INFORMATION TO USERS graph and reproduce this manuscript from the microfilm

INFORMATION TO USERS graph and reproduce this manuscript from the microfilm
INFORMATION TO USERS
The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm
master. UMI films the text directly from the original or
copy submitted. Thus, some thesis and dissertation copies
are in typewriter face, while others may be from any type
of computer printer.
The quality of this reproduction is dependent upon the
quality of the copy submitted. Broken or indistinct print,
colored or poor quality illustrations and photographs,
print bleedthrough, substandard margins, and improper
alignment can adversely affect reproduction.
In the unlikely event that the author did not send UMI a
complete manuscript and there are missing pages, these
will be noted. Also, if unauthorized copyright material
had to be removed, a note will indicate the deletion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the
upper left-hand corner and continuing from left to right in
equal sections with small overlaps. Each original is also
photographed in one exposure and is included in reduced
form at the back of the book. These are also available as
one exposure on a standard 35mm slide or as a 17" x 23"
black and white photographic print for an additional
charge.
Photographs included in the original manuscript have
been reproduced xerographically in this copy. Higher
quality 6" x 9" black and white photographic prints are
available for any photographs or illustrations appearing
in this copy for an additional charge. Contact UMI directly
to order.
UaM·I
University Microfilms International
A Bell & Howell Information Company
300 North Zeeb Road, Ann Arbor, M148106-1346 USA
313/761-4700 800/521-0600
Order Number 8915943
Comparison of free amino acid profiles in carrot cell suspension
cultures resistant to stress conditions
Alyousuf, Saeed Habib Hassan, Ph.D.
The University of Arizona, 1989
U·M·I
300 N. Zeeb Rd.
Ann Arbor, MI 48106
1
COMPARISON OF FREE AMINO ACID PROFILES IN CARROT CELL
SUSPENSION CULTURES RESISTANT TO STRESS CONDITIONS
by
Saeed Habib Hassan Alyousuf
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF PLANT SCIENCES
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
WITH A MAJOR IN HORTICULTURE
In the Graduate College
THE UNIVERSITY OF ARIZONA
1 989
2
THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have read
the dissertation prepared by __~S~a:=e~e~d~H~a~b~i~b~H~.~A~I~y~o~u~s~u~f________________
entitled
Comparison of Free Amino Acid Profiles in Carrot
Cell Suspension Cultures Resistant to Stress
Conditions
and recommend that it be accepted as fulfilling the dissertation requirement
for the Degree of
Doctor of Philosophy
----~~~~~-=-=-=-=~=-~------------------------------
Frank R. Katterman
,~
2/7/89
Date
2/7/89
Date
Date
2/7/89
Date
Date
Final approval and acceptance of this dissertation is contingent upon the
candidate's submission of the final copy of the dissertation to the Graduate
College.
I hereby certify that I have read this dissertation prepared under my
direction and recommend that it be accepted as fulfilling the dissertation
requirement.
2/7/89
Dissertation Director Frank R. Katterman
Date
3
STATEMENT OF AUTHOR
This dissertation has been submitted in partial
fulfillment of requirements for an advanced degree at The
University of Arizona and is deposited in the University
Library to be made available to borrowers under rules of the
Library.
Brief quotations from this dissertation are allowable
without special permission, provided that accurate acknowledgment of source is made.
Requests for permission for
extended quotation from or reproduction of this manuscript in
whole or in part may be granted by the head of the major
department or the Dean of the Graduate college when in his or
her judgment the proposed use of the material is in the
interests of scholarship.
In all other instances, however,
permission must be obtained from the author. ~
/
/
SIGNED:
/
/
/
/
----------~rM~~------------------
4
ACKNOWLEDGMENTS
I thank God, who created the universe and sustains
it.
I thank Him for bestowing upon mankind a mind that knows
right from wrong.
I thank Him for entrusting the human race
with the secrets of His creation.
I plead with Him to guide
me to the truth and help me bear it.
I
thank my advisor, Dr.
F.
R.
instructions, patience and cooperation.
Katterman,
for his
I am thankful to Dr.
P. G. Bartels, Dr. J. W. Moon, Dr. D. T. Ray, and Dr. A. K.
Dobrenz for being on my committee, and for
their .valuable
instructions, comments and suggestions.
I am grateful to Anna M. Hocker and Dr. Zimmerman for
providing the cell cultures.
I am grateful to Dr. Bobby L.
Reid and his coworkers for the amino acid analysis.
I am
also indebted to my colleagues, A. M. Albahrany, N. Y. Slail,
and
Q.
Dr. R.
Fu for their cooperation and company.
E.
I also thank
Biggs for his support and advice during the most
critical moments of my program.
5
TABLE OF CONTENTS
page
LIST OF TABLES
7
LIST OF ILLUSTRATIONS .
9
ABSTRACT
10
INTRODUCTION
12
LITERATURE REVIEW
14
Plant Cell Resistance to Amino Acid Analogs .
Resistance to Tryptophan Analogs. . . .
Resistance to Proline Analogs. . . . . . .
Resistance to More Than One Amino Acid
An a 1 og. . . . . . . . . . . . . . . .
Carrot and Other Plant Cell Line's
Resistance to NaCl. . . . . . . . . .
Crosspathway Regulation of Amino Acid
Biosynthesis. . . . .
........
MATERIALS AND METHODS . . .
16
17
18
20
22
Media composition and Preparation . . . . .
Initiation and Maintenance of Suspension
Cultures . . . . . . . . . . . . . . . . .
Isolation of Resistant cell Lines . . . • . . .
Tr ea tmen ts. . . . . . . . . . . . . . . .
Growth and Amino Acid Characterization of
Cell Lines for Different Treatments .
RESULTS .
Growth of the Suspension Cultures.
pyruvate Family of Amino Acids.
G1 uta mat e Fa mil y 0 f Am in 0 Ac ids
Aspartate Family of Amino Acids
Se r i n e Fa mil y 0 f Am i no Ac ids. .
Aromatic Family of Amino Acids.
14
14
22
23
23
24
25
31
. . . . ..
....
. . . .
. . . . ..
. . . . ..
31
31
41
47
55
58
6
TABLE OF CONTENTS--Continued
Page
Summary of Results..
.........
SNA Cell Line . . . .
.......
SNT Cell Line . . . .
. . ..
SNAT Cell Line. . . . . . . .
....
AD Treatment of SNAT Cell Line. . . . .
TD Treatment of the SNAT Cell Line.
TAD Treatment of the SNAT Cell Line
TAL Treatment of the SNAT Cell Line
ND Treatment of the SNAT Cell Line.
Free Amino Acid Trends During the Growth
Pe r i od. .
. ..
...
DISCUSSION
Growth of the Cultures. . . . . . . . . . .
Cellular Free Amino Acids . . . . . . .
Levels, Fluctuation, and Response to
Growth Periods. . . . . . . . .
...
Response to Light and NaCI Treatments .
Response to Analog Treatment and Cell
Line Resistance Characteristics .
REFERENCES
60
62
62
62
67
67
67
72
72
72
75
75
75
75
76
77
86
7
LIST OF TABLES
page
Table
1.
2•
3•
4.
5.
6.
7•
8.
9.
10.
11.
The effect of treatment and cell line combinations and growth period on the growth of
the suspension cultures
......... .
32
The effect of treatment and strain combination
and growth period on alanine concentrations
in carrot cell suspension cultures . . . . . . .
33
The effect of treatment and strain combination
and growth period on valine concentrati~ns
in carrot cell suspension cultures . .
35
The effect of treatment and strain combination
and growth period on leucine concentrations
in carrot cell suspension cultures . .
37
The effect of treatment and strain combination
and growth period on isoleucine concentrations
in carrot cell suspension cultures . .
39
The effect of treatment and strain combination
and growth period on glutamate concentrations
in carrot cell suspension cultures . .
42
The effect of treatment and strain combination
and growth period on proline concentrations
in carrot cell suspension cultures . .
44
The effect of treatment and strain combination
and growth period on arginine concentrations
in carrot cell suspension cultures . .
46
The effect of treatment and strain combination
and growth period on aspartate concentrations
in carrot cell suspension cultures. .
. ..
48
The effect of treatment and strain combination
and growth period on threonine concentrations
in carrot cell suspension cultures . .
50
The effect of treatment and strain combination
and growth period on methionine concentrations
in carrot cell suspension cultures . .
52
8
LIST OF TABLES--Continued
page
Table
12.
13.
14.
15.
16.
17.
18.
19.
The effect of treatment and strain combination
and growth period on lysine concentrations
in carrot cell suspension cultures . . . . . . .
53
The effect of treatment and strain combination
and growth period on serine concentrations
in carrot cell suspension cultures . . . . . . .
56
The effect of treatment and strain combination
and growth period on glycine concentrations
in carrot 'cell suspension cultures . . . . . . .
57
The effect of treatment and strain combination
and growth period on phenylalanine concentrations in carrot cell suspension cultures . . . .
59
The effect of treatment and strain combination
and growth period on tryptophan concentrations
in carrot cell suspension cultures . . . . . .
62
Free amino acid concentrations in carrot cell
lines resistant to amino acid analogs, as
compared to that of the control cell line . . .
63
Free amino acid concentrations resulting from
the treatment of SNAT cell line (resistant
to both A2C and 5-MT) with amino acid analogs,
compared to its treatment with plain medium
68
Free amino acid trends during three growth
periods . . . . . . . . . . . . . . . . . .
73
9
LIST OF ILLUSTRATIONS
Figure
1.
2•
3•
4.
5.
6.
page
Free amino acids from different amino acid
families affected in SNA cell line (resistant
to A2C), grown in darkness and treated with
A2C (AD); compared to DL cell line (control),
grown in darkness and treated with plain
medium (PD) . . . . . . . . . . . . . . . . . .
64
Free amino acids from different amino acid
families affected in SNT cell line (resistant
to 5-MT), grown in darkness and treated with
5-MT); compared to DL cell line.(control),
grown in darkness and treated with plain
medium (PD) . . . . . . . . . . . . . . . .
65
Free amino acids from different amino acid
families affected in SNAT cell line (resistant
to A2C + 5-MT), grown in darkness and treated
with A2C and 5-MT (TAD); compared to DL cell
line (control), grown in darkness and treated
with plain medium (PD) . . . . . . . . . . .
66
Free amino acids affected in SNAT cell line
(resistant to A2C + 5-MT), subjected to AD
treatment (grown in darkness and treated
with.A2C), compared to its treatment with
darkness and plain medium (PD) . . . . . . .
69
Free amino acids affected in SNAT cell line
(resistant to A2C + 5-MT), subjected to TD
treatment (grown in darkness and treated with
5-MT), compared to its treatment with darkness
and plain medium (PD) . . . . . . . . . . .
70
Free amino acids from different amino acid
families affected in SNAT cell line (resistant
to A2C + 5-MT), subjected to TAD treatment
(grown in darkness and treated with A2C +
5-MT), compared to its treatment with plain
medium and darkness (PD) . . . . . . . . . .
71
10
ABSTRACT
Plant cells resistant to specific amino acid analogs
have been reported to accumulate the corresponding free amino
acids.
The purpose of
this study was to determine the
concentrations of fifteen free amino acids:
alanine, valine,
leucine, isoleucine, glutamate, proline, arginine, aspartate,
threonine, methionine,
lysine, serine, glycine, tryptophan
and phenylalanine in Daucus carota cell lines, resistant
either to the proline analog azetidine-2 carboxylic acid
(A2C), or to the tryptophan analog 5-methyltryptophan (5-MT),
or to both the analogs combined.
This study also intended
to determine if these analogs influence the biosynthesis of
the above-mentioned fifteen amino acids in the cell line
resistant to A2C and 5-MT.
Carrot cell lines resistant to 5-MT, to A2C, or to
both the analogs were selected by incubating carrot cells in
liquid growth media containing either 0.3 mM 5-MT, or 0.5 mM
A2C for 6 to 16 weeks.
Free amino acid concentrations were
then determined in the extracts of the cells.
Resistance to 5-MT resulted in significant increases
in the intracellular concentrations of tryptophan, phenylalanine, leucine,
valine,
isoleucine, and proline.
Resistance
to A2C resulted in significant increase in proline only.
11
Resistance to both the analogs caused increases in proline,
lysine, phenylalanine, and tryptophan concentrations.
