
O. sativa GA20 ox2
O. rufipogon GA20 ox2
P. sativum GA20 ox
A. thaliana GA20 ox2
Z. mays GA20ox
L. perenne GA20ox
T. aestivum GA20ox1A
H. vulgare GA20ox1
E. tef 2
S. bicolor GA20 ox2
O. sativa sd1
O. sativa GA20 ox2
O. rufipogon GA20 ox2
P. sativum GA20 ox
A. thaliana GA20 ox2
Z. mays GA20ox
L. perenne GA20ox
T. aestivum GA20ox1A
H. vulgare GA20ox1
.............RRTSG--SHV..D..............V.YQN..NA...........I.V......S.Y.......S
..........FH.RAAAP----V.AD..S....P..A.M.R.YQK..EE...L..T...L.EL....E.G.Y.E....SS
............HANAAGNNSST.AD..S-...D...H....YQE..EA.E..TK...A...E.....GG.Y.E..E.SS
..........FH.RAAAP----V.AD..S....P..A.M.R.YQK...E...L..T...L.EL....E.G.Y.E....SS
.........QFS.EKNS---SNI.KD.LSN...E..QQF...YQE..EA.SKL..G...L..M.....KECF....EENK
.........QFSNDNSG---SRT.QD..SD...QE.EQF.K.YQ...EA.SSL..K...L..L....N.D.F.G..EEND
.........RYT.DDDGDKSKDV.AS..VDK..EGYRHH...YGR..SE.SRL..EL.....E.......HF.R..QGND
.........RSCPSE-----PDL..D.IVA...E.HRR....YAR..SE.SRL..E......E......AHY.R..EGNE
.........RSCPSD-----PAL..D.IVA...E.HRR....YAR..SE.SRL..E......E......AHY.R..EGND
.........RSCPSD-----PAL..D.IVA...E.HRR....YAR..SE.SRL..E......E......AHY.R..EGNE
SIMRCNYYPPCPEPDRTLGTGPHCDPAAHTLLLQDDDVDGLQ..............E...........S.L.V....G...---..............E...........T.L.I....-..G..EV
..............E...........S.L.V....G......V
..............E...........T.L.I....-..G..EV
....L......QK..L..........TSL.I.H..-Q.G...V
....L.H....QT..L..........SSL.I.H..-H.N...V
....L......QR.YD..........TSL.I.H..-..G...V
....L......QR.NE..........TSL.I.H..-..G...V
....L......QR.LE..........TSL.I.H..-N.G...V
....L......QR.LE..........TSL.I.H..-..G...V
112
112
117
118
122
118
119
119
122
117
117
117
Identity (%)
100
81
68
72
69
57
57
56
58
57
58
78
76
79
76
77
77
80
75
75
75
Figure 4.3 Derived amino acid sequence alignment of putative E. tef GA20ox2 sequence
(E. tef2) with orthologous GA20ox gene sequences from other grass species namely
sorghum (S. bicolour; Acc No. XP_002441117.1), maize (Z. mays; Acc No.
ACN25832.1), rice (O. sativa and O. rufogen, Acc No. AAT44252.1 and BAK39011.1),
wheat (T. aestivum; Acc No. 004707.1) barley (H. vulgare; Acc No. BAK04752.1), pea
(P. sativum; Acc No. AAF29605.1) and Arabidopsis (A. thaliana; Acc No. NP_199994.1).
The position of the 37 aa intron region is indicated in the first row 23aa downstream
LPWKETL motif between amino acids “G” and “E”. Identical and similar regions are
shown by light and dark shades respectively and numbers indicate the position of the
amino acid within the predicted peptide. Percentage of identity is shown in the
parenthesis.
113
L. perenne GA20-ox1
76 H. vulgare GA20-ox1
H. vulgare subsp. vulgare GA20-ox1
57
D. villosum GA20ox
96 T. aestivum GA20-ox1B
T. aestivum GA20-ox1D
T. aestivum GA20-ox1A
99
O. sativa GA20 -ox1
59
29
23
88
67
85
72
99
Z. japonica GA20-ox1
E. tef 3
92
E. tef 1
S. bicolor GA20-ox1
Z. mays GA20-ox1
P. vulgaris GA20-ox
P. sativum GA20 -ox
A. thaliana GA20-ox
A. thaliana GA20- ox2
98 O. sativa GA20- ox2 (Sd-1)
O. rufipogon GA20 -ox2
O. sativa GA20- ox2
E. tef 2
75
91
S. bicolor GA20- ox2
0.1
Figure 4.4 Molecular phylogenetic analysis of the homologous of E. tef GA20oxsequences (E.
tef GA20ox1, GA20ox1b and GA20ox2; underlined). The tree was inferred by using the
Maximum Likelihood method based on the JTT matrix-based model. Initial alignment was
done by mafft (http://mafft.cbrc.jp/alignment) software. The tree with the highest log
likelihood is shown. The percentage of trees in which the associated taxa clustered together is
shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically
as follows: when the number of common sites was < 100 or less than one fourth of the total
number of sites, the maximum parsimony method was used; otherwise BIONJ method with
MCL distance matrix was used. The tree is drawn to scale with branch lengths measured in the
114
number of substitutions per site. All positions containing gaps and missing data were
eliminated. There were a total of 105 positions in the final dataset.
4.4.3
EtGA20ox copy number
To determine the copy number of EtGA20ox gene in E. tef, Southern blot analysis of genomic
E. tef DNA restricted with different enzymes (BamHI, EcoRI and HindIII) and probed with a
450-base-pair coding sequence of EtGA20ox was carried out. The probe hybridized with four
fragments after the HindIII digest (one is very slightly visible); four fragments after BamHI
digest, and two very visible and two hardly detectable fragments after EcoRI digest (Fig. 4.5).
Therefore, four copies of EtGA20ox are possibly present in the allotetraploid E. tef genome.
Marker BamHI
EcoRI
Hind III
Figure 4.5 Detection of EtGA20 ox gene copies in the E. tef genome
after restriction enzyme digest using Southern blotting.
115
4.4.4
GA20ox expression in E. tef
In both E. tef genotypes, Gea Lammie (short phenotype) and DZ-01-196 (long phenotype),
GA20ox1 was expressed in germinating hypocotyls, leaf, stem and inflorescence. Relatively
higher expression was found at earlier stages in young stem and leaf (Fig. 4.17) with slight
differences between the two genotypes. Further, when semi-quantitative PCR (RT-PCR) was
used to study expression of the three E. tef GA20ox homologous sequences in leaf, leaf
sheath, uppermost two internodes, nodes from these internodes, peduncle and inflorescence
both EtGA20ox1a and EtGA20ox1b expression was highest in the uppermost internodes
followed by nodes (Fig. 4.7). However, EtGA20ox1b expression was greater than
EtGA20ox1a expression in the internodes, nodes, leaf sheath and panicle. In contrast,
EtGA20ox2 was expressed at relatively lower rates in the nodes and panicle but also in the
upper most internodes when compared to the other two genes.
5.0
Gea Lammie
4.5
DZ -01 -196
Relative expression
4.0
Control
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
A
B
C
D
E
F
Growth stages/Tissues
116
G
Figure 4.6 Relative expression of GA20ox1 in two E. tef genotypes DZ-01-196
and Gea Lammie in different plant tissues and growing stages. (A) Control; (B)
3rd week stem + leaf; (C) 5th week stem + leaf; (D) 8th week stem; (E) 8th week
leaf (F) Old (10th week) stem and (G) 10 days old inflorescence. The qRT-PCR
was repeated three times, and gene expression level was calculated following the
expression 2–DCt (Yang et al., 2005).
L
LS
IN
N
Pe
Pa
EtGA20ox1
32
cycles
EtGA20ox1b
32
cycles
EtGA20ox2
40
cycles
Et SDH
32
cycles
RNA
Figure 4.7 Semi quantitative RT-PCR Expression analysis of three EtGA20ox genes in
various plant tissues (L=Leaf; LS=Leaf sheath; IN= Internode; N= Node; Pe= Peduncle;
Pa= Panicle) from cultivar DZ-01-196 sampled at the stage of stem elongation and panicle
initiation (for panicle). RT-PCR was performed with 32 and 30 cycles for EtGA20ox and
EtSDH, respectively. The experiment was repeated three times with similar result. EtSDH
was used as an internal PCR amplification control.
117
4.4.5
In vitro enzymatic activity of GA20ox in a heterologous system
In E. coli expressed GA20ox1 protein catalyzed the conversion of the [14C]-labelled
gibberellin precursor GA12/ to GA9 which could be detected by full-scan GC-/MS. In some
clones only a partial conversion of the substrate was found, possibly due to sub optimal
reaction conditions, and intermediates of the reaction pathway, GA24 and GA15, were also
detected by HPLC analysis (Fig. 5.8). Clone 1 showed optimum activity by complete
conversion of GA12 into GA9>GA15>GA24. Clones 2 and 3 showed only partial activity.
Tef GA20ox1 in vitro activity
mV
AD2
150
125
100
Clone 1
75
50
.
GA24
GA9 Retention time
=15.6min
GA15 Retention time
=16.0min
Relative intensity
25
100
75
Cone 2
50
GA15
GA12
GA15
GA12
GA9
GA24
25
125
100
Clone 3
75
50
25
300
250
200
150
100
50
GA9
GA24
GA9
GA9 std
0.0
2.5
5.0
7.5
10.0 12.5
15.0
17.5
20.0
22.5
25.0
27.5 min
Retention time (min)
Figure 4.8 Radiochromatograms after HPLC of E. tef GA20ox1 activity products after
incubation with a 14C-labeled GA12 as a substrate (standard shown at the bottom).
118
4.4.6
Isolation of the Rht and other genes
For isolation and cloning of E. tef Rht, specific primers (SP) (Table A.1) were designed based
on a 344 -base-pair known sequence for the Rht gene. Primers were used for nested PCR to
amplify a putative Rht orthologous gene from E. tef similar to the procedure used for GA20ox
to obtain a full-length sequence. PCR amplification resulted in two 5´ and two 3´ contigs
providing a total fragment size of 1395 bp after assembling the contigs. The sequence
analysis of the amplified cDNA fragment (1395bp) consisted of an open reading frame
encoding a putative polypeptide of 465 amino acids. Comparison of the E. tef Rht amino acid
sequence with sequences from other plant species, such as sorghum, rice, maize, wheat and
barley, showed the characteristic conserved amino acids domains such as the DELLA and
GRAS domains for the Rht gene (Figs. 5.9 and 5.10). When the aligned sequences were
trimmed to core areas present in all sequences (438-465 aa) a high identity to sequences of Z.
mays (90%), O. sativa (86%), S. bicolor (85%), T. aestivum (84%) and A. thaliana (55%)
were found. The above gene sequence in E. tef was named EtRht according to the
nomenclature used by Cloes et al. (1999).