In the cell line resistant to both the analogs, the
treatment with 5-MT caused increases in leucine,
aspartate, threonine, lysine, and tryptophan.
proline,
The treatment
with A2C caused increases in isoleucine, arginine, threonine,
methionine, lysine, and glycine, whereas treatment with both
the
analog~
caused increases in threonine,
lysine, phenyl-
alanine, and tryptophan.
These results indicate the possibility of a common
biosynthetic control of a number of amino acids in carrot
cells, resembling that found in microorganisms.
It is also
evident from the results that the analogs play an active role
in the biosynthesis of amino acids in the resistant cell
lines.
12
INTRODUCTION
Amino
acid
biosynthesis
in
microorganisms
regulated by both specific control mechanisms
is
(controls
the regulation of a single enzyme) and general control mechanisms
(controls the biosynthesis of a number of regulatory
enzymes), in addition to feedback inhibition.
General and
specific control mechanisms seem to work through repression
and derepression of
1982).
biosyn~hetic
enzymes
(Jones and Fink
In higher plants, biosynthesis of amino acids is
regulated through feedback inhibition of the control enzymes
(Gilchrist and Kosuge 1980).
Through
the
induction of
regulatory changes in
microorganisms and plant cells, it is possible to affect the
biosynthetic activity of certain amino acids significantly.
The overproduction of essential amino acids by plant cells
could have. commercial feasibility.
If these traits are
stable enough, plants regenerated from variant cells may
also accumulate higher amounts of the target amino acids.
Furthermore, the regulatory links between biosynthesis of
different amino acids can also be studied.
Carrot cells
resistant to tryptophan analog, 5-MT, accumulate high amounts
of free tryptophan (Widholm 1977).
Yeast cells, starved for
tryptophan, derepress the biosynthetic enzymes for histidine
13
and arginine (Carsiotis et al.
1974a).
The biosynthetic
pathways for aromatic amino acids in plants and microorganisms appear
to be similar
Biosynthetic pathways
for
family amino acids, also,
(Gilchrist and Kosuge 1980).
aspartate family and pyruvate
in plants are similar to those in.
microorganisms (Bryan 1980).
Previous studies on plant cells focused on the
biosynthesis of a single amino acid,
corresponding to the
analog used for development of the resistant characteristics.
Furthermore, the effect of the analog on the biosynthetic
activity of the cell was not investigated.
The main objective of this study is to determine if
the biosynthetic regulation of other than the target amino
acids are affected as a result of resistance to the tryptophan analog (5-MT) or the proline analog (A2C).
In addition,
the effect of these analogs on the biosynthesis of different
amino acids, in the cell line resistant to both the analogs,
is evaluated.
Sodium chloride and light treatments were used
to contrast the effect of the treatment with the two amino
acid analogs in the dual resistant cell line.
analyzed using a statistical design.
Results are
14
LITERATURE REVIEW
Plant Cell Resistance to Amino Acid Analogs
Resistance to Tryptophan Analogs
Nicotiana tabacum and Daucus carota cell growth is
reported to be inhibited by a number of tryptophan analogs.
Different concentrations of 5-methyltryptophan, 4-methyltryptophan,
5-fluorotryptophan,
droxtryptophan,
6-fluorotryptophan,
5-hy-
3-beta-indole acrylic acid, 7-azatryptophan,
and 3-methyl anthranilic acid were found to
inhibit the
growth of carrot and tobacco cells in suspension,cultures.
Growth inhibition by some of the analogs could be reversed
by the addition of either anthranilic acid,
tryptophan.
indole, or L-
With the exception of 3-methyl anthranilic acid,
all the analogs seem to inhibit anthranilate synthetase,
thereby blocking tryptophan biosynthesis.
This blockage
results in the cessation of cell growth (Widholm 1972a).
Carrot cells resistant to growth inhibition by DL-5methyl tryptophan have been selected by continuous shaking
for a period of 30 to 60 days in medium containing 220 uM of
the analog.
These cells are observed to contain a different
form of anthranilate synthetase (AS) which is less susceptible to feedback
inhibition by 5-methyltryptophan (5-MT).
While AS from non-resistant cells is generally inhibited by
15
5.4
uM 5-MT,
it takes
five times this concentration to
inhibit AS from 5-MT resistant cells.
Low sensitivity of
this enzyme to feedback inhibition by 5-MT and tryptophan
causes the resistant cells to accumulate considerably higher
concentrations of free tryptophan (Widholm 1972b).
Tobacco cells are also found to contain two different
forms of AS, one of which is more abundant in 5-MT resistant
cells and is located within the particulates.
The trp-
sensitive form, on the other hand, is found in the cytosol.
Plants regenerated from both types of cells,
resistant and
susceptible to 5-MT, contain only the trp-sensitive form of
AS (Brotherton et. al. 1986).
5-MT resistant carrot cells have significantly higher
concentrations of free tryptophan (2170 nmoles/gram fro
compared to normal cells (81 nmoles/gram fro
1977).
wt.)
wt.)
(Widholm
A 5-MT resistant wild carrot cell line, producing
higher amounts of tryptophan, also contains high concentrations of indole-3-acetic acid.
As a result,
this line is
auxin habituated, has the 5-MT resistant form of AS and a low
regeneration potential (Sung 1979).
In some of the carrot and tobacco cell lines, resistance to 5-MT can probably be attributed to reduced uptake of
the analog (Widholm 1974a).
Cell lines from carrot plants,
regenerated from cells resistant to 5-MT, are also found to
be resistant to 5-MT.
As no resistant form of AS is found in
16
these cells, resistance to 5-MT in this case can also be the
result of reduced uptake of the analog.
These cells are also
observed to overproduce tryptophan (Widholm 1971b).
Resistance to Proline Analogs
Growth of carrot cells is reported to be completely
inhibited by 0.1 mM azetidine-2-carboxylic acid (A2C).
This
growth inhibition can be prevented by the addition of proline
to the cultures.
Carrot cells treated with the mutagen
ethylmethane sulfonate and selected for resistance to hydroxproline are found to have several times greater resistance to
the analog.
These cultures are reported to retain their
resistance when grown in the absence of the analog and accumulate proline considerably higher than normal cells (Widholm
1976).
Carrot cells resistant to A2C also overproduce pro-
line and are more resistant to NaCl (Riccardi et. ale 1983).
The proline tRNA in A2C resistant carrot cells binds
to A2C,
which competes with proline binding just as in the
normal cells (Nielsen et ale 1980).
A2C resistant carrot
cell lines accumulate considerably higher amounts of free
intracellular serine and alanine along with proline, which is
also excreted into the medium at elevated levels.
The rate
of A2C uptake by normal cells is ten times greater than the
resistant cells, which do not incorporate this analog into
their proteins as normal cells do.
Reduced uptake of A2C by
17
resistant cells is probably due to high proline concentrations in the media.
The overproduction of free proline in
these cells can probably be explained by a mutation resulting
in altered amino acid carriers (Cella et.
al.
1982).
A
comparison between Oryza sativa cells (resistant to A2C) and
carrot cells (susceptible to A2C) for uptake and incorporation into protein of A2C, and for intracellular free proline
levels did not indicate a significant difference among these
two cultures.
However, A2C was found to inhibit the bio-
synthesis of proline from glutamate in carrot and not in
rice cell cultures.
This finding probably indicates the
existence of an effective feedback
inhibition system for
proline in carrot cells as opposed to rice (Nielsen et al.
1986).
Resistance to More Than
One Amino Acid Analog
A carrot cell line l
resistance selection
to
when subjected sequentially for
analogs of alanine
(p-fluoro-
phenylalanine), methionine (ethionine), cysteine (aminoethylcysteine) and tryptophan
resistance
to all
the
(5-MT),
is reported to exhibit".
analogs simultaneously and also
accumulates the corresponding amino acids (Widholm 1978).
Some of the carrot cell lines, when treated with UV
light and selected for resistance to 5-MT, were also found to
be resistant to A2C as well.
These cell lines differed among
18
themselves in the amount of resistance to the two analogs and
also in the accumulation of the corresponding amino acids.
All the cell lines resistant to 5-MT were also observed to
accumulate large concentrations of free proline (Cella and
Iadarola 1983).
Strains resistant to 5-MT and A2C accumulate
higher amounts of free proline than those resistant to A2C
only (Riccardi et al. 1983).
Carrot and Other Plant Cell Line's
Resistance to NaCI
Sea water at concentrations of 10 to 20% (v/v) is
observed to decrease carrot seedling callus growth substantially.
At 30% sea water affects the green color of the
callus, while at 40% it inhibits growth completely.
NaCI
also affects callus growth and color like sea water, but
mannitol with identical osmotic pressures is less inhibitory
to growth and has no effect on the green color.
Therefore,
it is inferred that while osmotic pressures do affect growth,
toxicity by specific ions affects both;
i.e., growth and
chlorophyll development (Goldner et al. 1977).
Osmotic pressures generated by 555 mM mannitol causes
a 50% inhibition of growth of carrot cell suspension cUltures.
The same amount of growth inhibition also results
from 85 mM NaCI in
adapted,
these cultures.
Cells osmotically
using mannitol as osmoticum, are more resistant to
growth inhibition by iso-osmotic medium containing NaCI.
19
instead of mannitol.
In this case 50% growth inhibition
occurs at an NaCl concentration of 127 mM (Harms and Oertli
1985).
Tobacco cells, adapted to NaCl,
are reported to
undergo a substantive osmotic regulation in order to maintain
turgor.
But these cells do not exhibit any expansion or gain
in fresh weight in proportion to their turgor, which could
probably be attributed to alterations in the properties of
the cell wall (Binzel et ale 1985).
Tobacco cells, both resistant and sensitive to NaCl,
accumulate significantly higher
~mounts
of proline in the
presence of NaCl when compared to its absence in the medium.
Salt sensitive cells, however, in spite of their failure to
grow in 1.5% NaCl medium during ten days of culture, continued to accumulate considerably larger amounts of free proline
than the
resistant cells.
,
This probably indicates that free
proline accumulates due to stress and not as a sign of
resistance to NaCl (Dix and Pearce 1981).
Proline accumulating, A2C resistant strains of Spirulina platensis and D.
carota cells are observed to exhibit
sodium chloride tolerance.
proline in S.
However,
the accumulation of
platensis is correlated more to the degree of
salt tolerance than in D. carota (Riccardi et ale 1983).
20
crosspathway Regulation of
Amino Acid Biosynthesis
In bacteria and fungi, along with specific control,
general control of amino acid biosynthesis has been observed.
However, in plants no such observations have been reported.
In this section some examples of general control or crosspathway control regulation in both prokaryotes and eukaryotes
will be reviewed.
Activity of
the enzyme prephanate dehydrogenase
(involved in tyrosine biosynthesis) in the Braditrophic leaky
mutant of Bacillus subtilis has been found to be inhibited by
histidine and phenylalanine.
The growth of this bacterium is
inhibited by 10- 4 M histidine.
However, histidine resistant
strains overproduce tyrosine and their
enzymes are
repressed by aromatic amino acids (Nester 1968).
not
B. subtilis
mutants resistant to histidine or tyrosine analogs are found
to cdntain elevated levels of enzymes involved in the biosynthesis of both amino acids.
Due to confinement of the genes,
involved in resistance to both analogs to one locus, the
common control of both biosynthetic pathways may be attributed to the activity of an aporepressar (Nester et ale 1974).
The gene coding for a transaminase,
involved in the biosyn-
thesis of histidine and aromatic acids,
is found
to be
located within a gene cluster coding for
the synthesis of
aromatic amino acid biosynthetic enzymes.
This indicates the
21
possibility of crosspathway regulation between these two
biosynthetic pathways (Nester and Montoya 1976).
Starvation for histidine in the yeast Saccharomyces
cerevisiae results in derepression of a number of enzymes
involved in histidine,
biosynthesis.
arginine,
tryptophan,
and lysine
A short noncoding, repeated DNA sequence and a
protein coded by another gene are probably implicated in the
general cODtrol of these amino acids (Fink et ale 1983).
In Aspergillus nidulans, starvation for either arginine,
histidine,
proline or tryptophan derepresses
biosynthesis of ornithine carbamoyl transferase (OCT).
the
Star-
vation for either lysine, leucine or phenyl alanine does not
seem to affect the OCT activity (Piotrowska 1980).
In spite of high levels of histidine and arginine in
the cells,
tryptophan or histidine mutants of Neurospora
crassa depresses tryptophan, histidine and arginine biosynthetic enzymes when starved for either tryptophan (Carsiotis
and Jones 1974a) or histidine (Carsiotis et al. 1974b).
A
number of N.
crassa arginine auxotrophs, mutants at locus
CPC-l+,
to derepress arginine,
fail
histidine and lysine
biosynthetic enzymes when deprived of any of these amino
acids (Barthelmess 1982).
22
MATERIALS AND METHODS
Media Composition and preparation
In all of the cultures, modifications of revised MS
medium (Murashige and Skoog 1962) were used and prepared in
the following way:
A.
MS major salts and minor salts,
(0.0005 mM/l),
sucrose, and NaCl
Fe/EDTA,
2,4-D,
(used only in NaCl
treatments) were dissolved in double distilled water
and sterilized by autoclaving for
twenty min after
adjusting the pH to 5.7.
B.
MS vitamins and kinetin (0.0005 mM/l) were filtersterilized.
C.
65.49 mg 5-MT (5methyl-DL-tryptophan), was dissolved
in 2 ml 0.5 N HCl, filter-sterilized, pH adjusted to
5.7, and used per liter of the 5-MT medium (0.3 mM).
D.
50.·55 mg A2C
(L-azetidine-2-carboxylic acid)
dissolved in 2 ml of water,
filter-sterilized,
was
and
used per liter of A2C medium (0.5 mM).
For preparing plain medium (without 5-MT, A2C and
NaCl), components A (without NaCl) and B only were used.
5-MT medium,
components A,
medium, components A,
B,
B, and C were used.
and D were used.
medium, components A and B were used.
For
For A2C
For 0.5% NaCl
23
Initiation and Maintenance of
Suspension Cultures
Callus was initiated from carrot root explants on
callus induction medium composed of MS salts and vitamins,
2% sucrose, 0.005 mM/I 2,4-D, 0.0005 mM/l kinetin and agar
0.8%
(a modification of Tisserat and Murashige 1977).
Th~
callus was subcultured a number of times before it was
transferred to the liquid medium; hence callus existed as
about one gram aggregates, spherical, solid at the core and
friable outward.
One to two grams of callus was used as
inoculum per 50 ml of medium in 125 ml Erlenmeyer flasks.
The cultures were maintained on a rotary shaker at a speed of
100 rpm.
Initially they were subcultured only after exhi-
biting significant amount of growth, but later the transfers
were made about every 2 weeks.
Fine suspension cultures were
developed by avoiding the transfer of large aggregates.
Size
of the inoculum used for subculture was 10 ml per 25 ml of
medium in 125 ml Erlenmeyer flasks.
Isolation of Resistant Cell Lines
For the isolation of cell lines resistant to inhibitory concentrations of either of the two analogs (5-MT or
A2C), the following method (Widholm 1972b) was used:
about
2 gm of the actively growing normal carrot cells were transfer red to 50 ml of either 5-MT or A2C medium.
The resulting
suspension cultures were maintained in a temperature of 25 to
24
28 0 C,
100 lux of fluorescent light, and on a rotary shaker
set at the speed of 100 rpm.
Once these cultures exhibited a
significant amount of growth (after 1.5 to 4 months) they
were transferred to the new medium.
Cells resistant to both
5-MT and A2C were produced from A2C resistant cells by
implementing the above mentioned procedure.
Because of
contamination of our control cell line cultures (ScarletNantes), Danvers Half Long
Baltimore) was used instead.
(provided by Dr.
Zimmerman of
The following four cell lines
were used for the study:
1.
Danvers Half Long (DL) was used as the control cell
line.
2.
Scarlet Nantes resistant to 5-MT (SNT).
3.
Scarlet Nantes resistant to A2C (SNA).
4.
Scarlet Nantes resistant to 5-MT + A2C (SNAT).
Treatments
1.
Plain medium and darkness (PD), used for DL and SNAT
cell lines.
2.
5-MT medium and darkness (TD), used for SNT and SNAT
cell lines.
3.
A2C medium and dark (AD), used for SNA and SNAT cell
lines.
4.
5-MT + A2C medium and darkness (TAD), used for SNAT
only.
25
5.5-MT + A2C medium and fluorescent light intensity
of
300 to 500 lux (TAL), used for SNAT cell line only.
6.
0 . 5 % Na C'l me diu man d dar k n e s s
(N D),
use d for S NAT
only.
SNAT cell line cultures were subcultured 4 times in
the growth medium,
in all cases of amino acid analog and
plain medium treatments, before being used for the experiment.
This was done to insure that the cells were totally
under the influence of the medium at the time of experiment.
Growth and Amino Acid Characterization of
Cell Lines for Different Treatments
One week before commencement of the characterization
study for any of the treatments, stock cultures were prepared
in 250 ml Erlenmeyer flasks by dispensing 25 ml of cell
culture in 55 ml of medium (80 ml/flask).
On the seventh day
of the transfer, stock cultures were used in the experiment
by dispensing 10 ml aliquots per 25 ml medium in 125 ml
Erlenmeyer flasks.
Autoclaved glass tiltapets
(including
500 ml container and 10 ml automatic pipetter) were used for
inoculation to insure sterility of cultures and relative
uniformity of the inoculum.
Three growth periods were
designated tor harvesting the cultures, as follows:
1.
First harvest:
2.
Second harvest:
3.
Third harvest:
about two hours after inoculation.
four days after inoculation.
eight days after inoculation.
26
Temperature throughout the experiment was in the
range of 25 to 28 0c and the rotary shaker speed was adjusted
to 100 rpm.
All
treatments were conducted in darkness
(except TAL treatment of the SNAT cell line).
Cells were harvested using sieves of 170 mesh (pore
size <80 microns).
The medium trapped among the cells was
blotted out through the sieves using paper towels.
weight of
~ells
Fresh
was determined by the difference between the
weights of the sieves, with and without the cells.
Half a gram of the freshly harvested cells from each
replication was used to extract free amino acids according to
the following method (Bieleski and Turner 1966).
The cells
were ground with a similar amount of sea sand and 5 ml of
cold MCW (12 ml methanol:5 ml chloroform:3 ml water), which
was added intermittently while grinding with porcelain pestle
and mortar.
The mixture was centrifuged at 1460 g for 5 min.
The supernatant was saved and the pellet ground again with
4 ml of cold MCW, centrifuged at 1460 g for 5 min, and the
supernatant added to the previous one.
supernatant
~as
The accumulated
brought up to a known volume with MCW,
treated with similar amounts of chloroform and double distilled (d.d.) water, centrifuged at 4340 g for 10 to 30 min
(till all the visible particles were packed in a thin layer
between the extract and the chloroform), then the extract
(top layer) was separated from the rest, brought up to a
27
known volume with d.d.
determination.
water,
Absorbance of
and saved for
the extract
amino acid
from
the
two
replicates was determined at 280 nm, and if the difference
in absorbance between the two replications was found to be
greater than 10%, the extraction was repeated.
Cell extracts from different treatments were used to
determine the concentration of fifteen free amino acids in
micromoles/gram fresh weight (uM/gm fro wt.).
The concentra-
tion for 13 of them was determined chromatographically.
The
HPLC Spectraphysics SP8000A,. equipped with a Beckman detector
and Microsorb raisin column,
was used to determine the
concentrations of aspartate, glutamate, serine, glycine,
threonine, arginine, alanine, methionine, valine, phenylalanine, isoleucine, leucine, and lysine.
Proline concentrations in cell extracts were determined colorimetrically (Bates 1973).
In this case,
acid
ninhydrin reagent was prepared by dissolving 1.25 gm of
ninhydrin in a solution containing 20 ml of 6M phosphoric
acid and 30 ml of acetic acid.
Then equal proportions of the
sample, acetic acid, and reagent were mixed well and heated
in boiling water for 1 hr, thereby developing reddish brown
color.
The preparations were then cooled to room temperature
and absorbance determined at 520 nm.
Proline concentrations
were then estimated from a standard curve, prepared by using
different concentrations of proline.
28
Tryptophan concentrations were also determined
colorimetrically (Dalby and Tsai 1975).
The reagent was
prepared by mixing equal amounts of acetic acid (containing
about 8% (v/v)
acetic anhydride and 270 mg/l FeC13) and 30N
sulfuric acid.
0.5 ml of the sample was then mixed with 2 ml
of the reagent and incubated at 60
pink color.
°c for 15 min to develop
The preparation was then cooled to room tempera-
ture and absorbance measured at 575 nm.
Concentrations were
then calculated from a standard curve.
The results for free amino acid determinations are
presented in 15 tables.
Each table contains the values
determined for a single amino acid obtained from four different cell lines, subjected to different treatments.
The
values in each cell of the main body of the table is the mean
for two replications (except those for the second harvest of
the PD treatment of SNAT cell line are of one replication
only).
The 15 tables of the results are divided among five
amino acid families,
according to Miflin and Lea (1982),
which are presented in alphabetical order.
The
results
for
all
the amino acids have been
statistically analyzed using a completely randomized design
with two factors.
One of the two factors with 9 levels was
used for the amino acid analog, plain medium, light and NaCl
treatments to the 4 cell lines.
The other factor was used
for the growth periods at which the cultures were harvested.
29
Statistical tests were carried out at the 5% significance
level and the LSD (least significant difference) method was
used for mean separation.
The AVMF program of the MSUSTAT
was used for the statistical analysis.
The
results
in each table are
individually in three paragraphs.
first
explained
The first paragraph deals
with the main parameters and trends.
The second paragraph
attempts to compare between the four cell lines using DL as
the reference line, whereas the treatment TAD of SNAT is used
as a reference for this cell line comparison for all treatments applied to it.
TAD treatment
The reason for this choice is that the
is the medium to which this cell line is
adapted, as is the case with the specific treatments for the
other three cell lines.
The use of DL as the control cell line (due to last
minute contamination of the Scarlet Nantes control cultures)
is that the· distribution of free amino acids in this cell
line is not significantly different than those in the Scarlet
Nantes line.
advisor, Dr.
This assumption is based on the opinion of my
Frank Katterman, and Dr. J.
M. Widholm of the
University of Illinois, Urbana (personal communication with
Dr. Katterman, 1988).
The third paragraph attempts the comparison between
the different treatments of the SNAT cell line using PO as
the reference treatment for all treatments except TAL, which
30
is compared with TAD (to determine the effect of light on
this cell line).
Finally, a summary for the analysis of all
the results is presented.
During the presentation of the results the term cell
value is used for the means of two replications presented in
the table.
The term analog is used to mean amino acid
analog, whereas the term amino acid is used to mean the free
amino acid under consideration, unless stated otherwise.
31
RESULTS
Growth of The Suspension Cultures
Comparison between the cell lines DL, SNA, SNT and
SNAT (TAD treatment) indicates that at the third harvest the
increase in weight for DL and SNT is about the same, for SNAT
it is intermediate, and for SNA it is the lowest (Table 1).
Comparison between different treatments of SNAT at
the
third harvest
shows
that TAL recorded the highest
percentage .increase, ND the lowest, TD almost equal to PD,
TAD a little higher than the latter, and AD slightly lower
than PD.
pyruvate Family of Amino Acids
Alanine.
fr.
wt.
Alanine concentrations are above 1 uM/gm
in all the treatments (Table 2).
value is 6.999 uM/gm fr.
1.589 uM/gm fro wt.
wt.
The highest mean
(PD and DL) and the lowest is
(TD and SNT).
Concentrations fluctuate
up to 3.4 times, the lowest per cell value.
The general
trend indicates that the mean value does not change much
between the first and second harvests but drops by the
thir~.
Comparison between the four cell lines indicates that
alanine levels in all three resistant cell lines have been
adversely affected,
pronounced.
with the effect on SNT being most
32
Table 1.
The effect of treatment and cell line combinations
and growth period on the growth of the suspension
cultures (mg/ml).
The percent increase is based
on initial weight at 2 hours after inoculation.
Growth Period
1 Hour
4 Days
Strain a Treatment b Weight
Weight
AD
52.50 c
104.83
TD
48.33
TAD
Weight
Increase
99.68
132.50
52.38
85.50
76.91
136.00
181. 40
37.50
79.00
110.67
118.00
214.67
TAL
37.33
73.50
96.89
139.50
273.69
ND
35.00
43.50
24.29
57.33
63.80
PD
43.67
97.00
122.12
122.67
180.90
SNT
TD
56.50
93.33
65.19
194.67
244.55
SNA
AD
46.50
84.83
82.43
128.00
175.27
DL
PD
35.33
78.50
122.19
122.00
245.32
SNAT
Increase
8 Days
a Cell lines:
DL, control; SNA, resistant to A2C;
resistant to 5-MT; and SNAT, resistant to A2C + 5-MT.