Partial sequence for brassinosteroid inactivating gene that encodes the Cytochrome P450
monooxygenase family gene, was also cloned using semi-degenerate oligonucleotides. After
optimization of PCR conditions degenerate primers F1: 5´- ACA GCC GCA GCG TCT CGT
T(GCT) - 3´ and primer R1: 5´ - A(GA)(CG) (CG)(TC)C (CA)AC GGC GA(CGA)
(GC)(CG)(AT) GTT, were used to obtain 749 bp long fragment. Further sequencing and
comparison through BLAST-search showed homology range between 49 - 52% with ortholog
gene sequences of the Cytochrome P450 monooxygenase family for closely related species
including the rice CYP734A1 involved in BRs catabolism (Fig. A.10 and Fig. A.11).
119
Domain I
Domain II
EE-VDELLAALGYKVRSSDMADVAQKLEQLEMAMGMGGVP----AADDGFVSHLATDTVHYNPSDLSSWVESMLSELNAPPPPLPPAPAPPAPQL-VSTSSTVT-GGGSG
.D-....................................GGAGAT....................................A.....--T...R.-A.......S.AAA.
..-M..M...V............................GGAGAT.....I...............................--------...R.-A.......S.AAA.
.D-.................................A..SAPG-....................................L..I....--..ARH-A.......G....GNMD.....V........E..E..L......TM.SN--------VQE..-L.............E.Y..LDN......P..L.-------------A.SNGLDP-----..-.............A......................G-AGA.P..S.AT...........T.....................-----.....NA.......GS.---
103
106
100
104
82
100
AGYFDPPPAVDSSSSTYALKPIP-SPVAA-PADP----SADSAREPKRMRTGGGSTSSSSSSSSSMGGGGARSSVVEAAPP---ASAAANAPAVPVVVVDTQEAGIRLVH
.....L.................-.....PS...----.T..........................D..RT..........ATQ......G...................
.....L.................-...V.-S...S---.T..T.......................D..RT..........ATQ......G.......M..P........
-.F.EL.A.A.........R..S-L..V.-T...S---A.....DT...................L...AS.G........AMQGA........................
--VLPS.EICGFPA.D.D..V..GNAIYQF..IDS---.SS.NNQN..LKSCSSPD.MVT.T.TGTQI..VIGTT.TTTTT---TTT..AESTRS.IL..S..N.V....
-....L..S......I...R...-..AG.-T.PADL--....V.D........S...........L...-...........--V.A..NAT..L................
204
211
205
208
184
202
ALLACAEAVQQENFSAAEALVKQIPMLASSQGGAMRKVAAYFGEALARRVYRSPPPPPTAPSSTPPSPTSSTPHFYESCPYLKFAHFTANQAFLEAFAGCRRVHVVDFGI
....................................................FR...DSS-LLDAAFADLLHA...................I.................
.................D..................................FR.T.DSS-LLDAAVADFLHA...................I.................
..............A..........T..A.......................FR.-ADST-LLDAAFADLLHA...................I.................
..M.....I..N.LTL........GC..V..A.......T..A......I..LS.-.QNQ--IDHCLSDTLQM....T..............I....E.KK....I..SM
.............L...........L..A.....................F.FR.Q.DSS-LLDAAFADLLHA...................I.................
314
320
314
316
291
311
EQGMQWPALLQALALRPGGPPSFRLTGVGPPQPDETDALQQVGWKLAQFAHTIRVDFQYRGLVAATLADLEPFMLQPEGE-ENDEEPEVIAVNSVFEMHRLLAQPGALEK
K..............................................................................D-DT.D............L............
K..L............................H..............................................D-DK..............L............
K...............................................................................ADAN.............L............
N..L.....M......E....T.....I...A..NS.H.HE..C....L.EA.H.E.E...F..NS....DAS..ELRPS-----DT.AV.......L.K..GR..GI..
K...............................................................................EDPN..........................
Identity (%)
423
429
423
426
396
421
VLGTVRAVRPKIVTVVEQEANHNSGSFLDRFTQSLHYYSTMF 465
100
..........R..............T......E......... 471
90
..........R..............T......E......... 465
85
.....H....R.....................E......... 468
86
...V.KQIK.V.F......S...GPV......E.......L. 438
84
..........R..............T......E......... 463
55
120
E. tef Rht
Z. mays D8
S. officinarum GAI
O. sativa Indica GAI
A. thaliana GAI1
T. aestivum rht-D1a
Figure 4.9 Amino acid sequence alignment of the putative tef Rht sequences with orthologous
amino acid sequences from maize (Z. Mays; Acc No. AAL10325.1), rice (O. sativa; Acc No.
EAY91579.1), wheat (T. aestivum; Acc No. Q9ST59.1) and sugarcane (S. Officinarum; Acc
No. AAZ08571.1) and Arabidopsis (A. thaliana, Acc No. BAC42642.1). The putative
sequence shows the characteristic domains (dark shaded) for this gene: a conserved Nterminal: DELLA motif (Domain I), a VHYNP motif (Domain II) and VHVVD. The red
boxes in Z. maize and T. aestivum in domain I and domain II show positions of small internal
deletions and introduction of a stop codon (in wheat) in alleles that cause semi-dwarf
phenotype (Benetzen and Mulu, 2000). Identical and similar regions are shown by grey and
light grey shaded areas and dots whereas numbers indicate the position of the amino acid
within the predicted peptide.
121
Oryza sativa Indica GAI
97 Oryza sativa Japonica GAI
Oryza rufipogon GAI
46
Hordium vulgare –SLN1
40
99 Triticum aetivum rhtD1a
Zea mays subsp mays –Dwarf 8
100
Saccharum hybrid cultivar GAI
98 Sorghum bicolor
Eragrostis tef
Arabidopsis thaliana GAI
Populus trichocarpa
33
Vitis vinifera
60
Phaseolus vulgaris
99
Pisum sativum
0.05
Figure 4.10 Molecular Phylogenetic analysis of E. tef putative Rht gene by Maximum
Likelihood method. The evolutionary history was inferred based on the JTT matrix (Jones et al.,
1992). Initial alignment was done by mafft (http://mafft.cbrc.jp/alignment) software. The
percentage of trees in which the associated taxa clustered together is shown next to the branches.
Initial tree(s) for the heuristic search were obtained automatically as follows. When the number
of common sites was < 100 or less than one fourth of the total number of sites, the maximum
parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. The
tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
There were a total of 478 positions in the final dataset. Evolutionary analyses were conducted in
MEGA5 (Tamura et al., 2011).
122
4.4
Discussion
In this study full and partial coding sequences of three GA biosynthesis gene homologs for the
GA20ox has been isolated, characterized and the expression analyzed. The putative E. tef
GA20ox genes showed close homology with genes of other cereal species (sorghum, maize, rice,
wheat, barley) and a grass species Zyocia japonica. The putative GA20ox genes belong to the
GA 2-oxoglutarate-dependent dioxygenase (2-ODD) gene family as confirmed by the
heterologous expression assay producing final and intermediate products of the GA20ox
enzymatic actions. For convenience, gene nomenclature has been kept consistent with the
naming of genes used in several recent publications about GA-biosynthesis and signalling genes
in plant hormone metabolism (Thomas et al., 1999; Sakamoto et al. 2004; Spielmeyer et al.
2004).
The three E. tef GA20 oxidases also have characteristic conserved amino acid residues, namely
the 2-oxoglutarate co-factor, LPWKET motif, very likely involved in the binding to the GA
substrates (Xu et al., 2002), and the NYYPXCQKP motif for common co-substrate binding
(Miao et al., 2010). The conserved H and D residues are involved in Fe2+ binding at the active
site of isopenicillin N synthase (Hedden and Phillip, 2000 and Xu et al., 1995). In E. tef
GA20ox2 an intron (120 bp) has been further found 23 aa downstream of the LPWKET domain.
The three E. tef GA20ox genes (hereafter named EtGA20ox) showed differential transcription in
different plant tissues when semi-quantitative RT-PCR was applied. Generally, EtGA20ox1a and
EtGA20ox1b were similarly expressed in tissue with highest transcription in the uppermost
123
internodes followed by nodes. In contrast, EtGA20ox2 transcription was relatively lower than
transcription of either EtGA20ox1a or EtGA20ox1b. These two genes might therefore act to
promote internode elongation and panicle growth and might further also be involved in
reproductive growth of the panicle. Such overlapping transcription has also been found in
Arabidopsis where AtGA20ox1 and AtGA20ox2 act redundantly promoting elongation in the
hypocotyls and internode, flowering time, and elongation of anther filaments including seeds
number per silique (Rieu et al., 2008). Also, in Arabidopsis GA20ox1 greatly contributes to
internode elongation (Galun, 2010). Further, in rice OsGA20ox2 (SD-1) is a predominant
dwarfing gene with a role in height control (Spielmeyer et al., 2002, Monna et al., 2002). Results
in this study indicate, however, that EtGA20ox1b, and not EtGA20ox2, is the functional gene in
E. tef for height control equivalent to OsGA20ox2 (SD-1). However, a future study is required to
investigate whether EtGA20ox1b regulation will affect E. tef plant growth as previously found in
rice.
EtGA20ox1a also showed differential transcription when quantitative RT-PCR was used. This
was dependent on growth stages and plant parts in the two genotypes, Gea Lammie and DZ-01196. EtGA20ox1a transcription was found in DZ-01-196 in the immature (emerging)
inflorescence, germinating hypocotyls, young stem as well as leaf. However, EtGA20ox1a
transcript abundance was generally higher in the shorter genotype, Gea Lammie, than in the
taller genotype DZ-01-196. It is still unclear if higher transcript abundance in the short genotype
depends on a feedback response interfering GA biosynthesis since the level of bioactive GA
measured in Gea Lammie in this study was about half the amount in DZ-01-196. Increased
transcript levels (reduced sensitivity to exogenous GA application) have been reported for
124
response pathway mutants in different crops due to impairment in the response genes
(Olszewski, et al, 2002). Also a low bioactive GA amount despite relatively high transcription of
GA20ox has to be further investigated.
In a heterologous expression, EtGA20ox1a substrates are converted through several successive
oxidations at C-20 of GA12 to the alcohol and aldehyde intermediates GA15, GA24 and GA9
(Hedden and Kamiya, 1997 and Yamaguchi 2008). Further, it has been shown that GA9 is
converted by 3β-hydroxylation to the bioactive GA4 (Junttila et al., 1992 and Rood and Hedden,
1994). All these products could be detected when EtGA20ox1a was expressed in E. coli and
reacted also with GA12 as a EtGA20ox1a substrate instead of GA53 which is the natural substrate
in tef plants. This sequence was successfully expressed converting substrates to final
intermediates product except in few cases where GA15 was a more abundant product due to
incomplete downstream reactions.
In this study, a Rht ortholog and a partial sequence of an Eui and a BR deactivating gene were
also isolated from E. tef. However, none of them were further characterized regarding their
transcription in E. tef. The E. tef Rht gene has a conserved N-terminal DELLA motif (Alevy and
Harberd, 2005). In general, when the Rht gene is expressed it represses downstream genes in the
absence of GA inhibiting plant growth (Hedden, 2006). Other conserved Rht motifs which were
also found in E. tef Rht include the TVHYNP domain essential for the perception of an upstream
GA signal, VHVVD, and a C-terminal GRAS domain which are the functional domains
responsible for transcriptional regulation such as the suppressive function of DELLA proteins
against GA action (Peng et al., 1997; Silverstone et al., 2001; Ueguchi-Tanaka et al., 2005). Two
125
conserved N-terminus DELLAs domains (I and II) (Fig. 12; see Appendix) found in E. tef Rht
are highly conserved regions in angiosperms (Yasumura et al., 2007) presumably necessary for
DELLA interaction of the peptide during GA signalling process (Alevy and Harberd, 2005).
The Eui gene also isolated from E. tef is a single recessive gene responsible for culm length
modifications. Eui mutants have a longer culm length due to elongation of the uppermost
internodes (Zhang et al., 2008). Ectopic expression of the Eui coding sequence under the control
of the rice GA3ox2 and GA20ox2 gene promoters reduced plant height (Zahang et al., 2008).
Further, the isolated E. tef BR gene has been found to be closely related to the rice CYP724As
(Cytochrome P450 monooxygenases) involved in BR catabolism (Sakamoto et al., 2011).
Several of closely related sequences also identified through a BLAST search in this study were
also related to BR inactivation. Studies have shown that BR mutants develop a dwarf phenotype.
In rice, CYP734As control bioactive BRs by direct inactivation of castasterone (CS), a bioactive
BR, and by the suppression of CS biosynthesis thus decreasing the levels of BR precursors. In
Arabidopsis, CS and brassinolide (BL) are inactivated mainly by two Cytochrome P450
monooxygenases (CYP734A1/BAS1and CYP734A1/BAS1) that inactivate CS and BL by C-26
hydroxylation (Ohnishi et al., 2009).
In summary, genes were isolated in this part of the study allowing further characterization for
their function. Given the redundancy of the GA biosynthetic genes reported in this study for
tetraploid E. tef, it can be assumed that finding for instance a recessive dwarfing sd-1 homologue
mutant may be challenging since it requires independent mutations in both its A and B genomes.
In such a case the semi-dominant Rht mutation version (full coding region cloned) is an easier
126
alternative for TILLING application and easy to express phenotypically due to its semi-dwarf
nature. They can now be directly used for genetic engineering approaches using the
transformation protocol developed for E. tef, which is part of this PhD study. Further, sequences
might allow mutagenesis and selection through TILLING or Eco-TILLING and marker assisted
breeding in the conventional and modern E. tef development process targeting lodging resistance.
The genomic information developed through this part of the study therefore provides useful
information for further studies to understand and establish the precise roles in plant growth of
these isolated genes and for direct use in lodging resistance improvement through modern and
conventional techniques.
127
CHAPTER 5
EVALAUTION AND ANALYSIS OF MUTANT TEF (Eragrostis
tef) LINES FOR DWARFISM FOR LODGING RESISTANCE
128
5.1
Abstract
To evaluate E. tef plants generated via mutagenesis to induce dwarfism, selected mutant lines
were evaluated for traits including culm height and diameter, internode number and length,
panicle length, shoot biomass, tillering and grain yield under GA sprayed and non-sprayed
conditions. Semi-dwarfed phenotypes could be developed in E. tef through mutagenesis
approach and culm height was significantly reduced (23.1 cm - 41.7 cm) in three mutant lines.
These mutants were semi-dwarfed with short culm and peduncle length. Regardless of height,
grain yield was considerably reduced in all the mutants showing severe defects in fertility except
mutant GA-10 which gave a reasonable yield. Line GA-10 also had a significantly higher
diameter at 2nd - 5th and at 7th internodes contributing to the stiffness of the stem and also had the
highest panicle dry weight among mutant lines tested. Internode diameter showed consistent
increase acropetally with weak tapering. All semi-dwarfed mutants did not respond to GA
treatment. Plants of G-10 possibly harbours a mutation in GA signalling. Since biomass
production in mutant line GA-10 was not reduced this line might be used for crossings with other
parental lines to restore yield without losing its other useful traits.
129
5.2
Introduction
Several studies have shown morphological traits that are related to the lodging in E. tef to be
related to plant height, stem diameter of lower internodes, panicle length, biomass and seed
weight (Chanyalew, 2010; Hundera et al. 1999; Ketema, 1983; Mengesha et al. 1965).
Considerable efforts have been made over the last 50 years to incorporate by conventional
breeding desirable agronomic traits into E. tef. However, no lodging resistance traits, such as
reduced height and stiff straw, have been so far reported using conventional breeding (Assefa et
al., 2010).
Mutation breeding has been carried out in cereals to induce semi-dwarfness (Narahari, 1985) and
also to solve the lodging problem (Maluszynski and Szarejko, 2003). In general, inducing
mutation in target genes using various mutagens provides rapid generation and enhancement of
genetic variability (Nichteriein, 2000). Short stature mutants without changing the background
character of important traits have been beneficial not only to improve lodging resistance but also
to increase productivity. This has been possible because of a more efficient partitioning of the
dry matter resulting in high harvest index and increased grain yields in dwarf mutants in cereals
due to pleiotropic effects (Hanson et al., 1982; Hu, 1973; Nichteriein, 2000). In indica type rice,
a spontaneous mutation resulting in semi-dwarfs led to the development of the high-yielding
variety ‘IR8’. This variety, together with its descendants, was responsible for the Green
Revolution in Asia. More than 60 dwarf or semi-dwarf mutant lines have been so far reported for
rice. Some of these are allelic to sd1, but no mutation, other than in the sd1 locus, resulted in
improvement of agronomic performance. In barley several lines with induced mutation has
130
resulted in superior breeding material (Maluszynski and Szarejko, 2003) and in wheat, semidwarf phenotypes have been obtained through mutation in the Rht loci.
Recently a TILLING (Targeting Induced Local Lesion IN Genomes) approach has been carried
out for E. tef to generate variability in plant height and to develop semi-dwarf phenotype for
lodging resistance in E. tef (Esfeld and Tadele, 2010). In this part of the overall study, the first
objective was to select mutant lines from the TILLING M3 population to evaluate them for
morphological (plant stature) and yield traits to possibly identify any desirable mutation for
lodging resistance development in E. tef. The second objective was to characterize potential
candidate mutant lines for changes in the DNA sequence of particular target genes (GA20ox
homologues and Rht).
.
5.3 Materials and Methods
5.3.1 Plant material
Seed material of mutagenized tef (Eragrostis tef) lines (M3 progenies) of variety DZ-Cr-37
(fairly tall) was obtained from Dr. Zerihun Tadelle, University of Bern, Switzerland.
5.3.2 Plant growth and GA treatment
Plant growth experiments were carried out in an environmentally controlled greenhouse at
Rothamsted Research, UK, from May to August 2010. Up to 36 seeds per each mutated line ( 9
131
per pot) were germinated and thinned down to 3 per pot and grown on a compost mix consisting
of peat (75%), sterilized loam (12%), vermiculite (3%) and grit (10%). The mix was
supplemented with a slow releasing fertilizer containing 15-11-13 NPK plus micronutrients.
Selected seedlings were maintained in the same pot [15 cm diameter (top) x 12.5 cm (height) and
10 cm (bottom)] maintaining 3 seedlings per pot. Since these mutants were from M3 seeds
(successively selfed plants), uniformity was kept among the segregating plants by removing
seedlings with phenotypes that resembled the wild-type. When the mutant phenotype was very
close to the wild-type, selection was not possible and seedlings were selected at random Six
plants per treatment were hand-sprayed with 100 µM GA3 every week on the surface of the
leaves. Seedlings were grown for 14 wks until plant maturity in an environmentally controlled
greenhouse using a 16 h photo-period provided by natural light supplemented with light from
sodium lamps to maintain a minimum PAR of 350 µmol m-2s-1.
5.3.2.1 Growth measurements
Culm length was measured from main culms from the ground to the base of the spike. Culm
diameter was measured at harvest time from the main culm at the basal internode with a digital
calliper. Grain yield and shoot biomass were measured for each plant in each replication. Panicle
length of main tillers was measured from the node, where the lower base emerges above the
peduncle. Tiller height was determined by summing the culm height as well as peduncle and
panicle lengths. Internode and tiller number were determined by counting the number of
internodes or tillers per plant. Internode diameter was measured about 3 mm above the each node
using a standard digital calliper. Dry weight was determined from above-ground plant material
132
by drying fresh material at 80oC for 2 days in an oven. Grain yield was determined by measuring
the weight of seeds from all tillers.
5.3.2.2 Data analysis
Growth and yield data were analyzed using the SAS statistical package (SAS Institute Inc., Cary,
NC, USA) for Analysis of Variance (ANOVA) and Pearson Correlation Coefficients. Statistical
significance of difference between treatment means was determined using the Tukey's
Studentized Range (HSD) test. A P-value of ≤0.05 was considered as significant.
133
5.4 Results
5.4.1 Culm height, internode length and diameter
Three lines (3-7-4852-1, 3-7-315-12 and GA-10 ) had significantly shorter (p < 0.05) and two
lines (3-7-4682 and 3-7-5160) had significantly (p < 0.05) taller culm length than the wild-type
plants (Fig. 5.1). Most dwarfed lines (3-7-4852-1, 3-7-315-12 and GA-10) were not significantly
different (p > 0.05) from each other in culm length despite a 18.5cm culm length difference
between them. Culm height difference between plants of the tallest and shortest mutagenized
lines was about 70cm (Fig. 5.1). Decrease in culm length in plants of the three shorter lines
derived from either a decrease in internode number or length or a decrease in peduncle length
(Figs. 5.1 and Table 5.3). Lines 3-7-5131-2 and 3-7-4852-1 gave significantly (p < 0.05) higher
number of internodes (8.3) when compared to wild-type plants (6.9; Table 5.1).