SNT,
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and
darkness; TAL, 5-MT +A2C and light (300-500 lux); ND, NaCl
and darkness.
c Valves are means for 2 replications (except PD and SNAT at
4 days of growth period is one replication only).
33
Table 2.
The effect of treatment and strain combination and
growth period on alanine concentrations (uM/gm fro
wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fr. wt.)
Mean
AD
2.848 c
4.232
1.952
3.011 cd
TD
3.217
4.031
2.675
3.308 C
TAD
2.743
3.463
2.567
2.925 CB
TAL
2.663
3.014
1.653
2.443 B
ND
2.707
2.282
2.271
2.420 B
PD
2.66
3.627
2.157
2.652 CB
SNT
TD
1.664
1.589
2.38
1.877 A
SNA
AD
3.151
3.234
3.086
3.157 C
DL
PD
6.999
4.386
4.608
5.331 D
3.184 Bd
3.299 B
2.594 A
SNAT
MEAN
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and" SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
34
Comparison between SNAT treatments does not show a
significant effect for any of the treatments compared to the
PO treatment of this cell line.
Valine.
In all cases except two (second and third
harvests of TO and SNT), concentrations of valine are below
1.0 uM/gm fr.
wt.
1.106 uM/gm fro
uM/gm fr.
wt.
(Table 3).
wt.
The highest concentration is
(TO and SNT) and the lowest is 0.125
(TAL and SNAT).
The extent of fluctuation is
slightly below eight times the lowest concentration (i.e.,
7.8 times).
The .general trend indicates a slight increase in
valine level between the first and second harvests and its
substantial decrease at the third.
The TO and SNT combination exhibited the highest mean
value than any other combination.
In fact,
this combination
gave the highest values during the three harvests.
Compari-
son between the four cell lines OL, SNA, SNT, and SNAT shows
that TO and .SNT exhibited the highest mean value, AO and SNA
the lowest, whereas PO and OL showed a significantly higher
mean for valine concentration than both TAO and SNAT and AO
and SNA.
This would
indicate that
resistance
to
5-MT
resulted in a significant increase in valine content, whereas
resistance to A2C caused a significant drop.
Resistance to
both analogs, though showing higher a value than A2C resistant cell line,
control cell line.
resulted in lower valine level than the
35
Table 3.
The effect of treatment and strain combination and
growth period on valine concentrations ( UM/ gm f r .
wt. ) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fro wt. )
Mean
AD
0.533 c
0.615
0.384
0.511 Cd
TD
0.461
0.650
0.404
0.505 C
TAD
O. 373
o. 445
0.354
0.391 B
TAL
0.211
0.512
0.125
0.286 A
ND
0.348
0.228
0.228
0.268 A
PD
0.429
0.462
0.429
0.435 CB
SNT
TD
0.952
1.003
1.106
1.020 E
SNA
AD
0.307
0.271
0.217
0.265 A
DL
PD
0.851
0.790
0.767
0.802
0.497 ABd
0.558 B
0.446 A
SNAT
MEAN
D
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
b Treatments:
PD, plain medium and darkness; AD, A2C and
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
36
Comparison between different treatments of SNAT cell
line indicates that the presence of either one of the two
analogs in the medium does not affect valine levels significantly; but both analogs together cause its slight drop.
Light
NaCl also affect valine content significantly
an~
downward.
Leucine.
Leucine concentrations in all treatment and
cell 1 i n e comb ina t ion s we reb e 1 ow O. 5 u M/ g m f r. wt.
4).
( Ta b 1 e
The highest concentration is 0.405 uM/gm fro wt. (TD and
SNT) and the lowest 0.055 uM/gm fro wt.
(ND and SNAT).
The
extent of fluctuation is up to 6.4 times the lowest concentration.
The general trend does not show a substantial dif-
ference between mean values of the first and second harvests,
but at the third harvest the mean value drops considerably.
The comparison between the four cell lines DL, SNA,
SNT, and SNAT reveals a pattern identical to that of valine,
with TD and SNT giving the highest mean value of 0.349 uM/gm
fro wt., AD and SNA the lowest mean value of 0.083 uM/gm fro
wt., and TAD and SNAT's mean value of 0.162 uM/gm fro
higher than
fr.
~he
wt.,
AD and SNA mean, but lower than 0.231 uM/gm
wt., mean value for the normal cell line DL.
Here too
the 5-MT resistant cell line has exhibited a higher value and
the A2C resistant line a lower value.
Resistance to both
analogs also resulted in lower leucine values compared to the
normal cell line.
37
Table 4.
The effect of treatment and strain combination and
growth period on leucine concentrations (uM/gm fro
wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fro wt.)
Mean·
AD
0.174c
0.148
0.148
0.166 Bd
TD
o. 214
0.269
0.134
0.205 C
TAD
0.128
0.182
0.176
0.162 B
TAL
0.157
0.180
0.074
0.137 B
ND
0.105
0.055
0.059
0.073 A
PD
0.184
0.136
0.136
0.152 B
SNT
TD
O. 337
0.405
0.305
0.349 D
SNA
AD
0.101
0.092
0.056
0.083 A
DL
PD
0.248
0.224
0.221
0.231 C
0.183 Bd
0.191 B
0.145 A
SNAT
Mean
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
b Treatments:
PD, plain medium and darkness; AD, A2C and
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
38
Different treatments of the SNAT cell line compared
to its PD treatment show that the presence of 5-MT in the
medium causes a significant increase in leucine level, but
A2C alone, or with 5-MT in the medium does not significantly
affect the level of leucine.
Light and NaCl treatments of
SNAT had a negative trend on amino acid levels.
Isoleucine.
Like leucine, isoleucine concentrations
are also below 0.5 uM/gm fro wt. for any treatment and cell
line combination at any harvest (Table 5).
centration is 0.464 uM/gm
fr~
The highest con-
wt. (TD and SNT) and the lowest
0.0421 uM/gm fro wt. (ND and SNAT).
Fluctuation is up to ten
times the lowest concentration.
There is no significant
difference between overall mean values at the three harvests,
indicating relative stability of the cell's
isoleucine
content during this period of the growth curve.
Treatment and cell line combination mean values show
that as in leucine, TD and SNT has the highest mean value,
but unlike leucine, values for AD and SNA and TAD and SNAT
are not significantly different from one another, but both
are significantly lower than
th~
could be interpreted as follows:
control (PD and DL).
This
changes in resistance,
which in turn gave the second highest concentration as in
leucine and valine, characteristics of the cells (i.e., from
39
Table 5.
The effect of treatment and strain combination and
growth period on isoleucine concentrations (uM/gm
fro wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fro wt.)
Mean
AD
0.147c
O. 214
0.139
0.166 Da
TD
0.134
0.140
0.119
0.131 C
TAD
0.070
0.073
0.107
0.083 AB
TAL
0.119
0.125
0.051
0.098 BC
ND
0.078
0.045
0.042
0.055 A
PD
0.108
0.128
0.083
0.106 BC
SNT
TD
0.351
0.394
0.464
0.403
SNA
AD
0.090
0.077
0.064
0.077 AB
DL
PD
O. 214
0.197
0.221
0.211 E
0.146 Ad
0.154 A
0.143 A
SNAT
Mean
F
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 ~ux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
40
resistance to A2C only to A2C and 5-MT both) did not significantly affect their status for isoleucine concentration as
it did in the case of valine and leucine.
Variation between different treatments of the SNAT
cell line shows that AD treatment has significantly higher
mean value than that of PD treatment.
But unlike what was
found in leucine, the isoleucine AD treatment has a significantly higher mean value than that of TD.
By examining the
mean values of the TAD, TAL and PD treatments, it appears as
if the presence or absence of both analogs from the medium,
or the treatment with light do not affect isoleucine content
of the cells significantly.
ND and SNAT has the lowest
isoleucine mean value of any other combination, indicating
the adverse affect of NaCI in the medium on cell status for
free isoleucine.
The conclusion drawn from these results
is that
resistance to 5-MT is accompanied by an increase in free
isoleucine content of the cells, whereas resistance to A2C
results in a decrease of isoleucine.
In cells resistant to
both the analogs (TAD and SNAT), a significant decrease in
isoleucine status was detected
However,
t~eatment
compared
to PD and DL.
with A2C in the SNAT cell line causes
higher levels of isoleucine than treatment with 5-MT.
41
Glutamate Family of Amino Acids
Glutamate.
Glutamate concentration of the cells is
in most cases above one uM/gm fro wt. (Table 6).
concentration is 6.33 uM/gm fro wt.
est is 0.299 uM/gm fr. wt.
The highest
(PD and DL) and the low-
(TAL and SNAT).
up to 20.2 times the lowest concentration.
Fluctuation goes
The mean value
for the third harvest is considerably lower than for both the
first and .second harvests.
Therefore,
cellular glutamate
contents tend to drop during the late log phase in most of
treatment and cell line combinations.
The PD and DL mean value of 5.89 uM/gm fro wt. is the
highest among all the treatments.
Therefore,
acquiring
resistance to either of the two amino acid analogs or to both
of them simultaneously caused a decrease in cellular free
glutamate content.
The severest decrease is caused by
gaining resistance to 5-MT, the least by resistance to both
analogs, and a moderate decrease results from resistance to
A2C only.
There is also significant variation among different
treatments of SNAT.
Although the presence of either of the
two amino acid analogs or the absence of both does not affect
intracellular glutamate levels significantly, the presence of
both analogs in the medium results in a significant decrease
in the cell's free glutamate content.
The presence of NaCl
in the medium also affects cellular free glutamate content
42
Table 6.
The effect of treatment and strain combination and
growth period on glutamate concentrations (uM/gm
fro wt. ) in carrot cell suspension cultures.
Growth Period
Strain a
SNAT
Treatment b
2 hours
AD
3.698 c
5.15
3.875
4.241 Ed
TD
3.215
5.38
4.353
4.316 E
TAD
3.663
4.102
2.189
3.318 D
TAL
3.106
1. 72
ND
3.582
1. 912
1.984
2.493 C
PD
5.·493
4.387
3.085
4.322 E
.783
.457
4 days
8 days
(uM/gm fro wt. )
.5885
.299
Mean
1.708
B
SNT
TD
SNA
AD
3.077
2.3
2.243
2.54
DL
PD
6.327
5.285
6.33
5.98 F
3.639 Bd
3.446 B
2.757 A
Mean
.609
A
C
DL, control; SNA, resistant to A2C;
a Strains (cell lines):
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
43
negatively, but the effect of treatment with light is more
severe.
Proline.
fro wt.
The highest per cell value is 11.841 uM/gm
(TD and SNAT), and the lowest is 0.470 uM/gm fro wt.
(PD and DL)
(Table 7).
Fluctuation goes up to 24.2 times
that of the lowest mean value.
The overall mean value for
the third harvest is considerably lower than mean values for
the first and second harvests, indicating a drop in intracellular proline content in most treatment and cell line
combinations at the third harvest.
Comparison among the four cell lines shows that TAD
and SNAT and AD and SNA have significantly higher mean
values, PD and DL has the lowest mean value, and TD and SNT
has an intermediate mean value.
Therefore it seems that
resistance to either of the two analogs is accompanied by an
increase in intracellular free proline contents, but resistance to A2C results in significantly higher proline values
than resistance to 5-MT.
The TD treatment of the SNAT cell line has the highest mean value among the treatments performed on this cell
line and also among all treatment and cell line combinations.
AD treatment of the SNAT cell line, on the other hand, exhibits the lowest treatment mean value among treatments of this
cell line.
With TAD exhibiting an intermediate value between
the above two treatments, it would rather be appropriate to
44
Table 7.
The effect of treatment and strain combination and
growth period on proline concentrations (uM/gm fro
wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
AD
5.417c
4 days
8 days
(uM/gm fro wt.)
Mean
4.126
2.804
11.841
9.265
9.909
TAD
4.621
5.769
4.614
5.001 D
TAL
5.926
8.229
.777
4.997 D
ND
7.865
9.119
PD
7.915
6.968
6.521
7.135 E
SNT
TD
2.245
1. 825
2.085
2.051 3
SNA
AD
5.688
5.445
3.587
4.906 D
DL
PD
.497
. 47
.717
.561 A
SNAT
TD
Mean
5.7793 d
5.69 3
10.96
4.155 Cd
10.34 G
9.315 F
4.664 A
DL, control; SNA, resistant to A2C;
a Strains (cell lines):
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
b Treatments:
PD, plain medium and darkness; AD, A2C and
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
45
conclude that the two analogs have opposite effects on the
proline status of this cell line.