Application of GA did not significantly increase (p > 0.05) culm or internode length in plants of
the three dwarfed lines (3-7-4852-1, 3-7-315-12 and GA-10) when compared to the wild-type
control. GA treatment further did not significantly (p > 0.05) affect peduncle and panicle length
including the wild-type control. Dwarfed plants of mutant lines did not significantly (p > 0.05)
change culm, panicle or peduncle length when treated with GA (Figs. 5.2 and 5.3). Also, mutant
plants of line GA-10 had a significant (p < 0.05) increase in internode diameter when compared
to wild-type plants when either treated or not treated with GA3 (Table 5.4). Further, internode
diameter had a slight, but steady, increase upward in plants of all mutant lines (Table 5.4).
134
Table 5.1 Peduncle length, internode number, number of tillers, culm and panicle dry weight of
plants of different mutant E. tef lines and wild-type control (variety DZ-Cr-37).
Line
Peduncle
Internode
Tiller
Culm + Leaf
Panicle
length (cm)
number
number
DWT (g)
DWT (g)
(wild-type)
29.92a
6.8bc
13.08dc
25.8b
9.60a
3-7-4682-2
24.58ab
7.5ab
13.08dc
26.8b
9.12a
3-7-5160-1
25.72ab
6.9bc
16.23bc
32.5a
3.72c
3-7-5131-2
27.65ab
8.3a
10.23d
25.1b
5.06b
3-7-4852-1
13.08d
8.3a
20.75a
15.1c
0.31d
3-7-315-12
18.04cd
7.4abc
18.73ab
10.3d
0.40d
GA-10
23.67cb
6.4c
14.42c
25.5b
9.49a
Mean
23.24
7.37
15.22
23.1
10.22
***
**
***
***
***
DZ-Cr-37
P
DWT= dry weight; Letters within a column denote significance as determined by the Tukey's
Studentized Range (HSD) test. Data shown represent mean values ± SE of 12 individual
plants. Significance level was determined using ANOVA (*** P < 0.001; ** P < 0.01).
Means followed by the same letter are not significantly different.
135
Culm
180
Panicle
Seed
90
80
70
120
60
*
*
50
*
90
40
*
60
*
30
0
Control
4682
5160
5131
*
*
*
4853
315
30
20
*
*
Seed wt (g)
Culm/Panicle length (cm)
150
10
0
10
Mutagenized lines
Figure 5.1 Culm and panicle length and seed weight in plants of different mutant
lines and the control (DZ-Cr-37). Data represent the mean ± SE of 12 individual
plants.
136
200
(-) GA
180
(+) GA
Culm length (cm)
160
140
120
100
80
60
40
20
0
Control
4682
5160
5131
4853
315
10
Mutagenized lines
Figure 5.2 Culm length of plants of different mutagenized lines and wild-type plants
(DZ-Cr-37) with (+) GA and without (-) GA treatment. Data represent the mean ± SE
of 6 individual plants.
137
5.4.2 Panicle length, tillering, biomass and yield
Among the plants of the three dwarf mutant lines with the highest reduction in culm
length, plants of lines 3-7-4852-1 and 3-7-315-12 had significantly (p < 0.05) lower
panicle length when compared to wild-type control plants. However, plants of the most
dwarfed mutant line GA-10 had an identical panicle length when compared to the wildtype control (Fig. 5.1) regardless of additional GA treatment (Fig. 5.3). Plants of the two
mutant lines 3-7-4853 and 3-7-315 had further the shortest panicle length (Fig. 5.1) and
plants of these lines had also severe defects in fertility and produced only little grain.
Tillering in plants of the two mutant lines (3-7-4852-1 and 3-7-315-12) was further
significantly (p < 0.05) higher than in plants of the shortest mutant line (GA-10) and the
wild-type control (Table 5.1). Tillering was also not reduced by GA treatment (Table 5.3)
and tiller number was also not related to culm length (data not shown).
Plants of the two mutant lines with reduced height (3-7-4852-1 and 3-7-315-12) had
significantly (p < 0.05) lower panicle dry weight and reduced yield when compared to the
control or all other mutant lines (Table 5.3). Plants of mutant line GA-10 produced about
half the yield (2.5 g) of the wild-type control (4.8 g). In all plants of dwarf mutant lines
yield was not increased by GA treatment (Table 5.3).
138
Table 5.2. Response of mutant lines in terms of internode length to exogenously applied GA3
INT1
GA
INT2
-
+
Wild type
6.7
-
INT3
+
-
INT4
+
-
INT5
+
12.1 14.6 16.5 16.4 22.6 18.3
20.4 20.6 24.1 20.2 27.8 21.2 27.5 -
-
-
-
3-7-4682-2
10.2 11.5 18.7 16.3 17.8 18.0 18.8
24.8 22.3 24.1 24.1 23.3 28.6 27.3 -
23.7
-
23.0
3-7-5160-1
9.1
13.0 15.8 18.9 19.6 22.1 18.1
21.8 24.1 27.9 26.3 27.9 29.4 26.1 -
-
-
-
3-7-5131-2
7.7
7.1 15.4 9.46 15.8 15.8 16.3
17.0 19.9 17.3 24.0 20.9 25.6 15.6 25.0 11.4
-
16.5
3-7-315-12
5.3
6.5 10.5 8.8
14.9 18.5 16.1 17.8 16.2 16.6 13.1 17.1 15.3
-
-
3-7-4852-1
3.6
8.1 6.8
14.0 10.1 14.0 13.1 19.7 15.6 16.8 17.1 12.5
-
-
GA-10
5.1
4.1 9.58 9.4
13.7 16.1 18.3 18.1 19.7 20.6 23.7 -
-
-
12.4 12.5 13.8
-
+
-
+
INT9
-
12.1 9.5
+
INT8
+
10.8 8.8
-
INT7
-
13.0 10.6 15.5
+
INT6
-
GA Mean
7.0b 8.8a 13.3a 13.2a 15.4a 16.4a 16.5b 18.6a 19.5a 20.8a 21.2a 22.2 23.1a 21.8a 19.7a 17.9b 15.8a 19.7a
GA
*
NS
NS
**
NS
NS
NS
***
NS
LinexGA
NS
*
*
NS
NS
**
**
NS
NS
139
Significance level was determined using SAS GLM for ANOVA (*** P < 0.001; ** P <
0.01;*<0.05); L!= Line; Letters for mean denote significance as determined using the
Tukey's Studentized Range (HSD) Test. Data shown represent mean values ±SE of 6
individual plants. Means followed by the same letter are not significantly different. NS =
Non-significant; (-) = control or unsprayed; (+) = Sprayed with GA3 (100uM).
140
40.0
A
35.0
Peduncle GA-
Peduncle GA+
Length (cm)
30.0
25.0
20.0
15.0
10.0
5.0
0.0
Control
4682
5160
5131
4852
315
10
Mutagenized lines
70.0
B
Panicle GA-
Panicle GA+
60.0
Length (cm)
50.0
40.0
30.0
20.0
10.0
0.0
Control
4682
5160
5131
4852
315
10
Mutagenized lines
Figure 5.3 Effect of GA application on peduncle (A) and panicle (B)
length of mutant lines and the control (DZ-Cr-37). Data represent the
mean ± SE of 6 individual plants.
141
Table 5.3 Peduncle length, internode number, number of tillers, culm and panicle
dry weight of plants of different mutagenized E. tef lines and wild-type control
plants (cv. DZ-Cr-37).
Line
Internode
Tiller
Culm DWT
Panicle DWT
Seed WT
number
number
(g)
(g)
(g)
GA-
+
-
+
-
+
Wild-type
7.0
6.5
13.5
12.7
26.1
25.4
3-7-4682-2
7.0
8.0
12.7
13.5
26.4
3-7-5160-1
6. 7
7.2
15.8
16.4
3-7-5131-2
7.3
9.2
8.7
3-7-4852-1
9.2
7.5
3-7-315-12
7.3
GA-10
6.5
Mean
7.3a 7.3a 15.2a 14.6a 22.9a 23.3a 5.60a
-
+
-
+
10.2
8.9
4.9
4.6
27.2
9.1
9.1
4.5
4.9
33.8
31.2
3.4
4.1
0.4
1.1
11.8
21.6
29.3
4.3
5.9
1.3
2.4
22.7
18.8
14.2
15.9
0.4
0.3
0.07
0.08
7.4
21.7
14.6
11.4
9.1
0.3
0.5
0.00
0.01
6.3
13.7
15.2
24.7
26.2
8.8
10.2
2.2
2.8
6.10a
2.25a 2.70a
GA
NS
NS
NS
NS
NS
Line x GA
***
NS
***
***
***
DWT= dry weight; Significance level was determined using SAS GLM or ANOVA (*** P
< 0.001). Letters for mean denote significance as determined using the Tukey's Studentized
Range (HSD) test. Data shown represent mean values±SE of 6 individual plants. Means
followed by the same letter are not significantly different. NS = Non-significant; GA (-) =
control or unsprayed; GA (+) = sprayed with gibberellic acid (GA3 at 100 µM).
142
Table 5.4 Internode diameter of different mutant lines and wild-type control (cv. DZ-Cr-37) plants in response to
GA3 treatment.
Line
INTØ1*
INTØ2
INTØ3
INTØ4
INTØ5
INTØ6
INTØ7
INTØ8
INTØ9
GA-
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
Wild type
1.84
1.55
2.30
1.92
2.50
2.44
2.74
2.51
2.89
2.66
3.06
3.02
3.29
3.16
-
-
-
-
3-7-4682-2
1.97
1.61
2.67
1.43
2.95
2.24
3.04
2.73
3.48
2.74
3.42
2.80
3.65
2.83
-
2.52
-
1.98
3-7-5160-1
2.08
1.59
2.61
1.80
2.80
2.46
3.15
2.68
3.11
2.78
3.44
3.06
3.86
3.13
-
-
-
-
3-7-5131-2
1.93
1.52
2.25
1.86
2.89
2.34
3.23
2.57
3.28
2.69
3.84
2.88
3.92
2.84
3.63
3.02
-
1.92
3-7-4852-1
1.41
1.02
1.44
1.13
1.42
1.26
1.61
1.51
1.61
1.84
1.71
1.83
1.61
1.55
1.55
1.30
-
-
3-7-315-12
1.50
0.77
1.87
1.09
2.03
1.40
2.03
1.41
2.33
2.02
2.32
1.83
2.54
1.54
2.26
1.28
-
-
GA-10
1.90
1.85
2.74
2.45
2.86
2.80
3.24
2.95
3.55
3.11
3.24
3.34
3.71
2.90
-
-
-
-
Mean
1.82a
1.43b
2.28a
1.69b
2.51a
2.14b
2.74a
2.34b
2.92a
2.54b
3.01a
2.69b
3.19a
2.58b
2.48a
2.11a
1.44a
1.74
a
GA
***
***
***
***
***
***
***
**
NS
Line x GA
NS
*
NS
NS
NS
NS
NS
NS
NS
143
*IntØ1= internode No. 1diameter from the base of the stem; Significance level was
determined using SAS GLM for ANOVA (*** P < 0.001; ** P < 0.01;*<0.05). Letters for
mean denote significance as determined using the Tukey's Studentized Range (HSD) test.
Data shown represent mean values ±SE of 6 individual plants. Means followed by the same
letter are not significantly different. NS = Non-significant; GA (-) = control or unsprayed;
GA (+) = sprayed with gibberellic acid (GA3 at 100 µM).
144
5.5 Discussion
This study has shown that semi-dwarf phenotypes could be developed in E. tef through
mutagenesis. However, regardless of plant height grain yield was considerably reduced in all
the mutant lines, possibly because of other undesirable effects induced in the plants. Some of
the mutants showing considerable reduction in height were also found near infertile and
produced only a very little amount of grain except for plants of the most dwarfed mutant line
GA-10,whichgave a about half of the untreated wild type control..
Internodal diameter was not increased due to induced semi-dwarf phenotype with the
exception of semi-dwarf line GA-10 which had consistently higher intermodal diameter from
2nd to the 5th internode in the GA untreated plants compared to GA untreated wild type
control Such increase in internode diameter contributes to the stiffness of straw (stem) by
increasing the ratio for stem basal-diameter to height which would be of benefit for obtaining
lodging resistance. On the other hand, the weak tapering of control plants could not be
improved in any of the dwarf mutant plants since acropetally steadily increasing intermodal
diameter was found in all of the mutant plants. Based also on our previous results from GA
inhibition studies (Chapter 2) that plant height reduction did not increase stem basal diameter,
results of this study also confirmed absence of meaningful relationship between plant height
and stem diameter. However, this is under the assumption that the induced semi-dwarf
phenotype in the mutant plants in this study also involves mutation in the height controlling
genes. This, therefore, is subject to further verification. Therefore, it is possible to speculate
that factors, other than GA genes mediated controlling of culm height might also be involved
in controlling internode diameter. This speculation is in line with the recent report by
Ookawa et al. (2010) about enhanced stem strength obtained in japonica rice plants carrying
145
the STRONG CULM2 (SCM2) gene due to its effect on increasing culm diameter. Therefore,
mutagenesis and TILLING application can also be directed to induce change in such genes
involved in culm diameter control for lodging resistance improvement. It, however, is not yet
clear from the present study if mutant line GA-10 may harbor induced changes in such genes
involved in stem diameter control.
None of the semi-dwarf mutant including GA-10 plants showed a response to GA treatment
and did not recover height. This means the induced changes are not in the GA biosynthesis
since such mutants can easily recover height by exogenous GA application. The alternative is
change in GA signalling in which case studies have already shown that GA signalling
mutants are insensitive to GA treatment (Milach et al., 2002; Hedden et al., 1998). It might,
therefore, be possible that mutant plants not responding to exogenously applied GA harbour a
mutation in GA signalling components such as the Rht in GA metabolism.
Plants of semi-dwarf lines also had normal tillering but did not have identical biomass
amount as the wild-type control except mutant line GA-10. Hence, most tillers were rather
weak with slender stem having little biomass to support further growth and sustain grain
filling as evident from the very low yield. However, biomass in the semi-dwarf mutant GA10 was identical to the biomass in the control plants which could be of significant advantage
if this line is used for crossings with other parental lines to restore yield without losing its
other useful traits such as the semi-dwarf and higher internodal diameter. Plants of GA-10
were non-responsive to GA application implying that it may harbor a mutation possibly in
GA-response genes. It also have shown a higher GA content (data not shown: personal
communication Dr. Tadelle) which also suggests an impaired GA signalling component such
as changes in the Rht gene. GA signalling mutants have been shown to have elevated levels
146
of bioactive GA presumably because of a feedback regulation as a result of reduction in GAresponse (Alvey and Harberd, 2005). However, it is necessary to verify the assumptions in
future experiments to determine the agronomic importance of mutant line GA-10. Crossing
GA-10 with known tall varieties would also be necessary to determine the line’s potential as
a source of a semi-dwarf trait. Evaluation can be further supplemented with characterization
of the DNA sequences for target GA-signalling genes such as Rht.
Overall, this study has shown that a TILLING approach can be applied to obtain semi-dwarf
E. tef plants. Application so far has resulted in different semi-dwarf lines and one of these
lines, GA-10, has potential for further characterization including the cause for dwarfism.
147
CHAPTER SIX
GENERAL DISCUSSION AND FURTHER PERSPECTIVE
148
The study aimed to be investigate the role of GA in plant height control in E. tef and
associated changes in morpho-physiological parameters of the plant including yield . Results
showed the significance of GA genes as prime targets for plant height reduction in E. tef to
ultimately improve lodging resistance by reduction of culm length.
A first new aspect of this study was the high responsiveness of E. tef in terms of plant height
reduction following GA inhibition by anti-gibberellins chemical treatment (CCC and PBZ). A
further new finding was that growth of the E. tef panicle, which constitutes one third of the
total plant height, was not very sensitive to GA inhibition. At higher concentration of a potent
inhibitor, such as PBZ, stem elongation was severely reduced. Generally, the control of GA
inhibitors on stem height and panicle elongation provided strong evidence that targeting the
GA biosynthesis pathway is a realistic strategy for the control of plant height in E. tef.
Further, evidence was provided that decoupling plant height and yield could be achieved in E.
tef. This would allow developing dwarf lodging-resistant plants in high yielding cultivars.
Although PBZ had a much stronger GA inhibition on internode elongation in E. tef than CCC
and also acting at a much lower concentration than CCC, PBZ cost and persistence in the soil
would restrict its wider application. In addition, panicle bearing tillers was not increased by
PBZ or CCC treatment although PBZ increased tillering many-fold. Unfortunately, CCC
treatment did not increase stem diameter, but the diameter to height ratio was increased
improving plant standing. Therefore, CCC appears to be a suitable inhibitor and a candidate
for further plant height control for reducing the lodging problem under field conditions. Finetuning of CCC application and observing response of plants (with reduced height) under field
conditions are, however, required to prepare a practical guideline for wider application of the
PGR in E. tef. This will help to identify a good balance between vegetative (stem and tiller)
149
and reproductive (panicle and seed setting) growths for effectively reducing height (therefore
lodging) without compromising seed yield. The weak tapering in E. tef observed in this study
together with absence of any promising effect on stem diameter is still a concern suggesting a
weak transition between the shoot base, 1st lowermost internode and the root collar, in E. tef
plants. Emphasis should therefore also be given to understand the mechanism, other than
those regulated by GA genes, that might involve improving E. tef stem-base.
A further new aspect of this study was the optimization of E. tef regeneration to produce
putative transformed plants from immature somatic embryos via Agrobacterium-mediated
transformation for the induction of dwarfism over-expressing GA inactivating gene (GA2ox)
from Phaseolus coccineus. In this study, 8 putative transformed plants carrying the insert
(PcGA20 ox or nptII gene sequence) at the T0 generation were obtained. Regenerated plants
were successfully grown into mature fertile plants producing seeds. In the transformation
procedure, a combination of different previously reported media for various crops have been
successfully applied for embryogenic callus induction, Agrobacterium inoculation and cocultivation and plant regeneration. The success in embryogenic callus induction using less
than 1-week old zygotic immature embryos explants for regeneration into shoots was
dependent on the use of intermediate size embryos. It was further found that the antibiotic
geneticin (G418) fully controlled shoot growth from mature E. tef embryos which also
requires further optimization for E. tef callus.
Molecular assessment of the transgene PcGA2ox has been based on results of previous
studies where reduction in plant height in other cereals to be a key agronomic feature to limit
lodging (Rajala, 2003; Rademacher, 2000). Results in this study showed only a putative T0
transformants having a positive PCR results for the transgene. Selected putatively
transformed T0 plants were characterized further growing seeds (T1 progeny) for genotype
150
and phenotype and inconsistent results were obtained during PCR detection of the presence
of the transgene. Selected semi-dwarf T1 generation plants showed that the reduction in plant
height significantly varied even among the semi-dwarf plants. The reduction in height was
also associated with amounts of bioactive GA1. On the other hand, the accumulation of GA8
in the semi-dwarf plants was not proportional to the relative height differences or supposedly
deactivation of GA1. Such phenomenon was also observed in transformed Solanum nigrum
over-expressing same GA inactivating gene, PcGA2ox transgene (Dikstra et al., 2008).
Deficiency through GA deactivation decreases height in rice (Lo et al., 2008) with increasing
yield (Ookawa et al., 2010). However, due to the ambiguity of the PCR result, therefore,
these plants have to be further characterized to show if GA2ox transgene expression such as
GA2ox transcript abundance in the stem tissue is always associated with reduction in plant
height to exclude the possibility of somaclonal variation induced in the tissue culture process
using the auxin 2, 4-D (Banerjee et al., 1985) and not due to stable transgene integration.
The fourth new aspect of this study was the successful isolation three GA20ox homologous
genes from E. tef, involved in GA biosynthesis. Additionally, the Rht (Reduced height) and
Eui (Elongation Uppermost Internode) genes involved in GA biosynthesis and a Cytochrome
P450 monooxygenase gene in brassinosteroid deactivation were either fully or partially
isolated and cloned. All these genes are involved in plant height control. Genomic analysis
identified four copies of GA20ox to exist in E. tef. The EtGA20ox1 sequence was successfully
expressed in a heterologous bacterial system and allowed converting the substrate GA12 to
products GA9 ,GA15 and GA24. Further, in E. tef EtGA20ox1b was the functional equivalent
to the rice sd-1 gene. Alignment and phylogenetic relationship of the full coding region of
putative E. tef GA2ox1 and partial putative GA2ox1b and EtGA20ox2 sequences showed high
identity scores with orthologous genes from S. bicolor, Z. mays, O. sativa and L. perenne.
151
This gene is expressed with highest transcriptional abundance in the uppermost internodes
followed by node tissues and any mutation in this E. tef gene might specifically control plant
height. However, a further study is required to confirm any functional similarity between
EtGA20ox1b and EtGA20ox1 in different plant tissues.