As NaCl caused significant
increase in proline values, light does not seem to have any
effect on it.
Arginine.
With intracellular arginine mean values
per cell ranging between 11.85 uM/gm fr. wt.
0.0 uM/gm fro
wt.
(TD and SNT),
(PD and DL) and
it is probably one of the
most extensively fluctuating amino acids (Table 8).
The gen-
eral trend shows a decrease in mean arginine values between
the first and second harvests and a more substantial rise at
the third harvest.
This is probably compatible with its main
role as a nitrogen storage compound.
Comparison between different cell lines shows that DL
has the highest mean value of 8.861 uM/gm fro wt. and the SNT
cell line the lowest mean value of 0.0 uM/gm fro
wt., which
is not significantly lower than the mean value of 0.345 uM/gm
fro
wt. for SNA cell line.
But compared to 2.671 uM/gm fro
wt., the mean value for TAD and SNAT, they are significantly
lower.
This pattern resembles the situation encountered in
glutamate and probably
indicates a high demand or low
production of these two amino acids in resistant cell lines.
Comparison between different treatments of the SNAT
cell line shows that all treatments except AD have significantly lower mean value than the PD treatment.
This indi-
cates that only A2C has a positive effect on intracellular
46
Table 8.
The effect of treatment and strain combination and
growth period on arginine concentrations (uM/gm fro
wt.) in carrot cell suspension cultures.
Growth Period
Strain a
SNAT
Treatment b
2 hours
4 days
8 days
(uM/gm fro wt.)
5.738
AD
11.63
Mean
7.427 pd
TD
1.467
1.297
5.852
2.866 D
TAD
3.099
.879
4.036
2.671 D
TAL
1.262
. 349
1.221
0.944 C
ND
1.571
. 614
o
0.728 BC
PD
2.1 74
1.749
7.179
3.701 E
SNT
TD
o
o
o
o
SNA
AD
.226
.025
DL
PD
9.926
4.805
2.738 Bd
1. 715 A
Mean
.783
11. 85
A
0.345 AB
8.861 G
4.728 C
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replicatio~
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
47
arginine while 5-MT,
light, and NaCl affect it negatively,
with the effect of NaCl being most severe (contrary to what
was found in proline).
Aspartate Family of Amino Acids
Aspartate.
1.0 uM/gm fr.
wt.
Aspartate concentrations are mostly below
per cell
replication value is 1.672
(Table 9).
The highest mean
(PD and DL) and the lowest is
0.102 uM/gm fro wt. (TAL and SNAT).
15.5 times the lowest mean value.
The fluctuation is up to
Mean values for the second
and third harvests fall considerably below that of the first
harvest.
This probably means that intracellular aspartate
contents in most treatments rise right after the transfer of
cells to new medium,
then fall during the initial phase of
the growth curve and stay stable up to the third harvest.
Comparison between the four cell lines shows that the
mean value f6r the control cell line of 1.415 uM/gm fro wt.
is the highest, followed by that of SNA, while the means for
SNT and SNAT, which are not significantly different, are the
lowest.
This would imply that cell lines resistant to either
one or both of the amino acid analogs contain significantly
lower aspartate levels than normal cells.
resulted
in greater
Resistance to 5-MT
reduction in aspartate levels
than
resistance to A2C.
Comparison between treatments of the SNAT cell line
shows that the effect of A2C is not significant, 5-MT causes
48
Table 9.
The effect of treatment and strain combination and
growth period on aspartate concentrations (uM/gm
fro wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
8 .days
4 days
(uM/gm fro wt. )
Mean
~-------
AD
0.594c
0.568
0.553
0.572 Dd
TD
0.823
0.966
0.522
0.770 E
TAD
0.510
0.295
0.270
0.358 BC
TAL
0.324
0.173
0.102
0.200 A
ND
0.354
0.176
0.167
0.232 AB
PD
0.721
0.347
0.345
0.471 DC
SNT
TD
0.263
0.204
0.159
0.208 AB
SNA
AD
0.400
O. 440
0.496
0.445 DL
DL
PD
l. 486
1.088
1.672
1.415
0.608 Bd
0.473 A
0.476 A
SNAT
Mean
F
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCI and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and·SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
49
significant increase in intracellular aspartate content while
the two analogs together,
light and NaCl, affect aspartate
values negatively.
Threonine.
All threonine concentrations are below
one uM!gm fro wt. per cell (Table 10).
uM!gm fr. wt.
wt.
The highest is 0.618
(TAD and SNAT) and the lowest 0.073 uM!gm fr.
(ND and SNAT).
The values fluctuate up to 7.5 times the
lowest concentration.
There is no substantial difference in
the overall mean values of the three harvests, probably due
to the relative stability of cellular threonine contents
during the growth of the cultures.
Comparison between the four cell lines does not show
a significant difference between the means of DL, SNT and
SNAT, whereas the mean for SNA is significantly lower.
This
would mean that resistance to 5-MT only, or to A2C and 5-MT
together does not
result
in any significant changes in
threonine status, but resistance to A2C only causes a drop in
cellular threonine values.
comparison between different treatments of the SNAT
cell line shows that the presence of either A2C or 5-MT or
both of them together in the medium results in a significant
increase in threonine values.
The combined effect of the two
analogs in the medium, however, is greater than the effect of
50
Table 10.
The effect of treatment and strain combination and
growth period on threonine concentrations (uM/gm
fro wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fr. wt.)
Mean
AD
0.245 c
0.346
0.299
0.297 CBd
TD
0.244
0.300
0.259
2.660 B
TAD
0.396
0.618
0.227
O. 413 D
TAL
0.053
0.175
0.062
0.096 A
ND
0.130
0.082
0.073
0.095 A
PD
0.104
0.125
0.160
0.130 A
SNT
TD
0.339
0.349
0.420
0.369 CD
SNA
AD
0.165
0.155
0.123
0.147 A
DL
PD
0.351
0.282
0.440
0.358 BCD
0.225 Ad
0.270 A
0.229 A
SNAT
Mean
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
b Treatments:
PD, plain medium and darkness; AD, A2C and
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
51
either of them alone.
NaCl does not seem to have a signifi-
cant effect on threonine levels in this cell line whereas
light causes its significant drop.
Methionine.
Cellular methionine contents for dif-
ferent treatment and cell line combinations range between
1.133 uM/gm fr.
and SNAT,
frequent
wt.
(AD and SNAT) and 0.0 uM/gm fr. wt.
TL and SNAT),
f~uctuation
(AD
probably due to extensive and
of this amino acid (Table 11).
In most
treatments, methionine values increase continuously between
the first and third harvests, which indicates that methionine
is accumulated with the growth of the culture.
Comparison between the four cell lines indlcates that
cell lines resistant to either one or both the analogs contain lower levels of methionine.
Cell lines resistant to A2C
only, or to A2C AND 5-MT both seem to be more drastically
affected than that resistant to 5-MT only.
Comparison between different treatments of SNAT shows
that 5-MT by itself or combined with A2C in the medium has a
negative effect upon cellular methionine levels.
A2C by
itself in the,medium, on the other hand, seems to enhance the
cellular methione content.
Light treatment does not seem to
have a significant effect on methionine values,
while Natl
seems to decrease them significantly.
Lysine.
wt.
Lysine values range between 5.169 uM/gm fro
(TD and SNAT) and 0.070 uM/gm fro
wt. per cell (AD and
52
Table 11.
The effect of treatment and strain combination and
growth period on methionine concentrations (uM/gm
fro wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fro wt.)
Mean
AD
0.688 c
0.958
1.133
0.927 pd
TD
0.058
0.087
0.235
0.127 B
TAD
0.000
0.000
0.056
0.0.19 A
TAL
0.026
0.000
0.000
0.009 A
ND
0.227
0.227
0.218
0.224 C
PD
0.498
0.674
1.069
0.759 E
SNT
TD
0.134
0.121
0.241
0.165 B
SNA
AD
0.000
0.000
0.000
0.000 A
DL
PD
0.452
0.248
0.502
0.400 D
0.231 Ad
0.257 B
0.384 C
SNAT
Mean
a Strains (cell lines):
DL, control; SNA, resistant to A2C:
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
53
Table 12.
The effect of treatment and strain combination and
growth period on lysine concentrations (uM/gm fro
wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fr. wt.)
Mean
AD
2.886 c
2.567
1.426
2.293 Dd
TD
5.169
4.493
4.466
4.709 E
TAD
3.215
1.911
1.324
2.150 CD
TAL
1.157
1.231
0.804
1.060 ABCD
ND
1.780
1.559
1.106
1.482 BCD
PD
0.179
1.104
0.962
0.748 AB
SNT
TD
1.206
1.092
0.580
0.959 ABC
SNA
AD
O. 229
0.070
0.087
0.128 A
DL
PD
0.155
0.120
0.184
0.153 A
1.775 Ad
1.572 A
1.215 A
SNAT
Mean
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaC1 and
darkness.
'
c Values are means for 2 replications (except the value at 4
days growth period of PD and'SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
1etter(s) are not significantly different.
54
SNA) (Table 12).
These values fluctuate up to 73.4 times the
lowest concentration.
Although the overall mean values seem
to drop between the first and third harvests, the drop is not
substantial.
This indicates the relative stability of
cellular lysine content during growth of the cell cultures.
Comparison between the four cell lines shows that DL
and SNA have the lowest treatment mean.
The mean for SNT,
though considerably higher than that for DL and SNA, is not
significantly different from them.
The mean value for TAD
and SNAT, on the other hand, is significantly higher than the
mean for the control cell line PD and DL.
This probably
means that cell lines resistant to either A2C or 5-MT do not
exhibit asignificant change in their cellular lysine content
(though cells resistant to 5-MT only may contain relatively
higher amounts of this amino acid).
The cell line resistant
to both analogs, however, shows a statistically significant
increase in lysine content.
Comparison between different treatments of the SNAT
cell line shows that either A2C or 5-MT, by themselves or
combined, enhance cellular lysine content.
5-MT by itself, however,
is more pronounced.
The effect of
The light,
though causing considerable reduction in lysine values, does
not affect mean value significantly.
NaCl also causes a
relative increase in lysine values but its effect is not
significant.
55
Serine Family of Amino Acids
Serine concentrations range between 1.388
Serine.
uM/gm fro
SNAT)
wt.
(PD and DL) and 0.091 uM/gm fro
(Table 13).
wt.
(TAL and
Fluctuation goes up to 24.2 times the
lowest per cell value.
In most of the treatments serine
values seem to drop at the third harvest.
Comparison between the four cell lines indicates that
the normal cell line has the highest mean value, followed by
SNA, SNT, and SNAT, respectively.
This means that the cell
line resistant to both analogs suffered the most significant
drop in cellular serine levels, followed by that resistant to
5-MT only.
The comparison between treatments of SNAT indicates
that cellular serine contents are enhanced by A2C, depressed
by 5-MT, and not significantly affected by other treatments.
Glycine.
uM/gm fro
wt.
Glycine concentrations range between 0.418
(TAL and SNAT) and 0.0 uM/gm fro
wt.
(AD and
SNA) per cell (Table 14), which means that it is one of the
least abundant amino acids.
Although the mean value for the
first harvest is a little higher than that for the other two
harvests,
the difference is not substantial.
Therefore
glycine valQes are probably stable in most of the treatments
during the active growth of the cultures.
Comparison between the four cell lines reveals that
cellular glycine values are not affected in any of the three
56
Table 13.
The effect of treatment and strain combination and
growth period on serine concentrations' (uM/gm fro
wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Ii
Treatment b
2 hours
4 days
8 days
(uM/gm fr. wt.)
Mean
AD
0.495 c
0.712
0.492
0.566 DCd
TD
0.420
o. 373
0.204
0.332 A
TAD
0.559
0.557
0.439
0.518 BC
TAL
0.555
0.592
0.091
0.413 AB
ND
0.526
0.365
0.358
0.416 AB
PD
0.619
0.444
0.384
0.490 BC
SNT
TD
0.652
0.621
0.668
0.647 D
SNA
AD
0.778
0.791
0.768
0.779 E
DL
PD
1.218
1.187
1.388
1. 264 F
0.647 Bd
0.637 B
0.532 A
SNAT
Mean
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replicatiqn
on ly) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
57
Table 14.
The effect of treatment and strain combination and
growth period on glycine concentrations (uM/gm fro
wt.) in carrot cell suspension cultures.
-----Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fro wt.)