A fifth new aspect in this study was the potential usefulness of E. tef seed mutagenesis in
producing a semi-dwarfed phenotype using variety DZ-Cr-37, which is a fairly tall modern
cultivar. However, mutants combining reduced culm length and still good yield were not
identified among the mutants screened. Except for semi-dwarf mutant line GA-10, which had
a reasonable yield, low yield in tested dwarfed mutant lines was always associated with weak
and infertile panicle development possibly due to undesirable random genetic changes not
associated with height control. GA-10 also had a significantly higher diameter in most
internodes which could directly contribute to the stiffness of stem and lodging resistance.
This line also showed normal tillering and culm and panicle biomass and should therefore be
tested further. Since all mutants were insensitive to GA application, the mutation in GA-10
might possibly be due to a change in GA signalling genes. Further analysis is therefore
required if plants of this line harbour a GA signalling mutation. Also, crossing experiments
with tall varieties are also required to verify and predict agronomic significance of line G-10
for lodging resistance.
Overall, support of the original hypothesis that regulation of the GA amount in E. tef will
change pheno-morphic and also agronomic characteristics that would affect lodging and
further decoupling plant height from yield was found in this study. GA inhibition or
deactivation in E. tef produced a semi-dwarf phenotype without changing panicle
development and grain yield except under conditions of severe GA inhibition where panicle
152
growth was affected and grain yield was reduced. Other pheno-morphic features or plant
stand structures, such as diameter of basal internodes, the weak tapering (acropetally
increasing stem diameter) and panicle bearing tillers in E. tef plant were not improved by
reducing the endogenous GA amount.
Based on the findings of this study, any future following-up study might investigate stem
tissue specific down-regulation of height controlling genes such as down-regulation of the
already cloned EtGA201a in E. tef through a non-transgenic approach such as mutagenesis
and TILLING or through RNAi technology. Panicle elongation was unaffected by GA
inhibition, a future lodging-resistant improvement in E. tef also has to focus on obtaining
compact type panicles in a dwarf or semi-dwarf phenotype background. Moreover, it is
essential to further consider other potential threats in the future lodging resistant ideotype
development in E. tef such as the narrow stem base problem (weak stem -basal to root-collar
transition) that render E. tef plants weak in anchorage strength. Therefore, further study is
required to understand mechanisms involved in stem-base diameter regulation (with emphasis
on basal internodes), since the presumed weak transition from stem-base to the root-collar
(supposedly low for E. tef) appears not to be regulated by GA. Generally based on the
analyzed and implicated E.tef pheno-morhpic/architectural traits in relation to lodging, the
need for a more comprehensive intervention through gene regulation is clear and need to
combine traits for a short stature, thicker basal-diameter and shorter panicle form in the
lodging resistance E. tef ideotype development. The prime target genes related to stem height
control that have already been cloned in this study can now be used to employ a nontransgenic approach using mutagenesis and TILLING techniques to induce mutation and
select semi-dwarf genotypes in E. tef as an immediate future intervention strategy.
153
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Appendix
176
A
TGCGGCAGCGTGAAGAAGGCGTCCATTCGAGTGGAGTCTCGGAATCCTCAAACTCACTGCAG
CAGCAGCAGCAGTAGTAGTAGTAGTCCTCCTCCTAGTCAATCAGAGAGAGAGAGAGAGAGAG
AGAGAGAGAGAGAGAGCCAAGCCAGCTGCCCGTGATGGTGGTGGTAGCGCAGGCGCAGGCGC
AGGCGCAGATAGAAGTAGAAGCAGATGAGCCGCCGCAGCAGCTTGAGGTGGTGGTGTTCGAC
GCGGCCCGTCTGAGCGGGCTGAGTGACATCCCGGCCCAGTTTTTGTGGCCGGAGGAGGAGAG
CCCGACGCCTGACGCGGCGGAGGAGGAGCTGGACGTTCCTCTGATCGACCTCTCCGGCGACG
CGTCGGAGGTGGTCCGTCAGGTGCGTGAGGCCTGCGAGGCGCACGGCTTCTTCCAGGTGGTG
AACCACGGCATCGACGCCGGCCTCGTGGCGGAGGCGCACCGCTGCATGGACGCCTTCTTCAC
GCTGCCGCTGCCGGAGAAGCAGCGCGCCCAGCGCCAGCCCGGCGACTGCTGCGGCTACGCCA
GCAGCTTCACGGGCCGCTTCGCCAGCAAGCTGCCATGGAAGGAGACGCTCTCCTTCCGGGCC
AAGGCCAAGGCCACCGATGATGATGATGATGATGTGGTGGGCTACTACGTCAGCAAGCTGGG
CGAGGCCTACAGGCGGCACGGCGAGGTGTACGGGCGCTACTGCTCGGAGATGAGCCGGCTGT
CGCTGGAGATCATGGAGGTGCTGGGCGAGAGCCTGGGCGTGGGCCGCCGCTGCTTCCGCGAC
TTCTTCCAGGACAACGACTCCATCATGCGCCTCAACTACTACCCGCCGTGCCAGCGGCCCAT
GGAGACGCTGGGCACGGGCCCGCATTGCGACCCCACCTCCCTCACCATCCTGCACCAGGACC
ACGTCGCCGGCCTCCAGGTCTTCGCCGCCGGCCGGTGGCTCTCCATCCGCCCGCACGCCCAG
GCCTTCGTCGTCAACATCGGCGACACCTTCATGGCGCTCTCCAACGGCCGGTACAAAAGTTG
CCTGCACAGGGCGGTGGTCAACAGCAGCGTCCCGCGGAGGTCCCTGGCCTTCTTCCTCTGCC
CGGAGATGGACAAGCTCGTCACGCTCCCGCCGCAGCTTCTTCCTGATCTGCCCGGAGACAAC
CAAAGGAGGCGCCCCTACCCGGACTTCACGTGGCGCACCCTGCTCGAATTCACGCAGAAGCA
CACAGGGCCGACATGAAGACGCTCGAGGTCTTCTCCAACTGGCTCCGCCATGGCCAGGACAA
GGTAGCGCTACCTCCCCTATGCTAGTTATTCTCTCCATACCAATATGTAATGTAATGTAATG
AATGTAGTCATCCATGACCGCCTGTTCTGTTCAAAAAAAAAAGTCGACATCGATACGCGTGG
TCCGCCTCCTTTGGTTGTCTCC
B
RQREEGVHSSGVSESSNSLQQQQQ****SSS*SIREREREREREREPSQLPVMVVVAQAQAQ
AQIEVEADEPPQQLEVVVFDAARLSGLSDIPAQFLWPEEESPTPDAAEEELDVPLIDLSGDA
SEVVRQVREACEAHGFFQVVNHGIDAGLVAEAHRCMDAFFTLPLPEKQRAQRQPGDCCGYAS
SFTGRFASKLPWKETLSFRAKAKATDDDDDDVVGYYVSKLGEAYRRHGEVYGRYCSEMSRLS
LEIMEVLGESLGVGRRCFRDFFQDNDSIMRLNYYPPCQRPMETLGTGPHCDPTSLTILHQDH
VAGLQVFAAGRWLSIRPHAQAFVVNIGDTFMALSNGRYKSCLHRAVVNSSVPRRSLAFFLCP
EMDKLVTLPPQLLPDLPGDNQRRRPYPDFTWRTLLEFTQKHTGPT*RRSRSSPTGSAMARTR
*RYLPYASYSLHTNM*CNVMNVVIHDRLFCSKKKVDIDTRGPPPLVVS
177
Figure A.1 (A) E. tef GA20ox1 full coding region (nucleotide sequence) and (B) deduced
amino acid residues of E. tef GA20ox1 sequence with 482 aa with conserved domains
-TCGATTGACTTCGTGGTGGAGGACTGCAAGAACATCTACTTCGCGGGGTACGAGACCACCGCGGTGACCGCGGCCTGGT
GGT.GAG..G.....C.....CA......................C..............G...........C.......
CA.CG.GA..GA.A.A........G.......CGT...T.........CA.GC.......GT..AACCT.CT.A.A...A
GC..GAC..............CA..........................C.......G.G...C..C.....CA......
GC..GAC..............CA......................C...C.......G.G......C......A.G....
GC..GAC..............CA..........................C.......G........C.....CA......
79
80
80
80
80
80
GCCTGATGCTCCTGGCGCCGCACCCGGAGTGGCAGGACCAGGTGCGCGACGAGGCGTGCCAGGCGTGCG----------..AC..............T....................G................C.G..........----------C.ACAG.C..G..C..CAT......C...........G.TC.CC..TC.G....T.CT...A.TC....G---------....C.....G..C..CG..........C.........A...C.....C.....TCCT.G.C.TCCT..GCACCGGCACC
....C.....G..C..CG.....................GC.CC....C.....T.CT.G...TC....GCGGCGACGGC
....C.....G..C..CG.C..T..C.............GC.C.....C.....T.CT.G.C.TC....GCGGCG-----
148
149
150
160
160
155
-CAGGTGGCGCCG---------CGGCGCCAGACTTCACCTCTCTCCAGAGGATGAAGA-GCTGACGATGGTGATCCAGGA
-.G.CC.......---------..---..G......G.A..C..G..C.A........A.....................
--G.CGC.T.A.A------------TC..TTC.CG.GAGCAG...ACC.A.C.C....CA..CGG....A.CC.GA.C..
GGCACC.......GGGATCCCCT......G......GAGG..G.GTC...........C....GGC......G.G.....
G.C.CC.C.C...---------.C.....G......GA.ATGG.GTCCC.....CG..C.G..GG.......G.G.....
-.C.CC..T....---------.A.AC..G......GA.GTGA..TCC........A.C.G..GGT.....TG.G.....
217
216
216
240
231
225
AACGCTGCGGCTGTAC
G...............
G...............
G.............T.
..............T.
G.............T.
233
232
232
256
247
241
Identity (%)
66
60
49
39
40
30
E. tef Eui
S. bicolor
Z. mays
T. sylvaticum Eui
O. sativa EUI
T. aestivum
indicated (bold and underlined). Conserved domains include (i) the consensus sequence
NYYPXCXXP of 2-oxogltarate dependent dioxygenase (2ODDs) for binding the common
co-substrate; ii) sequence LPWKET, which binds to the GA substrates, and iii) three
histidine residues HCD for binding Fe2+.
C
GCTGCCGTGGAAGGAGACGCTCTCCTTCGGCCACCGCGACGTCGTGGAGTACTTCACATCCA
CCCTCGGCAGCGACTTCAAACCCCTAGGGTAAACATAAACTGTTGAAATAGCTAGCTTCGCT
ACGTTTTTTTTTGTACGTACAAGAGATCGGTCATCGGAGAGCTTTTACAATGACTAATGCAC
CGATCGAGCCATGCATGGACGCAGGGAGGTGTTCCGAGACTACTGCCAATCGATGAAGGAGG
TGTCGCTGGCGATCATGGAGGTGCTGGGCGCGAGCCTGGGCGTGGGGAGGCGCTACTGCAGG
GACTTCTTCGCCGACGGCTGCTCCATCATGAGGTGCAACTACTACCCGCCGTGCCCGGAGCC
GGACCGGACGCTGGGCACGGGGCCCCACTGCGACCCGGCGGCCCACACCCTCTTGCTCCAGG
ACGACGACGTGGACGGGCTCCAGGACGACGACGTGGACGGGCTCCAGGTGCTCGTCGACGGC
GAGTGGCGGCCCGTGCGGCCCAAGCCGGGAGCCATCGTCGTCAACATCGGCG
178
D
LPWKETLSFGHRDVVEYFTSTLGSDFKPLG*T*TVEIASFATFFFVRTRDRSSESFYND*CT
DRAMHGRREVFRDYCQSMKEVSLAIMEVLGASLGVGRRYCRDFFADGCSIMRCNYYPPCPEP
DRTLGTGPHCDPAAHTLLLQDDDVDGLQVLVDGEWRPVRPKPGAIVVNIG
E
CGATTGCTGCCGTGGAAGGAGACGCTCTCCTTCCGGGCCAGCCCAACGTCGCCGGCCTTGGTGGAGGA
CTACCTGGTGGGCCGCCTTGGCGACGAGTACAGGCGGCACGGCGAGGTGTACGGGCGCTACTGCTCGG
AGATGAGCCGGCTGTCGCTGGAGATCATGGAGGTGCTGGGCGAGAGCCTGGGCGTGGGCCGGCGCTGC
TTCCGCGACTTCTTCCAGGACAACGACTCCATCATGCGGCTCAACTACTACCCGCCGTGCCAGCGGCC
CAYGGAGACGCTGGGCACGGGCCCGCATTGCGACCCCACCTCCCTCACCATCCTGCACCAGGACCACG
TCGCCGGCCTCCAGGTCTTCGCCGGCGGCCGGTGGCTCTCCATYCGCCCGCACGCCGCCGCCTTCGTC
GTCAACATCGGCGA
F
RLLPWKETLSFRASPTSPALVEDYLVGRLGDEYRRHGEVYGRYCSEMSRLSLEIMEVLGESLGVGRRC
FRDFFQDNDSIMRLNYYPPCQRPETLGTGPHCDPTSLTILHQDHVAGLQVFAGGRWLS?RPHAAAFVV
NIG
179
Figure A.2 Homologous sequences of E. tef GA20ox1a and GA20ox2. (C) represents a E. tef
GA20ox2 partial coding nucleotide region, (D) deduced amino acid residues of E. tef
GA20ox2 sequence with 152 aa (excluding the sequence in grey shade) with conserved
domains indicated (bold and underlined) and stop codons (*); (E) E. tef GA20 ox1b partial
coding nucleotide region and (F) deduced amino acid residues of E. tef GA20ox1b sequence
with 140 aa with conserved domains indicated (bold and underlined). Sequence in box is in
“C” and “D” is an intron between the concerned region in tef GA20ox2 sequence.
Table A.1 Primers used for PCR amplification of gene fragments of Rht, Eui and BR using
E. tef genomic DNA
Target gene
Primers
Degenerate/specific primers
Rht
F1
GTGG(TCA)GGACACGCAGGAGGC
F2
TTCTACGAGTCCTGCCCCTACCT
R1
T(TCG)GCGGTGAAGTGGGCGAAC
R2
TCGGGCTCGTCATCCGTGTCAT
F1
A(GA)(CG)(CG)(TC)C(CA)ACGGCGA(CG)(AG)AT
R1
ACAGCCGCAGCGTCTCGTT(GCT)
F1
ACAGCCGCAGCGTCTCGTT(GCT)
R1
A(GA)(CG)(CG)(TC)C(CA)ACGGCGA(CGA)(GC)(C
G)(AT)GTT
Eui
BR
180
Table A.2 Rht primers used in RACE- PCR
Target gene
Primers
Degenerate/specific primers
Rht
Rht SPR3
ATCCCGGCCTCCTGCGTGTCCA
Rht SPR2
CGGTGGCGGGCGGAAGCGATACA
Rht SPR1
GGGCGAACTTGAGGTAGGGGCAG
Rht SPF1
TGTATCGCTTCCGCCCGCCACCG
Rht SPF2
CTGCCCCTACCTCAAGTTCGCCC
G
TCGATTGACTTCGTGGTGGAGGACTGCAAGAACATCTACTTCGCGGGGTACGAGACCACCGC
GGTGACCGCGGCCTGGTGCCTGATGCTCCTGGCGCCGCACCCGGAGTGGCAGGACCAGGTGC
GCGACGAGGCGTGCCAGGCGTGCGCAGGTGGCGCCGCGGCGCCAGACTTCACCTCTCTCCAG
AGGATGAAGAAGGTACTGCAGGTCAAATGAATCACACAGTTCCATGCATGCACGTTCAGTAC
ACACGCGCGCGCGCTGATCTGACCAACCTTGTGCATCCAAATATTTGTTTTTTCGGCAGCTG
ACGATGGTGATCCAGGAAACGCTGCGGCTGTACA
H
SIDFVVEDCKNIYFAGYETTAVTAAWCLMLLAPHPEWQDQVRDEACQACAGGAAAPDFTSLQ
RMKKVLQVK*ITQFHACTFSTHARALI*PTLCIQIFVFSAADDGDPGNAAAV
Figure A.3 Partial coding region of the Elongated Uppermost Intenode (EUI) gene in tef. (G)
represents a E. tef EUI partial coding nucleotide region (344bp), (H) deduced amino acid
residues of E. tef EUI sequence with 115 aa. The underlined region (109 bp) is a predicted
intron.
181
Figure A.4. Derived nucleotide sequence alignment of the putative tef Uppermost Elongated
Internode (EUI) to orthologous monocot sequences from sorghum (S. bicolour; Acc No.
XM_002439928.1), maize (Z. Mays; Acc No. BT043273.1), rice (O. sativa Acc No.
AY987040.1), wheat (T. aestivum Acc No. AL816398) and brachypodium (T. sylvaticum).
Identical regions (>80%) are shown by dark shaded areas and dots whereas number indicates
the position of the nucleotide within the sequence.
182
44
62
O. sativa EUI
T. aestivum EUI
T. sylvaticum Eui
Z. mays
E. tef Eui
98
S. bicolor
0.05
Figure A.5 Molecular phylogenetic analysis of the rice EUI ortholog gene in tef. The tree
was inferred by Maximum Likelihood method using partial gene sequence. Initial alignment
was done by mafft (http://mafft.cbrc.jp/alignment) software. The phylogeny was inferred by
using the Maximum Likelihood method based on the Tamura-Nei model (1993). The
percentage of trees in which the associated taxa clustered together is shown next to the
branches. Initial tree(s) for the heuristic search were obtained automatically as follows. When
the number of common sites was < 100 or less than one fourth of the total number of sites,
the maximum parsimony method was used; otherwise BIONJ method with MCL distance
matrix was used. The tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. There were a total of 368 positions in the final dataset. Evolutionary
analyses were conducted in MEGA5 (Tamura et al., 2011).
183
I
GAGGAGGTGGACGAGCTGCTGGCCGCGCTCGGGTACAAGGTGCGCTCGTCGGACATGGCGGA
CGTGGCGCAGAAGCTGGAGCAGCTCGAGATGGCCATGGGGATGGGCGGCGTCCCCGCCGCGG
ACGACGGGTTCGTGTCGCACCTGGCCACGGACACCGTGCACTACAACCCCTCCGACCTGTCG
TCCTGGGTGGAGAGCATGCTGTCCGAGCTCAACGCCCCGCCGCCGCCGCTCCCGCCCGCGCC
CGCGCCGCCGGCCCCGCAGCTGGTTTCCACCTCGTCCACCGTCACGGGCGGCGGCTCCGGCG
CCGGGTACTTCGATCCCCCGCCCGCCGTCGACTCCTCCAGCAGCACGTACGCGCTGAAGCCG
ATCCCCTCGCCGGTGGCGGCGCCGGCCGACCCGTCCGCGGACTCCGCGCGGGAGCCGAAGAG
GATGCGCACTGGCGGCGGCAGCACGTCGTCTTCCTCGTCCTCGTCTTCGTCCATGGGCGGCG
GCGGCGCCAGGAGCTCCGTGGTTGAGGCTGCCCCGCCCGCATCCGCGGCGGCGAACGCGCCC
GCGGTGCCTGTGGTGGTGGTGGACACGCAGGAGGCCGGGATCCGGCTCGTGCACGCGCTGCT
GGCGTGCGCGGAGGCCGTGCAGCAGGAGAACTTCTCCGCCGCGGAGGCGCTGGTGAAGCAGA
TCCCCATGCTGGCCTCGTCGCAGGGCGGCGCCATGCGCAAGGTGGCCGCCTACTTCGGCGAG
GCTCTCGCTCGCCGCGTGTATCGCTCCCCCCCCCCGCCCCCGACAGCTCCCTCCTCGACGCC
GCCTTCGCCGACCTCCTCCACGCCCCACTTCTACGAGTCCTGCCCCTACCTCAAGTTCGCCC
ACTTCACCGCGAACCAGGCCTTCCTCGAGGCGTTCGCCGGCTGCCGTCGCGTCCACGTCGTC
GACTTCGGCATCGAGCAGGGGATGCAGTGGCCGGCGCTCCTCCAGGCCCTCGCCCTCCGCCC
CGGCGGCCCCCCGTCCTTCCGCCTCACCGGCGTCGGCCCACCGCAGCCTGACGAGACCGACG
CCTTGCAGCAGGTGGGTTGGAAGCTCGCCCAGTTCGCTCACACCATCCGCGTCGACTTCCAG
TACCGCGGCCTCGTCGCCGCCACGCTCGCAGACCTGGAGCCGTTCATGCTGCAACCGGAGGG
CGAGGAGAATGACGAGGAGCCCGAGGTGATCGCCGTCAACTCGGTGTTCGAGATGCACCGGC
TGCTGGCGCAGCCCGGCGCCCTGGAGAAGGTCCTGGGCACGGTGCGCGCGGTGCGGCCCAAG
ATCGTGACCGTGGTGGAGCAGGAGGCCAACCACAACTCCGGCTCGTTCCTGGACCGCTTCAC
GCAGTCTCTGCACTACTACTCCACCATGTTCGA
J
EEVDELLAALGYKVRSSDMADVAQKLEQLEMAMGMGGVPAADDGFVSHLATDTVHYNPSDLS
SWVESMLSELNAPPPPLPPAPAPPAPQLVSTSSTVTGGGSGAGYFDPPPAVDSSSSTYALKP
IPSPVAAPADPSADSAREPKRMRTGGGSTSSSSSSSSSMGGGGARSSVVEAAPPASAAANAP
AVPVVVVDTQEAGIRLVHALLACAEAVQQENFSAAEALVKQIPMLASSQGGAMRKVAAYFGE
ALARRVYRSPPPPPTAPSSTPPSPTSSTPHFYESCPYLKFAHFTANQAFLEAFAGCRRVHVV
DFGIEQGMQWPALLQALALRPGGPPSFRLTGVGPPQPDETDALQQVGWKLAQFAHTIRVDFQ
YRGLVAATLADLEPFMLQPEGEENDEEPEVIAVNSVFEMHRLLAQPGALEKVLGTVRAVRPK
IVTVVEQEANHNSGSFLDRFTQSLHYYSTMF
Figure A.6 Full coding region of the E. tef RHT gene. (I) represents a E. tef Rht near full
coding nucleotide sequence (1397 bp), (J) deduced amino acid residues of E. tef Rht sequence
(465 aa) with characteristic domains (bold and underlined): the DELLA motif, VHYNP and
VHVVD of the GRAS domain.
184
K
TAAGGCCCACGGCGAGGAGTGGGCGCGCCGCCGCAAGATCCTCACCCCCGCCTTCCACACCGAGAACC
TCAAGCTGCTGGTGCCGTTCGTCGGCGAGACGGTGCAGCGGATGCTGGAGGAGCGCGTGCTCTCGCCG
TCGGCGTCGGCGGCGAACGGCGGCGAGGTGGAGGTGGACGTCGCGGAGTGGTACCCGCGGCTGCCGCA
GGAGGCCATCACGCTCGCCACGTTCGGCCGGAACTACGCCGAGGGCAGCGTCGTGTTCCGGCTGCAGG
GCGAGCACGCCAGCCACGCCACGGTGGCGCACAGCAAGGTCTTCATCCCGGGGTACAGGTTCATCCCG
ACAAGGCGGAACCGGCGCGTGTGGCAGCTGGACAGGGAGATCAAGAGCACCCTGGCCAAGTTCGTCGT
CGCCCTGCAGAGCCGCGGCGGCGGCGGTGACCACCACCACCGCCGGGACGAGGGGCGAGCGGACGACG
GCTTGAGGGACTTCATGAGCTTCATGGCGCCGGCCATGACGGCGGACGAGATCATAGAGGAGTGCAAG
AACTTCTTCTTCGCCGGCAAGGAGACCCTGACCAGCCTCCTCACCTGGGCCACCGTCGCGCTCGCCAT
GCACCCGGAGTGGCAGGACCGCGCGCGCCGGGAGGTCGTCTCCGTCTGCGGCCACCGCGGCCTCCCGA
CGAGAGACCACCTTCCCAAGCTCAAGACCCTGGGGATGATCGTGAACGAGACGCTGCGGCTGTAATCG
A
L
KAHGEEWARRRKILTPAFHTENLKLLVPFVGETVQRMLEERVLSPSASAANGGEVEVDVAEWYPRLPQ
EAITLATFGRNYAEGSVVFRLQGEHASHATVAHSKVFIPGYRFIPTRRNRRVWQLDREIKSTLAKFVV
ALQSRGGGGDHHHRRDEGRADDGLRDFMSFMAPAMTADEIIEECKNFFFAGKETLTSLLTWATVALAM
HPEWQDRARREVVSVCGHRGLPTRDHLPKLKTLGMIVNETLRL
Figure A.7 Putative E. tef brassinosteroid deactivation related Cytochrome P450
monooxygenase gene partial sequence. (K) represents brassinosteroid deactivation gene
partial coding region (749 bp), (L) deduced amino acid residues of the sequence (247 aa).
185
KAHGEEWARRRKILTPAFHTENLKLLVPFVGETVQRMLEERVLSPSASAANG-GEVEVDVAEWYPRLPQEAITLATFGR-NYAEGSVVFRLQGEHASHAT
NL..D.............NA......A...AD...........L..S...G.S.......V...Q...K....V.....-.SD...A.....A....Y..
NL...R.....RV.........HRMIA...AG..T...D.---LAERAR.G.A..A.......FQ.V......F.A...R..DD.AA.....D.L.GY..
NL...R.....RV.........-----------------------------------......FQ.V......F.A...R..DD.AA.....D.L.GY..
SL..DK..LH.RV.V...YPD..NR...H..RS.AALA.R----WR.M.CASG..........FQAVAE....R.....-S..S.R....M..RLMAF.S
SL.DDK..LH.RV.....YPD..NR.A.H.ARS.VALA.R----WR.M.SAAG...........QAVAE....R....S-S.DS.R....M.ARLMAF.S
SLR.DK..H...V......M......L....R..VDVVDK----WHDM..AAS....I..S..FQVVTED...RTA...-S.ED.KA..K..TQLMAF.S
SLR.DK..H...V......M......L....R..VDVVDK----WHDM..AAS....I..S..FQVVTED...RTA...-S.ED.KA..K..TQLMAF.S
SLK..K..HH...I..T.YI...R.MI.MM.KSMKE..DK----W.KMSN-AS.K..IE.S.MFST.AEDV..RIV..N-S.ED.KAI.E..AQQMIY..
SLK..K..HH.R.IS.T..M......I.VMATS.VE...N----W.EMS--HK....IE.S.CFQT.TEDV..KTA..S-S.QD.KAI....AQQMVL.A
98
99
97
65
95
95
95
95
94
93
VAHSKVFIPGYRFIPTRRNRRVWQLDREIKSTLAKFVVALQSRGGGGDHHHRRDEG-RADDGLRDFMSFMA------------PAMTADEIIEECKNFFF
E............L...............RRL...L.AG...---.D..R..GRDP-..G-.M.N......------------...........S.....
E.....Y......L...K...........R.H.....TG...--CSSS.GDDA.D.GDGGG.M.E......------------.....G.....S.....
E.....Y......L...K...........R.H.....TG...--CSSS.GDDA.D.GDGGG.M.E......------------.....G.....S.....
E.FR..LV.....L..KK..MS.G.....RRG.VQLIGRRSD---AAEEREAEIKDKG---.F..LLGL.INARDKK-----SQP.PVE.MV....T...
E.FR...V.....L..KK..LQ.S.....RRG.VTLIGHRNDE--AAQDDDSEPNDKGSSN.F..LLGL.INASDKKKKQEEAR..PVEDML....T...
E.FR.........L..KK.TTS.K..K..RKN..TLIGRR.EA--AD.EKLSG--------CAK.LLGLLINAGSNG---GKVSPI.VND.V....T...
E.FR.........L..KK.TTS.K..K..RKN..TLIGRR.EA--AD.EKLSG--------CAK.LLGLLINAGSNG---GKVSPI.VND.V....T...
E.YQ.........L.SKK..IC.R..KQVRKS.M.LIEERRKK--EE-VLSEE--------CPN.LLEV.IKAGSDD---EYRNTI.VND.V....TI..
D.FQ.........F.....IKS.K..KQ..KS.V.LIERRREN--SN-ERIEK--------.PK.LLGL.IQASN-------KTNV.V.D.VG...S...
185
182
183
151
184
193
182
182
180
175
AGKETLTSLLTWATVALAMHPEWQDRARREVVSVCGHRGLPTRDHLPKLKTLGMIVNETLRL
..L...N.........................D...R..V..K....R.R....V.......
......SN....T...........E.......A...RGD...K............L......
......SN....T...........E.......A...RGD...K............L......
...Q.T.N.......L.....D..A...Q..LA...PGE...KE..H........L......
...Q.T.N.......L.....D..E...Q..LA...ADE..SKE...........L......
...Q.TSN....T..L........EL..Q..LQ...A.DI.S.EQ.T........L...--...Q.TSN....T..L........EL..Q..LQ...A.DI.S.EQ.T........L......
...H.TSN....T.IL.....K..EL..D..LT...A.DP.SKQQIS........I..SV-...Q.TSN....T.IL.....Q..VQ..D..LKM..S.DV..K..VV.....N.....S---
186
247
244
245
213
246
255
241
244
240
234
Identity (%)
100
50
52
50
52
50
51
42
42
48
E. tef P450 monooxygenase
S. bicolor
O. sativa CYP734A1/BAS1
O. sativa P450 monooxygenase
H. vulgare
Z. mays P450 monooxygenase CYP734A7
Z. mays
Z. mays P450 monooxygenase CYP734A8
S. lycoperiscum P450 monooxygenase
P. sativum P450 monooxygenase
Figure A.8 Amino acid sequence alignment of the putative brassinosteroid deactivating
gene sequences from E. tef with amino acid sequences from other species. sorghum
rice (O. Sativa; Acc No. EAY84935.1), barley (H. vulgare; Acc. No. BAK00002.1),
sorghum (S. bicolor; Acc No. XP_002453514.1), maize (Z. mays; Acc No.
ACG29333.1), pea (P. sativum; Acc No. BAF56240.1), and tomato (S. Lycoperiscum;
Acc. No. BAF02550.1). Identical and similar regions are shown by dark (100%) and
light shaded areas and dots whereas number indicates the position of the amino acid
within the predicted peptide.
187
Z. mays P450 monooxygenase CYP734A8
99
0.0
S. lycoperiscumCastasteron 26-Hydroxylase
P. sativum P450 Monooxygenase
100
H. vulgare subsp. Vulgare P450 monooxygenase
100
Z. mays cytochrome P450 CYP734A7
99
E. tef P450 monoxygenase
S. bicolor hypothetical protein
100
O. sativa CYP734A1/BAS1
0.1
Figure A.9 Molecular Phylogenetic analysis of putative E. tef brassinosteroid deactivation
gene sequence. The evolutionary history was inferred by using the Maximum Likelihood
method based on the JTT matrix-based (Jones et al., 1992). Branches corresponding to
partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage
of replicate trees in which the associated taxa clustered together in the bootstrap test (1000
replicates) are shown next to the branches. When the number of common sites was < 100
or less than one fourth of the total number of sites, the maximum parsimony method was
used; otherwise BIONJ method with MCL distance matrix was used. The tree is drawn to
scale, with branch lengths measured in the number of substitutions per site. There were a
total of 269 positions in the final dataset. Evolutionary analyses were conducted in
MEGA5 (Tamura et al., 2011).
188
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