Mean
AD
0.239
0.415
0.405
0.353 cd
TD
0.224
0.203
0.196
0.208 B
TAD
0.149
0.152
0.296
0.200 B
TAL
0.418
0.134
0.052
0.201 B
ND
0.203
0.077
0.085
0.122 AB
PD
0.1114
0.092
0.110
0.108 AB
SNT
TD
0.155
0.140
0.189
0.148 AB
SNA
AD
0.101
0.095
0.000
0.065 A
DL
PD
0.157
0.113
0.122
0.131 AB
0.191 Ad
0.162 A
0.162 A
SNAT
Mean
a Strains (cell lines):
DL, control; SNA, resistant to A2C;
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
58
cell lines compared to the control, although mean value for
TAD and SNAT is significantly higher than that- of AD and SNA.
Comparison between treatments of the SNAT indicates
that glycine values are enhanced by A2C only and not affected
by any other kind of treatment.
Aromatic Family of Amino Acids
Phenylalanine.
between 2.794 uM/gm fro
Phenylalanine concentrations range
wt.
(TAD and USNAT) and 0.067 uM/gm
fro wt. (PD and DL) per cell (Table 15).
to 41 times the lowest per cell value.
Values fluctuate up
There were continuous
decreases in cellular phenylalanine content between the first
harvest and third in most of the treatments.
that cellular phenylalanine
increases
This indicates
right atter
the
transfer of cells to the new medium then decreases during
active growth of the cells.
Comparison
between cell
lines shows that cells
resistant to 5-MT alone, or 5-MT and A2C both, accumulate
significantly higher amounts of phenylalanine.
A2C resistant
cells do not seem to be significantly affected.
Comparison between different treatments of SNAT does
not show a significant effect for either of the two analogs
.
on the phenylalanine status of the cells.
Both analogs
combined, however, seem to have a positive effect on accumulation of this amino acid.
hand, affect it negatively.
Light and NaCI, on the other
59
Table 15.
The effect of treatment and strain combination
and growth period on phenylalanine concentrations
(uM/gm fro wt.) in carrot cell suspension
cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fro wt.)
Mean
AD
1.722c
0.594
0.274
0.863 Cd
TD
1.165
0.741
0.390
0.765 C
TAD
2.794
1.699
1.224
0.906 D
TAL
1. 465
1. 264
0.091
0.940 C
ND
0.444
0.418
0.279
0.380 B
PD
0.897
0.907
0.387
0.730 C
SNT
TD
0.817
0.687
0.825
0.776 C
SNA
AD
0.522
0.215
0.182
0.306 AB
DL
PD
0.084
0.C67
0.071
0.074 A
1.101 Cd
0.732 B
0.413 A
SNAT
Mean
DL, control; SNA, resistant to A2C;
a Strains (cell lines):
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
b Treatments:
PD, plain medium and darkness; AD, A2C and
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
60
Tryptophan.
Tryptophan concentrations range between
2.714 uM/gm fro wt.
(AD and SNA)
(TAD and SNAT) and 0.129 uM/gm fro wt.
(Table 16).
Cellular content for tryptophan
fluctuated up to 20.1 times the lowest per cell value.
In
most treatments values tend to rise between the first and
second harvests then
fall
between the second and third
harvests, which probably means that tryptophan accumulation
is proportional to cellular activity.
Comparison between cell lines indicates that tryptophan levels of A2C resistant cell lines are not significantly
affected.
Cell lines resistant to 5-MT only or to 5-MT and
A2C together, on the other hand, exhibit significantly higher
tryptophan values.
however,
Cell lines resistant to both analogs,
showed significantly higher tryptophan concentra-
tions than that resistant to 5-MT only.
Comparison between different treatments of SNAT shows
that A2C by itself has no significant effect on free tryptophan levels of this cell line.
5-MT by itself or with A2C
affect it positively, whereas light and NaCI have a significantly depressing effect on it.
Summary of Results
In the summary, alphabetical letters will be used to
code for the five amino acid families mentioned above, as
follows:
A
= pyruvate family, B = glutamate family, C =
aspartate family, D
=
serine family, and E
=
aromatic family.
61
Table 16.
The effect of treatment and strain combination and
growth period on tryptophan concentrations (uM/gm
fro wt.) in carrot cell suspension cultures.
Growth Period
Strain a
Treatment b
2 hours
4 days
8 days
(uM/gm fro wt. )
Mean
------AD
0.785 c
1.389
1.495
1.223 Cd
TD
1.654
1.962
2.629
2.082 F
TAD
2.036
2.714
2.171
2.307 G
TAL
2.269
2.673
O. 447
1.796 E
ND
0.510
0.595
0.637
0.581 B
PD
0.796
0.945
1.527
1.118 C
SNT
TD
, 1. 379
1.846
1.294
1.507 D
SNA
AD
0.129
0.239
0.235
0.201 A
DL
PD
0.150
0.231
O. 362
0.248 A
1.079 Ad
1.426 B
1.200 A
SNAT
Mean
DL, control; SNA, resistant to A2C;
a Strains (cell lines):
SNT, resistant to 5-MT; SNAT, resistant to 5-MT + A2C.
PD, plain medium and darkness; AD, A2C and
b Treatments:
darkness; TD, 5-MT and darkness; TAD, 5-MT + A2C and darkness; TAL, 5-MT + A2C and light (300-500 lux); ND, NaCl and
darkness.
c Values are means for 2 replications (except the value at 4
days growth period of PD and SNAT is for one replication
only) .
d Numbers in the same column or row followed by the same
letter(s) are not significantly different.
62
SNA Cell Line
Compared to the control cell line, DL, SNA cell line
had higher concentration of B: proline only
Figure 1).
(Table 17 and
Concentrations of A: alanine, valine, leucine and
isoleucine; B: glutamate and arginine: C: aspartate, threonine and methionine; and D: serine were lower in this cell
line.
The concentrations of C: lysine; D: glycine; and E:
tryptophan and phenylalanine, on the other hand, were not
significantly affected.
SNT Cell Line
Compared to the control cell line, DL,
had higher
concentrations of A:
valine,
this strain
leucine,
and
isoleucine; B: proline; and E: phenylalanine and tryptophan
concentrations for A: alanine; B:. glutamate and arginine; C:
aspartate and methionine; and D: serine were lower (Table 17
and Fig u r e 2).
Am 0 un t s
0
f C: t h reo n i n e and 1 y sin e ; and D:
glycine were not significantly affected in this cell line.
SNAT Cell Line
Compared to the control cell line, DL,
SNAT (TAD
treatment) had higher concentrations of B: proline, C: lysine
and E: phenylalanine and tryptophan (Table 17 and Figure 3).
Concentrations of A: alanine, valine, leucine, and isoleucine: B: glutamate and arginine; C: aspartate and methionine;
63
Table 17.
Free amino acid concentrations in carrot cell
lines resistant to amino acid analogs, as compared
to that of the control cell line.
Treatment a and Cell Line b
Amino
Acids
PD and
DL
AD and
SNA
TD and
TAD and
SNT
SNAT
(uM/gm fro wt.)
ALA
VAL
LEU
ILE
5.331 c D
0.802 D
0.231 C
0.211 E
3.157
0.265
0.083
0.077
Ld
L
L
L
GLU
PRO
ARG
5.980 F
0.561 A
8.861 G
ASP
THR
MET
LYS
1.415
0.358
0.400
0.153
SER
GLY
TRP
PHE
0.349 H
0.403 H
2.925
0.391
0.162
0.083
2.540 L
4.906 H
0.345 L
0.609 L
2.051 H
0.000 L
3.318 L
5.001 H
2.671 L
4.322 L
7.135 H
3.701 L
0.445 L
0·.147L
0.000 L
0.128 S
0.208
0.369
0.165
0.959
0.358
O. 413
0.019
2.150
0.471
0.130
0.759
0.748
1.264 F
0.131 AB
0.779 L
0.065 S
0.647 L
0.148 S
0.518 L
0.200 S
0.490 L
0.108 S
0.248 A
0.074 A
0.201 S
0.306 A
1.507 H
0.776 H
2.307 H
1.906 H
1.118 H
0.730 H
F
CBD
D
A
1. 877 L
1. 020 H
PD and
SNAT
L
S
L
S
L
L
L
L
L
S
L
H
2.652
0.435
0.152
0.106
L
L
L
L
L
L
H
S
a Amino acid analog treatments with:
PD, plain medium and
darkness; AD, A2C and darkness; TD, 5-MT and darkness; TAD,
A2C + 5-MT and darkness._
b Cell lines:
DL, control; SNA, resistant to A2C;
resistant to 5-MT; and SNAT, resistant to A2C + 5-MT.
c Values are means for three growth periods:
and 8 days after inoculation ..
d S:
H:
L:
SNT,
2 hours, 4 days
not significantly different than the PD and DL.
significantly lower than PD and DL (significance level
of 5%).
significantly higher than PD and DL (significance level
of 5%).
64
l
6
~
PD
~.!:
00
tQQ1
AD &
DL
St·~.A
~
.J
3:
·.
~
U-1
e
0'1
'C)
.--!
~
0
l-<
u
·.-i
:::
3
0
i::<l
..:l
H
Figure 1.
Free amino acids from different amino acid
families affected in SNA cell line (resistant to
A2C), grown in darkness and treated with A2C
(AD); compared to DL cell line (control), grown
in darkness and treated with plain medium (PD).
-- Amino acid concentrations in uM/gm fr. wt. are
means for three growth periods:
2 hours, 4 days,
and 8 days after inoculation.
65
9
6
...;
:::
~
PD & DL
~
TO & St·-.JT
~
t'
~
1.1-4
E
ti'
t
"Q)
.-i
0
E
0
~
u
::;:
-~
3
Figure 2.
Free amino acids from different amino acid
families affected in SNT cell line (resistant to
5-MT), grown in darkness and treated with 5-MT;
compared to DL cell line (control), grown in
darkness and treated with plain medium (PD).-Am i n o a c i d con cent r a t i on s i n u MI g m f r . wt . a r e
means for three growth periods:
2 hours, 4 days,
and 8 days after inoculation.
66
9
l
6
~
~
.....
k'·
e0>
r
"
···-~
CIJ
~
PD
~
TA.D &!: St··J AT
j!:
DL
~
..
'-.
.--l
0
'··...
'·
e
0
1-1
'·
u
·.-4
~
3
'·.
0
Figure 3.
Free amino acids from different amino acid
families affected in SNAT cell line (resistant to
A2C + 5-MT), grown in darkness and treated with
A2C and 5-MT (TAD); compared to DL cell line
(control), grown in darkness and treated with
p 1 a i n me d i u m ( P D ) . - - Amino a c i d con cent r a t i on s
in uM/gm fr. wt. are means for three growth
periods:
2 hours, 4 days, and 8 days after
inoculation.
67
and D: serine were lower.
Concentrations of C: threonine and
D: glycine were not significantly affected.
AD Treatment of SNAT Cell Line
Compared to the PD treatment of SNAT cell line AD
treatment resulted in increases in the concentrations of A:
isoleucine; B: arginine, C: threonine, methionine and lysine;
and D: glycine (Table 18 and Figure 4).
the decrease of B: proline only.
AD treatment caused
It did not affect the con-
centrations of A: alanine, valine and leucine; B: glutamate;
C: aspartate; D: serine and E: tryptophan and phenylalanine.
TD Treatment of the SNAT Cell Line
Compared to PD treatment of SNAT cell line, the TD
treatment caused increases in A:
aspartate,
threonine and lysine;
leucine,
and E:
B:
proline; C:
tryptophan.
It
caused decreases in B: arginine; C: methionine; and D: serine
(Table 18 and Figure 5).
valine and. isoleucine;
The concentrations of A: alanine,
B: glutamate; D: glycine;
and E:
phenylalanine were not affected.
TAD Treatment of the SNAT Cell Line
Compared to PD treatment of the SNAT, TAD resulted in
increases in C:
phenylalanine.
threonine and lysine and E:
tryptophan and
It caused decreases in B: glutamate, proline
and arginine; and C: methionine (Table 18 and Figure 6).
did not affect the concentrations of A:
alanine,
TAD
valine,
68
Table 18.
Free amino acid concentrations resulting from the
treatment of SN'AT cell 1 ine (resistant to both A2C
and 5-MT) with amino acid analogs, compared to its
treatment with plain medium.
Treatment a
Amino Acid
PD
AD
TD
(uM/gm fro wt. )
AL
VAL
LEU
ILE
2.652 b BC
0.435 BC
0.152 B
0.106 BC
3.011
0.511
0.166
0.166
GLU
PRO
ARG
4.322 E
7.135 E
3.701 E
4.241 S
4.155 L
7.472 H
ASP
THR
MET
LYS
0.471 CD
0.13·A
0.759 E
0.748 AB
O. 572
0.297
0.927
2.293
SER
GLY
0.49 BC
0.108 AB
TRP
PHE
1.118 C
0.73 C
SC
S
S
H
3.308
0.505
0.205
0.131
TAD
S
S
H
S
2.925
0.391
0.162
0.083
S
S
S
S
4.316 S
10.34 H
2.866 L
3.318 L
5.011 L
2.671 L
S
H
H
H
0.77 H
0.266 H
0.127 L
4.709 H
0.358 S
0.413 H
0.019 L
2.15 H
0.566 S
O. 353 H
O. 332 L
0.208 S
0.518 S
0.2 S
1.223 S
0.863 S
2.082 H
0.765 S
2.307 H
1. 906 H
PD, plain medium and
a Amino acid analog treatments with:
darkness; AD, A2C and darkness; TD, 5-MT and darkness; TAD,
A2C + 5-MT and darkness.
b Values are means for three growth periods:
and 8 days after inoculation.
c S:
H:
L:
2 hours, 4 days
not significantly different than the PD treatment.
significantly higher than the PD treatment (significance level of 5%).
significantly lower than the PD treatment (significance
level of 5%).
69
8
l
~
PD
~!.:
~
.AD & St-··.JAT
St··.J.AT
6
2
Figure 4.
Free amino acids affected in SNAT cell line
(resistant to A2C + 5-MT), subjected to AD treatment (grown in darkness and treated with A2C),
compared to its treatment with PD (grown in
darkness and plain medium).
70
15
~
PD & St· .J.AT
t8Sj TD &
s t·.JAT
10
.J
~
~
"-l
5,
'Q)
.-I
0
e:
0
io-1
u
·.-t
::<:
5
Figure 5.
Free amino acids affected in SNAT cell line
(resistant to A2C + 5-MT), subjected to TD treatment (grown in darkness and treated with 5-MT),
compared to its treatment with darkness and plain
medium (PD).
71
W
~
PD ·~.• •;t·JAT
\..,;4
K.X1 TA. .CJ lr-U2j
·-
••
c....J-.
t··JAT
6
~
:::
~
~
E
0'>
........
4
Q)
~
~
0
1-4
u
·.-I
::.:
Figure 6.
I
~~~·
Free amino acids from different amino acid
families affected in SNAT cell line (resistant to
A2C + 5-MT), subjected to TAD treatment (grown in
darkness and treated with A2C + 5-MT), compared
to its treatment with plain medium and darkness
( PD) •
72
leucine and isoleucine; c: aspartate;
and D:
serine and
glycine.
TAL Treatment of the SNAT Cell Line
Compared to the TAD treatment of the SNAT cell line
TAL treatment did not result in an increase in any of the
free amino acids
(Tables 2-16).
It caused decrease in A:
val; B: glutamate and arginine; C: aspartate and threonine;
and E: phenylalanine and tryptophan.
alanine,
The amino acids: A:
leucine and isoleucine; B: proline, C: methionine
and lysine; and D: serine and glycine were not affected.
ND Treatment of the SNAT Cell Line
compared to the PD treatment of the SNAT, ND caused
increase in B: proline only (Tables 2-16).
It caused de-
creases in A: valine, leucine, and isoleucine; B: glutamate
and arginine; C: aspartate and methionine; and E: phenylalanine and tryptophan.
The amino acids A: alanine; C: threo-
nine and lysine; and D: serine and glycine were not affected.
Free Amino Acid Trends During
the Growth Period
(Based on the mean values for all of the treatments
at 2 hours, 4 days, and 8 days after inoculation.)
The amino
acids A: alanine and leucine; B: glutamate and proline; and
D: serine appeared in high concentrations at the first and
second harvests and drop at the third (Table 19).
The amino
73
Table 19.
Free amino
periods.
acid
trends during
three growth
Growth Period
Amino Acid
2 hours
ALA
VAL
3.184 a Bb
0.497 AB
0.183 B
0.146 A
3.299
0.558
0.191
0.154
3.639 B
0.779 B
2.738 B
3.446 B
5.690 B
1.715 A
2.757 A
4.664 A
4.728 C
MET
LYS
0.608
0.225
0.213
1. 775
0.473
0.270
0.257
1.572
0.476
0.229
0.384
1. 215
SER
GLY
0.647 B
0.191 A
0.637 B
0.162 A
0.532 A
0.162 A
PHE
1.101 C
1. 079 A
0.732 B
1. 426 B
0.413 A
1.200 A
LEU
ILE
GLU
PRO
ARG
ASP
THR
TRP
B
A
A
A
4 days
(uM/gm fro wt.)
B
B
B
A
A
A
B
A
8 days
2.594
0.446
0.145
0.143
A
A
A
A
A
A
C
A
a Values are means for different treatment and cell line
combinations (PD and DL, AD ~nd SNA, TD and SNT, PD and
SNAT, AD and SNAT, TD and SNAT, TAD and SNAT, TAL and SNAT,
and ND and SNAT).
b Numbers in the same row followed by the same letter(s) are
not significantly different.
74
acids A: isoleucine;
c:
threonine and lysine; and D: glycine
maintained the same levels during all the three harvests.
The amino acids C: aspartate and E: phenylalanine appeared in
high concentrations at the first harvest and were low at the
second and third.
The amino acids A: valine; and E: trypto-
phan appeared in high concentrations at the second harvest
only.
The amino acids B:
arginine;
and C:
methionine
accumulated in high concentrations at the third harvest.
75
DISCUSSION
Growth of the Cultures
Resistance to A2C (AD and SNA) caused a reduction in
percentage increase in cell fresh weight compared to the
control cell line, PD and DL (Table 1).
In the SNAT cell
line, treatment with NaCl (ND) and A2C (AD) resulted in lower
percentage increase in cell fresh weight compared to control
treatment, PD (Table 1).
The negative effects of NaCl and A2C on culture
growth can probably be attributed to their involvement in
physiological besides biochemical changes in the cellular
system (Cella et ale 1982, Binzel et ale 1985).
Light,
on
the other hand, caused higher percentage increase in cell
fresh weight compared to PD treatment of the SNAT cell line
(Table 1).
This could probably be attributed to greater
biosynthetic activity.
Cellular Free Amino Acids
Levels, Fluctuation, and Response
to Growth Periods
Some of the amino acids like alanine, glutamate and
arginine generally appeared in higher concentrations, probably because of their wider metabolic activity.
On the other
hand, amino acid content fluctuations probably reflects the
76
amount of turnover and also the level of response to different treatments.
For example, alanine and valine exhibited
little fluctuation and at the same time did not show any
significant response to different analog treatments.
Regarding their
response to growth periods,
fifteen amino acids can be divided into three groups.
the
The
first group, comprised of nine amino acids, exhibited lower
concentrat~ons
at the third harvest
(or the second and the
third harvests), which would indicate that these amino acids
were accumulated during early growth period.
group,
consisting of four amino acids,
The second
did not show any
significant variation during all of the three growth periods.
This is probably indicative of a relatively stable rate of
turnover.
The third group, consisting of two amino acids,
accumulated at the third growth period.
to metabolic
r~quirements
This could be due
like storage of nitrogen
(e.g.,
arginine), or because of some regulatory adjustments, as in
the case of methionine.
Response to Light and NaCI Treatments
Light and NaCI treatments were used to stimulate and
stress growth of the cultures to determine their effect qn
free amino acid levels.
From Table 1, it is evident that the
effect of these two factors on culture growth was the opposite of each other, but ironically, their overall effect on
77
concentrations of many of the amino acids was almost identical.
However, there are some specific differences between
the effects of light and NaCI on amino acid concentrations.
While light caused a significant decrease in the amount of
free proline, particularly at the third harvest, NaCI caused
its increase, which extended throughout the growth period.
Light
also caused increases in concentrations of many
amino acids at the second harvest and a large decrease in
concentrations of most at the third harvest.
NaCI, on the
other hand, did not cause any significant differences in the
concentrations of most of the amino acids between the second
and the third harvests.
The behavior of free amino acids
under both treatments can probably be attributed to increased
protein synthesis for either growth in the case of light, or
for resistance to stress,
in the case of NaCI.
This indi-
cates that increase or decrease in growth rate does not
necessarily affect the cellular levels of most of the amino
acids.
Response to Analog Treatment and Cell
Line Resistance Characteristics
The Effect of AD, TD and TAD Treatments Compared to
That of PD on SNAT Cell Line.
As mentioned in the results,
AD caused significant increases in six amino acids from four
different families but also caused a decrease in proline.
The TD treatment caused increases in the concentrations of
78
six amino acids, also from four different families,
and
caused decreases in three amino acids from three different
families.
TAD caused increases in four amino acids from two
different families, among which was phenylalanine, which did
not show any significant increase in the former two treatments.
The TAD treatment also caused decreases in levels of
four amino acids from two different families.
Only threonine
and lysine increased in response to all three treatments.
Alanine and valine, on the other hand, were not affected by
any of these treatments.
The above results probably indicate that the effect
of each of the two analogs is not limited to specific sites
of action,
i.e.,
false feedback inhibition of the corres-
ponding amino acid, but a more extensive role in the cell's
biosynthetic process.
It appears that these analogs exert
their influence either through physiological, metabolic, or
enzymatic means or through a combination of these processes.
The cellular system of SNAT cells is probably more predisposed
to
channel
one analog at a time with greater
efficiency, as the use of both analogs simultaneously seems
to adversely affect the biosynthesis of several free amino
acids.
The Expression of Free A A Biosynthesis by the Three
Resistant cell Lines.
(In this part of the discussion SNAT
will not be represented by a single treatment, as with the
79
results, but all of its treatments except TAL and ND will be
considered. )
The amino acids found in lower concentrations
in the resistant cell lines as compared to the normal strain
are:
alanine (Table 2) from the pyruvate family, glutamate
(Table 6) and arginine (Table 8) from the glutamate family,
aspartate (Table 9) from the aspartate family,
and serine
(Table 13) from the serine family, even when all the pertinent SNAT treatments were considered.
One possible explanation for this phenomenon could be
a direct effect of a specific regulatory mechanism that
simultaneously affects the biosynthesis of these amino acids,
but to different degrees in each of the three
stra~ns.
Al-
though this possibility has been demonstrated in yeast (Jones
and Fink 1982), it has not been shown to exist in higher
plants.
Another explanation could lie in the realm of metabolic or physiological variations within these cell lines.
The fact that all five amino acids involved are metabolically
active gives some credence to this possibility.
The involve-
ment of both regulatory and physiological factors,
however,
could be another distinct possibility.
The amino acid found either in similar or lower, concentrations as compared to the normal cell line is threonine,
from the aspartate family.
80
Threonine levels in SNA (Table 10) were significantly
lower, and this decrease was maintained in the PD and SNAT
treatment.
Therefore,
it seems that the decrease in threo-
nine concentrations is probably linked to A2C resistance.
The resistance to 5-MT and A2C together, however, seems to
prepare the cells for a positive response to A2C treatment.
The amino acids found in higher concentrations in any
of the resistant strains as compared to the normal cell line
are:
valine, isoleucine, and leucine from the pyruvate fam-
ily; proline from the glutamate family; methionine and lysine
from the aspartate family; glycine from the serine family;
and phenylalanine and tryptophan from the aromatic family.
The amino acids valine (Table 3), leucine (Table 4),
and isoleucine (Table 5) are affected identically by resistance to either of the two analogs alone and by resistance to
both combined.
In all three amino acids, resistance to 5-MT
results in an increase in their levels; resistance to A2C
causes their decrease; and resistance to both analogs has an
intermediate effect on them.
Thus the biosynthetic regula-
tion of these three amino acids are identically affected by
the regulatory systems of each of the three resistant strains
in a
specifi~
way.
Therefore, the biosynthetic regulation of
these three amino acids seems to be linked to the resistant
characteristics of the three strains.
81
Proline levels (Table 7) are higher in all treatment
and cell line combinations of resistant strains than in the
normal cell line.
Proline is also reported to accumulate in
cells exposed to saline medium
(Dix and
Pearce 1981).
Considering the fact that proline levels in SNA are significantly higher than in SNT, and that A2C is a proline analog,
the involvement of an enzymatic regulatory process in proline
accumulati9n cannot be ruled out.
Therefore, the accumula-
tion of proline could be due to a combination of stress and
specific resistance.
Methionine
(Table 11)
is only found in the AD and
SNAT and PD and SNAT treatments in higher concentrations than
in the DL cell line.
This could indicate that methionine
accumulation is the result of resistance to both analogs.
But the different treatments of SNAT show that methionine
levels are low wherever
5-MT is present in the medium.
Therefore 5-MT seems to have an inhibitory effect on methionine biosynthesis.
This seems
to
indicate that
the
overproduction of methionine could have been the result of
resistance to,5-MT, and that the lower methionine levels in
TD and SNT as compared to PD and DL could be due to the
inhibitory effect of 5-MT on its accumulation.
In other
words, the regulation of methionine biosynthesis is probably
linked to resistance to 5-MT, but 5-MT itself has an inhibitory effect on methionine biosynthesis.
82
Higher
levels of lysine are
found
only in the
treatments of SNAT where either 5-MT or A2C or both of these
compounds are present in the medium (Table 12).
Therefore,
lysine accumulation is probably in response to analog treatment, besides the resistance characteristics of the SNAT cell
line.
But resistance to 5-MT results in considerably higher
lysine values than resistance to A2C.
Thus higher lysine
values in SNAT cell line can primarily be the result of
resistance to 5-MT.
Glycine concentratio·ns (Table 14) are only significantly higher in the AD and SNAT treatment as compared to DL.
There is no significant difference in glycine levels between
the treatments of PD and SNAT and PD and DL.
Therefore,
glycine accumulation seems to be the result of resistance to
both analogs combined and treatment with A2C.
When .compared to DL, phenylalanine concentrations
(Table 15) are significantly higher in TD and SNT and all
treatments of SNAT.
Therefore, phenylalanine accumulation is
probably linked to 5-MT resistance.
phenylalanine
is
Due to the fact that
a member of the aromatic family,
its
accumulation is probably the consequence of high tryptophan
concentration (Jones and Fink 1982).
Tryptophan's behavior
that of phenylalanine.
(Table 16)
is identical
to
Therefore, tryptophan accumulation is
also probably linked to 5-MT resistance.
High tryptophan
83
concentrations could be the result of either derepression of
tryptophan biosynthetic enzymes (Carsiotis and Jones 1974b),
an altered anthranilate synthetase
(Widholm 1972b),
or
reduced uptake of 5-MT (Widholm 1974b).
In the results, it was observed that analog treatments can influence the cellular levels of most of the free
amino acids.
It was also noted that a number of amino acids
have different levels in cell lines resistant to one analog
than that resistant to both of them.
Some of the amino acids
were also found to be identically affected by all the three
cell lines.
Based on these findings,
the criteria for the
determination of the effect of resistance on amino levels
were developed.
According to these criteria, the PD treat-
ment of the SNAT cell line was used to determine the linkage
of biosynthetic regulation of individual amino acids to the
resistance characteristics of the SNA and SNT cell lines.
The amino acid analog treatments of SNAT, on the other hand,
were used to determine the response of the resistant characteristics of this cell line to these treatments compared to
this cell line's response to the PD treatment.
these guidelines, the results were discussed.
Following
Based on these
findings, it can be concluded that changes in the levels of
alanine,
glutamate,
arginine,
aspartate,
and serine are
probably not linked to any specific characteristics of any of
84
the three resistant cell lines.
On the other hand, depres-
sion in threonine levels and possibly the increase in proline
concentrations seem to be linked to A2C resistance.
Eleva-
tions in methionine, lysine, phenylalanine, and tryptophan
levels are probably linked to 5-MT resistance.
Variations in
the concentrations of valine, leucine and isoleucine seem to
be identically linked to specific resistance characteristics
of each of SNA, SNT and SNAT cell lines in different ways.
The overproduction of glycine is probably contingent upon the
combined resistance to both analogs and also the presence of
A2C in the media.
Therefore, it appears appropriate to
conclude that the regulatory effect of these analogs is not
limited to their corresponding amino acids or their families,
but carries over to the regulatory systems
~f
other amino
acid families as well.
In bacteria the activity of an aporepressor has been
suggested to' be responsible for the common control of the
divergent biosynthetic pathways (Nester et al. 1974).
In
yeast, the general control of intrafamily amino acids has
been attributed to repressive and derepressive mechanisms
(Jones and Fink 1982).
Results from past experiments with
higher plants suggest that repression is not involved in the
regulation of amino acid biosynthesis (Bryan 1980).
The outcome of this study indicates that in the
analog resistant cell lines, other control systems for amino
85
acid biosynthesis as well as specific regulatory mechanisms
can also be affected.
It also indicates that the analog used
for selecting the' resistant cell line can play an active role
in the biosynthesis of several amino acids from different
families.
Based on these considerations and the consider-
ations derived from other related studies,
it might be
appropriate to suggest that the regulatory changes in the
biosynthesis of either tryptophan or proline is
refl~cted
upon the biosynthesis of a number of amino acids, from the
same or different families.
This could probably be attrib-
uted to a common regulatory system affecting the biosynthesis
of a number of amino acids, or to the presence of a number of
altered biosynthetic enzymes in the selected cell lines.
86
REFERENCES
Barthelmess, I. B. Mutants affecting amino acid crosspathway control in Neurospora crassa.
Genet. Res. 39:
169-185. 1982.
Bates, L. S. Rapid determination of proline for water stress
studies:
A short communication.
Plant and Soil
39:205-207. 1973.
Bieleski, R. L. and N. A. Turner.
Separation and estimation
of' amino acids in crude plant extracts by thin layer
electrophoresis chromatography. Analyt. Biochem. 17:
278-293. 1966.
Binzel, M. L., P. M. Hasegawa, A. K. Handa and R. A.
san.
Adaptation of tobacco cells to NaCl.
Physiol. 79: 118-125. 1985.
BresPlant
Brotherton, J. E., R. M. Hauptmann and J. M. Widholm.
Anthranilate synthase forms in plants and cultured
cells of Nicotiana tabacum L.
Planta 168: 214-22l.
1986.
Bryan, J. K. Synthesis of the aspartate family and branchedchain amino acids.
In:
Miflin, B. J. (ed.) Amino
acids and their derivatives.
The biochemistry of
plants, vol. V. Academic Press, London New York.
Carsiotis, M. and R. F. Jones.
Crosspathway regulation.
Tryptophan-mediated control of histidine and arginine
biosynthetic enzymes in Neurospora crassa. J. Bacteriol., 119(3): 889-892. 1974a.
Carsiotis M., ,R. F. Jones and A. C. Wesseling. Cross pathway
regulation. Histidine-mediated control of histidine,
tryptophan, and arginine biosynthetic enzymes in
Neurospora crassa.
J. Bacteriol., 119( 3): 893-89S.
1974b.
Cella, R. and P. Iadarola.
Characterization of carrot cell
lines resistant to 5-methyltryptophan obtained by
irradiating suspension cultures with UV-light. Plant
Science Letters 29: 327-337. 1983.
87
Cella, R., B. Parisi and E. Nielsen.
Characterization of a
carrot cell line resistant to azetidine-2-carboxylic
acid. Plant Science Letters 24: 125-135. 1982.
Dalby, A. and C. Y. Tsai.
Acetic anhydride requirement in
the colorimetric determination of tryptophan.
Analytical Biochemistry 63: 283-285. 1975.
Dix, P. J. and R. S. Pearce.
Proline accumulation in NaCl
resistant and sensitive cell lines of Nicotiana
sylvestris.
z. Pflanzenphysio1. Bd. 102.S. 243-248.
1981.
Fin k, G. R., G. Lu c chi n ian d A. G . Hi nne bus c h .
Po sit i ve
regulation of genes under the general amino acid
control in yeast.
Found. Biotech. Ind. Ferment.
Res., (Publ.), 1 (Gene Expression Yeast): 11-18.
1983.
Gilchrist, D. G. and T. Kosuge. Aromatic amino acid biosynthesis and its regulation. In: Miflin, B.J. (ed.),
Amino acids and their derivatives.
The biochemistry
of plants, vol. V.
Academic Press London New York.
1980.
Goldner, R., N. Umeil and Y. Chen.
The growth of carr'ot
callus cultures at various concentrations and composition of saline water.
z. Pflanzenpysio1. Bd. 85.
S. 307-317. 1977.
Harms, T. C. ~nd J. J. Oertli. The use of osmotically adapted cell cultures to study salt tolerance in vitro.
J. Plant Physiol. 120: 29-38.
1985.
Jones, E. W·. and G. R. Fink.
The molecula r biology of the
yeast Saccharomyces:
Metabolism and expression.
Cold Spring Harbor, New York. 1982.
Katterman, F. R.
Professor of Plant Science, Department of
Plant Science, University of Arizona, Tucson, Arizona
85721.
Miflin, B. J. and P. J. Lea.
Biosynthesis and metabolism of
protein amino acids and proteins.
In:
D. Boulter
and B. Parthier (eds.), Nucleic acids and proteins in
plants.
1.
Encyclopedi a of Plan t Physiology, vol.
l4A. Springer-Verlag Berlin Heidelberg. 1982.
88
Murashige, T. and F. Skoog.
A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiol. Plant., 15: 473-497. 1962.
Nester, E, W.
Crosspathway regulation:
Effect of histidine
on the synthesis and activity of enzymes of aromatic
acid biosynthesis in Bacillus subtilis.
J.
Bacteriol., 96(5): 1649-1657. 1968.
Nester, E. W., B. Dale, A. Montoya and B. VoId. Crosspathway
regulation of tyrosine and histidine synthesis in
Bacillus subtilis.
Biochem. Biophys. Acta, 361(1),
59-72. 1974.
Nester, E. W. and A. L. Montoya. An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus
subtilis. J. Bacteriol., 126(2), 699-705. 1976.
Ni e 1 sen, E., A. R . Am i 1 e n i, S. Ron chi and B. Par is i .
A
carrot cell line resistant to azetidine-2-carboxylic
acid. Dev. plant BioI., 5(Plant Cell Cult.: Results
Perspect.), 151-6. 1980.
Nielsen, E., G. Forlani, R. Cella and B. Parisi. Biochemical
characterization of the natural resistance of rice to
the proline analog azetidine-2-carboxylic acid.
Plant Sci (Limerick, Ire.), 44(3), 147-54. 1986.
Piotrowska, M.
Crosspathway regulation of ornithine carbamoyltransferase synthesis in Aspergillus nudilans. J.
Gen. Microbiol.: 116(2): 335-9. 1980.
Riccardi, G., R. Cella, G. Camerino and o. Cifferi.
Resistance to azetidine-2-carboxylic acid and sodium
chloride tolerance in carrot cell cultures and
Spirulina platensis.
Plant and Cell Physiol. 24(6):
1073-1078. 1983.
Sung, Z. R. Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophanresistant cell lines of wild carrots.
Planta,
145(4): 339-345. 1979.
Tisserat, B. and T. Murashige. Repression of asexual embryogenesis in vitro by some plant growth regulators. In
Vitro 13: 799-805. 1977.
89
Widholm, J. M.
Tryptophan biosynthesis in Nicotiana tabacum
and Daucus carota cell cultures:
Site of action of
inhibitory tryptophan analogs.
Biochemica. Biophysica. Acta: 44-51. 1972a.
Widholm, J. M.
Anthranilate synthetase from 5- methyltryptophan-susceptible and -resistant cultured Daucus
carota cells.
Biochim. Biophys. Acta. 279: 48-57.
1972b.
Widholm, J. M.
Selection and characteristics of biochemical
mutants of cultured plant cells. Tissue cult. Plant
Sci., Proc. Int. Congr. Plant Tissue Cell Cult., by:
Street, H. E. Academic: London, Engl. 1974a.
Widholm, J. M.
Cultured carrot cell mutants.
5-Methyltryptophan-resistance trait carried from cell to plant
and back. Plant. Sci. Lett., 3(5): 323- 30. 1974b.
Widholm, J. M.
Selection and characterization of cultured
carrot and tobacco cells resistant to lysine, methionine, and proline analogs.
Can. J. Bot. 54: 15231529. 1976.
Widholm, J.
Selection and characterization of amino acid
analog resistant plant cell cultures.
Crop Science
17: 597-600. 1977.
Widholm, J. M.
Selection and characterization of Daucus
carota L. cell line resistant to four amino acid
analogues.
Journal of Experimental Botany, vol.
29(112): 1111-1116. 1978.
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement