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THESIS

 
 

This is to certify that the

thesis entitled

Development of Selection Strategies for the Isolation
of Methionine Accumulating Cell Lines in Solanum Tuberosum L.

presented by

John Paul Hunsperger

has been accepted towards fulfillment
of the requirements for

 

 

Doctor of Philosophy degree in Crop and Soil Sciences

and

 

Date ~7fjjgf/8 2-—

0-7 639

 

Jllllll'llllllllllllfllfllllllllflllll L

3 1293 01733 9049

 

 

 

 

 

RETURNING MATERIALS:
1V153l_1 Place in book drop :of m
ve this chec ou .ro
w 53?}: record. FINES.will
be charged if book 15

returned after the date
stamped below.

 

2%?» 0020305

 

 

DEVELOPMENT OF SELECTION STRATEGIES FOR THE ISOLATION OF

METHIONINE ACCUMULATING CELL LINES IN SOLANMM TUBEFOSUM L.

By

John Paul Hunsperger

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Science
and

Program of Genetics

1982

ABSTRACT

DEVELOPMENT OF SELECTION STRATEGIES FOR THE ISOLATION OF

METHIONINE ACCUMULATING CELL LINES IN SOLANUM TUBEROSUM L.

By

John Paul Hunsperger

Lg yitgg selection strategies were defined which are capable of
identifying cell lines that accumulate methionine, the first nutrition-
ally limiting amino acid in potato. Inhibitory levels of cysteine
plus threonine, feedback regulators of homoserine dehydrogenase, were
not overcome by methionine, homoserine, or homocysteine. Methionine
relieved growth inhibition due to lysine plus threonine, end product
inhibitors of aspartate kinase and homoserine dehydrogenase. Selene-
methionine toxicity was not overcome by equimolar concentrations of
methionine or precursors. Ethionine toxicity was relieved by low
levels of methionine and homocysteine. Kinetic analysis of methionine-
ethionine antagonism indicated competitive interaction between these
compounds. Glutamyl- 7- methyl ester was established as a higher plant
methionine analog by virtue of methionine- mediated reversal of growth
depression. The presence of 6.8 mM glutamine conferred tolerance to
ten fold higher concentrations of growth inhibiting amino acids and

analogs.

John Paul Hunsperger

Stable ethionine-resistant cell lines were isolated from a

dihaploid clone at a frequency of 9 x 10-10

. Among ten ethionine
tolerant clones, resistance level correlated negatively with molar
percent free methionine, but positively with total free pool amino
acids. However, one isolate expressed 44 Z greater free methionine than
its progenitor line. Stability of ethionine resistance was monitored
for 450 days in a second isolate continuously cultured in the presence
or absence of ethionine. Resistance decreased from high to moderate,
and high level resistance was not reacquired upon reselection.
Spontaneous resistance to selenomethionine in cultures of tetraploid
cultivar 'Superior' occured at a frequency of 2 x 10—8. One variant
which.was cross resistant to ethionine contained 2.29 times as much
free pool.methionine and 1.37 times as much total methionine as
unselected cells.

Analog resistant clones could not be regenerated from callus
cultures; however, a protocol for regenerating potato shoots from true
roots was designed which involves zeatin, gibberellic acid, and

ahacisic acid.

ACKNOWLEDGMENT

The writer gratefully acknowledges past and current members of
his guidance committee for time spent in planning an academic program,
for helpful suggestions concerning the thesis project, and for
thoughtful evaluation of the resulting dissertation. Special grati—
tude is extended to Peter Carlson for maintaining a stimulating
environment for research and discussion and for encouraging exploration
of research areas beyond the thesis project.

Dr. Werner Bergen is acknowledged for providing access to ion
exchange facilities used in amino acid determinations.

Acknowledgment is also made to fellow students and post—doctoral
fellows for thoughtful discussion and laboratory interactions and to
Brenda Floyd for unfailing attention to laboratory needs.

Finally, the author expresses his sincere appreciation to Mary

for her love, understanding, encouragment, and assistance during the

course of these studies.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . iv
LIST OF FIGURES ' 0 O o o o o o o o o o o o o o o o o o o o o o o o v

I. THE POTATO AS AN EXPERIMENTAL SYSTEM FOR IN VITRO SELECTION
OF ENHANCED METHIONINE PRODUCTION. . . . . .
Introduction. . . . . . . . . . .

Plant Cell in vitro Selection Systems .

Nt—‘t—‘H

Manipulation of Solanum speciesin.vitr0 . . . . . . . . . .

The Nutritional Value of Potato Tubers: A Problem and
a Proposal. . . . . . . . . . . . . . . . . . . . . . . . . 5

II. REGULATION OF METHIONINE SYNTHESIS IN HIGHER PLANTS . . . . . . 9
III. DEVELOPMENT OF IN VITRO PROCEDURES FOR SELECTION OF
METHIONINE-ACCUMULATING POTATO CELL LINES. . . . . . . . . . . 19

Introduction. . . . . . . . . . . . . . . . . . . . . . . . 19

Optimization of Cell Growth in Dihaploid Potato Clone
"V-973' o o o o o o o o o o o o o o o o o o o o o o o o o o 19

Optimization of Cell Growth in Tetraploid Potato Clone
'Superior'. . . . . . . . . . . . . . . . . . . . . . . . . 21

Growth Assay. . . . . . . . . . . . . . . . . . . . . . . . 22

Effects of Aspartate Amino Acids on Growth of Potato
Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . 30

Methionine. . . . . . . . . . . . . . . . . . . . . . . 3O
Lysine. . . . . . . . . . . . . . . . . . . . . . . . . 34
Threonine . . . . . . . . . . . . . . . . . . . . . . . 37
Cysteine. . . . . . . . . . . . . . . . . . . . . . . . 37
Selective Systems Based upon Methionine Analogs . . . . . . 42
Preliminary Considerations. . . . . . . . . . . . . . . 42
Selenomethionine. . . . . . . . . . . . . . . . . . . . 44
Ethionine . . . . . . . . . . . . . . . . . . . . . . . 45
G1utamy1-‘Y-methyl ester. . . . . . . . . . . . . . . . 57

iii

Selection Strategies Utilizing Feedback Inhibition . . . .
Preliminary Considerations . . . . . . . . . . . . .
Cysteine-Threonine Selection . . . . . . . . . . . . .
Lysine—Threonine Selection . . . . . . . . . . . . . .

Interactions Among Lysine, Threonine, Cysteine, and
Methionine . . . . . . . . . . . . . . . . . . . . . . .

Reversal of Analog and Feedback Inhibitor—Induced
Growth Rate Depression by Methionine and Aspartate

Pathway Intermediates. . . . . . . . . . . . . .
IV. SELECTIONS AND RESISTANCE PROPERTIES OF RECOVERED VARIANT
CELL LINES . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . .

Ethionine Selection and Variant Characterization . . .
Selenomethionine Selection and Variant Characterization. .
Cysteine-Threonine Selection . . . . . . . . . .

V. AMINO ACID ANALYSIS OF WILD TYPE AND VARIANT CLONAL
SELECTIONS . . . . . . . . . . . . . . . . . . . . . . . . . .

Methionine-Specific Assays . . . . . . . . . . . . . . . .
Ion Exchange Chromatography. . . . . . .
Total Amino Acid Profiles.

VI. SHOOT AND ROOT MORPHOGENESIS IN POTATO TISSUE CULTURES .

APPENDIX A. PATTERNS OF AMINO ACID DISTRIBUTION IN WILD TYPE
TUBERS, LEAVES, AND CALLUS CULTURES AND IN VARIANT CALLUS CULTURES
OF TETRAPLOID S. TUBEROSUM CV 'SUPERIOR'. . . . . . . . . . .

APPENDIX B. PATTERNS OF AMINO ACID DISTRIBUTION IN WILD TYPE
TUBERS, LEAVES, AND CALLUS CULTURES AND IN VARIANT CALLUS CULTURES
OF DIHAPLOID S. TUBEROSUM CLONE 'WIS AG-231 US-W973'. . . .

BIBLIOGRAPHY. . .

iv

60
60
61
63

67

69

73
73
73
80
82

84
84
85
91
100

104

109

119

LIST OF TABLES

Relief of ethionine-induced growth inhibition by
methionine in 'W-973' callus cultures. . . . . . . . . .

Influence of various concentrations of cysteine and

threonine on growth rate in 'W-973' potato cell cultures .

The influence of lysine, threonine, cysteine, and
methionine and their interactions on growth in 'W—973'
potato cell cultures . . . . . . . . . . . . . . . . . .

Relief of analog and feedback inhibitor-induced growth
depression in 'W-973' potato cell cultures by methionine
and methionine precursors. . . . . . . . . . . . . . . .

Long term resistance to growth inhibition by ethionine-
resistant potato clones. . . . . . . . . . . . . .

Resistance of selenomethionine-selected potato clones
to growth inhibition by selenomethionine, ethionine,
and lysine plus threonine. . . . . . . . . . . . .

Correlations among ethionine stressed or nonstressed
growth rates, methionine concentrations, and total
amino acid levels for wild type and ethionine resistant

variant cell lines derived from cultures of clone 'w-973'.

Measures of free and total methionine in 'W—973' cell
line derivatives and whole plant organs. . . . . .

Measures of free and total methionine in 'Superior' cell
line derivatives and whole plant organs. . . . . . . . .

. 52

. 63

. 68

7O

75

, 81

, 95

97

98

10.

11.

12.

13.

14.

LIST OF FIGURES

Regulation of aspartate family amino acid synthesis
in higher plants . . . . . . . . . . . . . . . . . .

Time course of the effect of ethionine concentration
on fresh weight increase in 'W—973' potato cell
cultures 0 O O O O O O O O O C O O O O O O O O O O O O 0 O 0

Time course of cell doubling in 'W—973' potato cell
cultures as affected by ethionine concentration . . . . . . .

Effect of methionine concentration on growth rate in
'w-973' potato cell cultures. 0 O O O O O C O O O O O O O O O

Lysine-induced growth inhibition in 'W—973' potato
cell cultures . . . . . . . . . . . . . . . . . . . . . . .

Threonine-induced growth inhibition in 'W-973' potato
cell cultures . . . . . . . . . . . . . . . . . . . . . . .

Effect of cysteine on growth rate in 'W-973' potato
cell cultures . . . . . . . . . . . . . . . . . . . . . . .

Selenomethionine toxicity in 'W-973' potato cell cultures .

Growth rate inhibition in 'W—973' potato cell cultures
as a function of ethionine concentration. . . . . . . . . .

Competitive interactions between ethionine and
methionine in 'W—973' potato cell cultures. . . . . . . . .

Reversal of ethionine—induced growth inhibition in
'W—973' potato cell cultures. . . . . . . . . . . . . . .

Inhibition of growth rate in 'W—973' potato cell
cultures by glutamyl-'y-methy1 ester. . . . . . . . . . . .

Response surface topography of growth inhibition in
'W-973' potato cell cultures induced by lysine plus
threonine . . . . . . . . . . . . . . . . . . . . . . . . . .

Stability of resistance to ethionine among clone E1 deri-
vatives as a function of duration and intensity of selection.

10

24

26

. 32

35

38

4O

. 46

. 49

. 53

55

58

65

77

I. THE POTATO AS AN EXPERIMENTAL SYSTEM FOR IN VITPO SELECTION OF

ENHANCED METHIONINE PRODUCTION

Introduction

 

The potential for crop improvement through in vitro selection of
specific traits has been much heralded in the literature (56, 80). The
prospect of generating de novo a useful character in an agricultural
crop for which an appropriate gene does not exist is very appealing.
Such a character might overcome a specific defect in a crop or adapt
the crop to a new environmental situation. In recent years interest in
somatic cell genetics has accelerated as the number of realistically
manipulatable plant species has expanded, as new applications and the
means for achieving those applications are recognized, and as examples

of successful in vitro genetic modifications accumulate.

Plant Cell in vitro Selection Systems

 

For plant cells as well as for microorganisms successful selection
systems require that rare variants be easily and unambiguously
distinguished from a wild type background. While physiological
advantage is the usual basis for selection, other distinguishing
properties such as morphology, fluorescence, or production of a
colored product might be used. Most selection systems require the
creation of a stress to which resistant genotypes are sought. Growth

advantage is the usual criterion for assessing resistance; however,

1

for selection strategies based upon lethal synthesis, it is the absence
of a function or a state of metabolic quiescence which confers ‘
resistance.

It is axiomatic that resistance to any stress can be selected
without prior knowledge of the mechanism underlying such resistance.
Selection for resistance to stresses such as sustained high or low
temperature, high osmoticum, anoxia, heavy metals, pathotoxins, and
many herbicides and antibiotics is performed with only a rudimentary
knowledge of how a resistant cell might cope with the stress.
Alternatively, an understanding of the biochemistry involved in a
metabolic process can provide a basis for devising a straightforward
strategy capable of selecting a particular phenotype directly. The
latter approach is especially well suited to selecting for resistance
to analogs of normal metabolites as well as to the products of lethal
syntheses. In these instances the target molecule is frequently an
enzyme. Tests for altered regulatory properties or catalytic function

are direct and can often indicate the nature of a mutational change.

Manipulation of Solanum species in vitro

 

Until recent years the potato has not been a particularly strong
candidate for tissue culture manipulations. Early reported difficulties
with potato tissue cultures are primarily attributable to investigators'
restricted use of cultivated varieties. Once the focus was broadened
to include wild germplasm and hybrid derivatives between S. tuberosum
and wild or less developed species, almost all of the in vitro
techniques established for other species could be performed with one

or more tuber-bearing SOZanum clones. This technology has since been

extended to material which is taxonomically S.tuberosum and in certain
instances to agronomically important cultivars as well.

In vitro propagation through axillary shoot proliferation is now a
routine procedure for all potato cultivars (103). A refinement of this
procedure has proved useful for virus elimination (93). Shoot regene-
ration from unorganized tissues, however, is somewhat more difficult.
Shoot regeneration from tuber explants has been reported for several
cultivars (47,54), and regeneration from callus cultures has been
achieved with certain clones (5, 55). Experiments leading to shoot
regeneration from true roots will be described in a subsequent chapter.

Considerable effort has been invested in defining conditions
conducive to protoplast production and culture. Upadhya (99) first
described procedures for reproducible protoplast release and prolifera—
tion. Grun (38) later succeeded in regenerating an entire plant from
protoplast-derived cultures from a S.phureja X S.chacoense hybrid.
Subsequently,shoot regeneration was described for dihaploid S.tuberosum
clones (7, 101).

The elaborate protocols described by Shepard (82-84) now permit
high frequency regeneration from protoplasts originating from
commercial tetraploid cultivars. Concomitant with advances in proto-
plast technology, systems have been developed for somatic cell fusion.
Butenko has utilized fusion between protoplasts produced from regenera-
tive leaf mesophyll from S. chaooense and nonregenerative callus cell
protoplasts from S.tuberosum as a means for rescuing the S. tuberosum
cell line from culture (14). Potato protoplasts have also been fused
with tomato protoplasts to produce plants exhibiting features of each

progenitor species (64).

Interest in breeding systems utilizing dihaploid germplasm
extracted from tetraploid clones has encouraged the development of
anther culture procedures suitable for potatoes (29, 44, 92). Many
species, including S. tuberosum,have been demonstrated to be capable
of producing dihaploids and/or monohaploids through anther culture
but response is highly genotype dependent. Indeed, heritable
components conferring androgenic response in anther culture have been
recombined among dihaploid S. tuberosum clones to produce clones with
exceptionally high rates of haploid embryoid production (46).

As a consequence of the in vitro techniques developed for potato,
selection of variant cell types has already been performed. A cell
line of S. stenotomum which is resistant to 5-methyltryptophan, an
analog of tryptophan, has been described (45). Callus and regenerated
roots from this line exhibited diminished feedback sensitivity for
anthranilate synthetase. A second S-methyltryptophan cell line derived
from 'Merrimac' which accumulates free tryptophan to nearly 50 times
wild type levels has also been described (15). Recently, potato
variants have been selected which are resistant to semi-purified
pathotoxin filtrates. Matern et al. (6) report differential sensiti-
vity among protoplast regenerates of 'Russet Burbank' to Alternaria
salami toxins. Resistant clones have remained resistant through two
tuber generations. Several 'Russet Burbank' clones of similar origin
express differential sensitivity to inoculation with Phytophthora
infestans (83). Behnke (6) has succeeded in selecting dihaploid S.
tuberosum clones in vitro which are resistant to culture filtrates of
Phytophthora infestans and Fusarium oxysporum. These examples demon—

strate the utility of potato cultures as subjects for in vitdw>selection.

The Nutritional Value of Potato Tubers : a Problem and a Proposal

 

Among world food crops the potato ranks fourth in total production
(96). Its principal contribution to human nutrition lies in its value
as a source of calories and high quality protein. Despite widely held
beliefs,the protein value of potatoes exceeds its caloric value. The
recommended National Academy of Science's daily caloric requirement for
adult males can be satisfied by approximately 3.3 kg of potatoes, a
quantity which also supplies 115% of the daily amino acid requirement
(3).

Various measures of protein quality support the potato as the
highest quality source of all major food crops (96). Schuphan (79)
has reported a biological value of 80 for potato. Rexen (72) has
calculated essential amino acid indices, EAAI, ranging from 55 to 84
for European potato varieties. Surprisingly, clones cultivated for
starch production tended to rank higher in EAAI than did clones raised
for human consumption. Kaldy (48) has calculated protein scores for
several potato clones. These varieties ranged in value from 60 to 78.

Amino acid analyses of whole potato tubers reveal methionine as the
first limiting amino acid (48,72,75). Kaldy (48) has determined an
average chemical score of 69 for potato tubers. While a score of this
magnitude is exceptionally high for a food of plant origin, improvement
of tuber methionine levels can well be expected to enhance utilization
of the remaining amino acids. Evidence that more efficient amino acid
utilization is possible derives from feeding studies with small
animals. Voles fed methionine—supplemented potato diets exhibited

markedly improved growth relative to nonsupplemented controls (73).

Calculated PER values of methionine supplemented diets were as much as
twice those of unsupplemented controls.

Partitioning the tuber into nitrogenous fractions is useful in
identifying sources of nutritional inadequacy. Kapoor et al.(50) have
separated tuber proteins into albumin, globulin, prolamin, glutelin,
and residual fractions and have performed amino acid analysis on each.
The EAAI for all fractions except prolamin were equivalent at a value
of 83. The prolamin fraction with an EAAI of 53 was identified as
limiting. To focus upon improvement within the prolamin fraction is
nonetheless unjustified since it represents no more than four percent
of the total tuber protein. The relative contributions of each protein
fraction to total protein have been assessed in S. andigena and
S. phureja X S. tuberosum progeny (90). Despite a greater than two
fold difference in total protein among the clones examined, the
proportions of each fraction were constant. Selection based upon
total protein content will therefore not improve protein quality.

The nonprotein nitrogen fraction contains a very substantial
proportion of the total amino acids within a potato tuber. The free
amino acid pool has been determined to contribute from 23% (24) to over
50%(13) of the total amino acids present in the tuber. It is noteworthy
that the nutritional value of the nonprotein fraction is inferior to
that of the protein fraction (24). The proportion of nonprotein
nitrogen to protein nitrogen is relatively constant for both high and
low protein clones cultured under similar conditions (89, 91).

Fertilization practice strongly influences the proportion of free
to protein bound amino acids. Hoff (42) nearly doubled the free pool

by increasing nitrogen application from 40 to 376 kg/ha. Under the same

conditions of fertilization, total protein increased by 36% (107).
Among free amino acids the amides increased most dramatically. Free
methionine was observed to increase by 40%; however, its relative
proportion declined. Rexon (72) also observed amino acid increases in
whole tuber digests as a result of increased nitrogen fertilization.
Very significantly, he noted that not all amino acids increased in
response to higher nitrogen levels. Among the stable amino acids was
methionine. As a consequence, the EAAI of tubers harvested from plots
receiving more fertilizer was diminished.

Variability of methionine levels among potato clones is high.

Free pool methionine levels vary as much as two fold among commonly
cultivated British clones (21). Protein bound methionine among progeny
of several S. phureja X S. tuberosum families has ranged from 0.19 to
2.69 mg/g dry tuber weight, an almost 14 fold difference (24). Luescher
has shown that tuber methionine concentration is a selectable trait
(58). His heritability estimates of 79% to 94% indicate strong
potential for a breeding approach as a means for enhancing methionine
levels in potatoes.

The preceding discussion demonstrates that from a nutritional
perspective, methionine limits the value of potatoes as an amino acid
source. Selection for increased total protein will certainly increase
the abundance of methionine, but can have only a modest effect on the
relative proportion of methionine. Overall quality will therefore not
be improved. It is presently not feasible to identify particular
polypeptides which are high in methionine content with the objective of
selecting for enhanced expression of such a gene product. Plant

breeding techniques, however, can be used to concentrate genes which,

by as yet unrecognized mechanisms, enhance methionine content in some
clones. Despite real improvement that might be realized, this approach
is constrained by the natural occurrence of genes which contribute to
methionine accumulation. These limitations have prompted an alterna-
tive proposal to directly alter cellular metabolism in such a way as

to favor methionine accumulation. The structural and regulatory
features of the metabolic pathways leading to methionine synthesis are
now sufficiently well understood as to permit the design of in vitro
selection systems capable of conferring a growth advantage to methionine
accumulating cells. The potato tuber is already highly adapted to
storing much of its amino acid content in the free pool; consequently,
it should not be necessary to require processing of additional
methionine into a polypeptide product. Finally, the techniques
necessary to transform a selected cell into a whole plant have now been
worked out for several S. tuberosum clones. For these reasons a
program with the objective of selecting potato cells which accumulate

methionine by a deregulated synthetic mechanism was initiated.

II. REGULATION OF METHIONINE SYNTHESIS IN HIGHER PLANTS

The regulation of aspartate family amino acid synthesis has
received considerable attention in recent years. The sulfur amino
acids in particular have been intensively investigated since it is
partly through the aspartate pathway that net assimilatory reduction
of sulfur occurs. While much has been learned of the mechanisms by
which inorganic sulfur becomes incorporated into cysteine and
methionine, neither the structural pathways nor the regulation of
these pathways are as yet fully understood. Yet despite occasional
inconsistencies in what is known of sulfur amino acid synthesis in
higher plant species, a fairly cohesive model of the pathway can be
constructed.

Figure 1 encompasses much of what is generally recognized con-
cerning the synthesis and regulation of the protein bound sulfur amino
acids, cysteine and methionine. Cysteine is produced by the reaction
of activated serine with a sulfhydryl donor originating from
reductively assimilated sulfur (108). The convergence of cysteine
with an activated derivative of aspartate, O—phosphohomoserine, is
mediated by cystathionine-1'-synthase. With cleavage of cystathionine
by cystathionine-B -lyase, the transfer of a sulfur atom from cysteine
to an aspartate derived carbon backbone is achieved. Methylation of
homocysteine completes the synthesis of methionine. Unlike bacteria
animany fungi in which direct sulfhydrylation from S- to activated

9

10

Figure 1. Regulation of aspartate family amino acid synthesis in

higher plants.

 

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12

homoserine is possible, transsulfuration from cysteine to O-phospho-
homoserine is the predominant, if not sole, route by which methionine
acquires its sulfur atom in higher plants. This course of methionine
biosynthesis is corroborated by 35302” tracer studies (35, 36, 59) and
by relief of inhibition at different points along the pathway with
specific intermediates (19, 96).

Although the structural aspects of sulfur amino acid synthesis
are reasonably well characterized, regulation is less clearly under-
stood. Feedback inhibition of several key enzymes serves to coordinate
sulfur amino acid synthesis with the production of lysine, threonine,
and isoleucine. Features of aspartate family feedback inhibition
which are recognized by most researchers are depicted in Figure 1,
and regulated enzymes on the course to methionine will be discussed
below.

Aspartate kinase, the first enzyme on the path to methionine,
exhibits an elaborate feedback mechanism. In most species aspartate
kinase activity is expressed by two distinct isozymes. In carrot one
isozymic species is inhibited by high concentrations of lysine
while the other loses activity in response to threonine (22, 75).
Concurrent inhibition of both activities results in a strictly
additive reduction of activity. In pea as well, lysine sensitive (74)
and threonine sensitive (2) aspartate kinase enzymes exist. Barley
also contains both isozymes although the threonine sensitive form is
not strongly expressed (74). Maize is characterized by a lysine
sensitive aspartate kinase (32). The lysine sensitive aspartate
kinases of pea, broad bean, maize, barley, and melon have been shown

to be sensitive to a product of methionine, S-adenosylmethionine,

13

which interacts synergistically with lysine to inhibit activity (74).
The consequence of these interactions with aspartate kinase isozymes is
that lysine, threonine, and methionine jointly direct the flow of
carbon through their own and through each other's synthetic pathways.

Homoserine dehydrogenase also comes under regulatory control. All
homoserine dehydrogenase activity purified from pea or barley is
sensitive to cysteine inhibition; however, forms which are either
sensitive or insensitive to threonine inhibition can be separated
(1, 2). Sensitivity to threonine occurs in maize (25, 32) and soy-
bean (106). In these species several bands of activity which include
threonine-sensitive low molecular weight forms and threonine-insensitive
high molecular weight forms can be resolved. These bands appear to be
related as an oligomeric series of the same basic unit. Inhibition
of homoserine dehydrogenase by threonine in concert with lysine inhibi-
tion of lysine-sensitive aspartate kinase is a potent technique for
inducing methionine deprivation .

The positioning of O-phosphohomoserine at a metabolic branch
point implies a regulatory property for at least one of the enzymes
which utilizes this substance as substrate. It is therefore not sur-
prising that threonine synthase is highly sensitive to the methionine
derivative, S-adenosylmethionine. Yet regulation of threonine synthase
by S—adenosylmethionine is not by inhibition. S-adenosylmethionine
acts as a strong promotor of threonine synthase activity (60). Since
O-phosphohomoserine occurs at a cellular concentration approximately
two orders of magnitude below the Km of either threonine synthase or
cystathionine—'y-synthase, promotion of threonine synthase by

S~adenosylmethionine sharply enhances threonine synthesis at the

14

expense of methionine (36). In many species enhancement of threonine
synthase activity by S-adenosylmethionine is inhibited by cysteine,
thereby providing further modulation of regulatory control (35).

The regulatory controls described for aspartate kinase, homoserine
dehydrogenase, and threonine synthase are generally recognized features
of the aspartate amino acid family in higher plants. However,
observations from other studies suggest that additional or alternative
controls exist. Soluble O-acetylserine sulfhydrylase from wheat is com-
petitively inhibited by methionine (4) while pea and clover chloroplast
O-acetylserine sulfhydrylase can be competitively inhibited by
cystathionine (70). In maize suspension cultures or in partially
organized maize callus cultures B-aspartylsemialdehyde dehydrogenase
is suppressed by methionine (32). In Pisum isoleucine enhances
activity of threonine-sensitive aspartate kinase (2) but strongly
inhibits homoserine kinase (36). It is particularly interesting that
O-phosphohomoserine dependent cystathionine-Y -synthase-activity in
Lemma increases several fold in response to lysine plus threonine
treatment (96). This observation is consistent with enzyme induction.

Much of the information concerning regulatory properties in the
aspartate amino acid pathway is based upon in vitra characterization
of partially purified enzymes. While information of this nature is
useful, it does not necessarily reflect the situation in viva. One of
the first assessments of in viva aspartate family regulation was per-
formed with Mimulus seedlings (39). In this system lysine or threonine
alone decreased 14C-leucine incorporation into protein while their
combination led to the rapid cessation of all protein synthesis.

Addition of methionine, however, totally negated inhibition of protein

15

synthesis. By feeding with 14C-aspartate and analysing radioactive
products, it could be shown that lysine plus threonine had suppressed
methionine synthesis.

Barley and wheat embryos as well are subject to growth inhibition
by lysine plus threonine (10). That intermediates leading to methionine
production are depleted is substantiated by reversal of growth inhibi—
tion when homoserine or homocysteine are provided. Similarly, growth
inhibition in maize embryos (37) and Lemma (19) induced by lysine plus
threonine is relieved by methionine or methionine precursors. Growth
inhibition in Lemma induced by propargylglycine, an inactivator
cystathionine-(B-synthase, or by aminoethoxyvinylglycine, an inhibitor
of cystathionine--y-lyase, are also relieved by methionine or the
appropriate methionine precursors (19).

Intracellular regulation can be complicated by compartmentation
of substrate pools or of isozymes with distinct regulatory properties.
In actively photosynthesizing chloroplasts, aspartate family amino acid
synthesis proceeds rapidly, indicating light stimulation of the process
(65). Isolated chloroplasts appear to contain the requisite enzymes
for synthesis of all aspartate family amino acids as well as for
cysteine. Many of these enzymes are subject to feedback inhibition.
While few reports concerning isozyme localization are available, the
conjunction of specific isozymes with particular substrate pools can
influence synthetic rates. Homoserine dehydrogenase occurs in both
chloroplasts and cytosol (lOO). Differential regulation is indicated
by threonine inhibition of the chloroplast enzyme while the cytosol
enzymes remains insensitive. In contrast cytoplasmic homoserine

dehydrogenase is subject to cysteine inhibition while chloroplast

16

activity is relatively insensitive. A second enzyme, O-acetylserine
sulfhydrylase, has been isolated from the chloroplast stroma of some
species and from the cytoplasm of others (70). Finally, homocysteine
methyltransferase has been identified in both mitochondria and
chloroplast preparations (36). Multiple substrate pools also occur.
Free cysteine has been identified in the chloroplast, the cytosol, and
the vacuole (59). The pool which most likely contributes to net
methionine synthesis occurs in the chloroplast. Additionally,
S-adenosylmethionine is found in the vacuole where it may act as a
methionine reserve (36).

A further complication to aspartate family regulation relates to
regulatory differences associated with different developmental states
of tissues and organs. The relative susceptibility of carrot aspartate
kinase to lysine or threonine inhibition is a direct consequence of the
proportions of threonine-sensitive and lysine-sensitive isozymes
present (22, 75). These levels vary among fresh root tissues, cultured
root discs, arid embryogenic suspension cultures at different times
after subculture. Aspartate kinase activities in soybean cotyledons
and suspension cultures also differ in their sensitivity to lysine
inhibition (106). Differences among shoots, roots, kernels, callus and
suspension cell cultures of maize to methionine inhibition of
B-aspartylsemialdehyde dehydrogenase have also been described (32).
Threonine sensitivity of homoserine dehydrogenase is dependent upon
developmental stage in wheat (100) and maize (25) such that progressive
desensitization occurs shortly after germination.

A particularly poignant example of the dependency of enzyme

activity upon physiological state is given by anthranilate synthetase,

17

an enzyme in the tryptophan synthetic pathway. A tobacco cell line
isolated as resistant to 5-methyltryptophan accumulated tryptophan to
levels 25 times those occurring in wild type cells. Whole plants
regenerated from this line fail to express the tryptophan accumulator
phenotype, yet cultures reinitiated from leaf explants from these
plants resume tryptophan overproduction (106).

The regulatory features of the aspartate amino acid family
discussed in this section point to a highly integrated system of
controls which coordinately buffer the cell against wide fluctuations
in the synthesis of any particular end product. Resistance to
fluctuation is expressed by control redundancy in certain branches of
the pathway. Further complicationg the situation are higher level
controls which compartmentalize substrate pools, localize differen-
tially regulated isozymes to separate regions of the cell, and alter
the production or sensitivity of particular enzyme activities as the
cell varies in its physiological mode.

Despite the intricate regulation of end product synthetic rates
in the aspartate family pathway, the system is nonetheless vulnerable
to genetic manipulation. With respect to methionine synthesis,
enhanced production might result by altering sites of allosteric
interactions between enzymes and their feedback inhibitors or pro-
motors. Pertinent targets might be aspartate kinase isozymes,
homoserine dehydrogenase, or threonine synthase. Alternatively, the
activity of methionine adenosyltransferase might be reduced so as to
concurrently alleviate S-adenosylmethionine enhanced inhibition of
lysine sensitive aspartatekinase and its promotion of threonine

synthase. Each of these mechanisms should facilitate the flow of

18

carbon to methionine and lead to expression of a methionine accumulator

phenotype.

III. DEVELOPMENT OF IN VITRO PROCEDURES FOR SELECTION OF METHIONINE-

ACCUMULATING POTATO CELL LINES

Introduction

 

The determination of optimal in vitra growth conditions is a
necessary prerequisite to any study which relies upon cell growth rate
as a measure of the effectiveness of treatments designed to repress
growth or to relieve growth inhibition. For potato cell cultures
requirements for optimal growth vary from clone to clone and must be
empirically determined for each clone investigated. The development
of conditions promoting rapid growth rate in two potato clones

follows.

Optimization of Cell Growth in Dihaploid Potato Clone 'W-973'

Several dihaploid (2x = 24) potato selections recovered as
parthenogenic offspring from tetraploid S. tuberasum clones were pro-
vided by the USDA, ARS, North Central Region Potato Introduction
Station at Sturgeon Bay, Wisconsin. Potato plants were raised in the
greenhouse, and newly mature leaves were harvested for callus initia-
tion. As a first approximation of an appropriate culture medium,
surface disinfested tuber cortex, petiole, and lamina sections were
placed on media prepared with Murashige and Skoog inorganic components
(68), Nitsch and Nitsch vitamin components (69), 30 g/l sucrose, and

either 15 mg/l indoleacetic acid (IAA) or 2 mg/l 2,4-dichlorophenoxy-

19

20

acetic acid (2,4-D). Cultures were placed in the dark at 250 C and
evaluated after ten weeks of growth. Among six dihaploid clones
examined, two clones derived from tetraploid seedling clone 'Wis AG—
231' proliferated as uniformly soft, cream-colored callus. One of
these clones,'US-W 973', hereafter referred to as 'W-973', prolifera-
ted especially well on medium containing 2,4—D. This clone was used
in all subsequent work involving dihaploid potato cultures.

Growth factor requirements were optimized by examining growth
rate in a factorial design composed of benzylaminopurine (BAP) at
0, 0.02, 0.1, and 0.5 mg/l and 2,4-D at 0, 0.5, l, 2, 3, 4, and 5
mg/l. Growth assessment was performed as described in a later
section titled 'Growth Assay'. The generation time varied from 8.7
days to 14.4 days. The growth rate deemed optimal was provided by
3 mg/l 2,4—D without cytokinin. This hormone level resulted in a
9.4 day generation time, equivalent to a growth rate of 0.106
doublings per day. Callus on this medium was soft, bright, cream—
colored, and free from large cell aggregates. Although treatments
containing BAP as well as 2,4-D grew somewhat more rapidly, the
resultant callus morphology was hard, compact, and therefore inap-
propriate for growth rate assessments. Results of further growth
optimization experiments fixed sucrose at 30 g/l and nitrogen provided
as ammonium and nitrate at concentrations recommended by Murashige
and Skoog. A complete amino acid mixture equivalent to 0.75 times
that found in the free amino acid pool of potato tubers further
accelerated growth; however, 1000 mg/l glutamine proved a simpler
substitute and yielded a similar response.

The optimized basal medium on which all 'W-973' callus cultures

21

were maintained consisted of Murashige and Skoog inorganic salts,
Nitsch and Nitsch vitamins, 30 g/l sucrose, 3 mg/l 2,4-D, and 1000
mg/l glutamine and was designated D3 + Glnlooo medium. Growth on
this medium proceeded at a rate of 0.17 doublings per day for a
generation time of 5.8 days. While a generation time of this magni-
tude appears slow compared to 24 hours for well established soybean
suspension cultures, the rate is quite rapid for potato cultures.
Over the course of three years the growth rate of 'W—973' callus on
D3 + GlnlOOO medium gradually declined. Within this period the ploidy
status of these cells had also changed. By two years of culture the
chromosome number of the majority of cells had increased to approxi-

mately the tetraploid level with aneuploids predominating.

Optimization of Cell Growth in Tetraploid Potato Clone 'Superior'

 

The 'Superior' clone of S. tuberasum was selected because of its
culturability and its reported regenerability following up to one year
in culture (54,55). This feature was deemed critical if whole plants
were later to be established from selected variant cell lines.

Cultures initiated from 'Superior' originated from greenhouse
grown plants or tubers derived from premier certified stock 'seed'.
Explants from either tuber cortex or stem produced the callus used for
growth optimization. As an initial approximation of satisfactory
culture conditions, the complex medium described by Lam was examined
(54,55). This medium contained 0.5 mg/l benzylaminopurine (BAP),

0.01 mg/l kinetin, 0.2 mg/l gibberellic acid, and 10 mg/l naphthalene
acetic acid (NAA). Under our conditions mcre rapid growth rates were

consistently observed when cytokinins and gibberellic acid were

22

omitted and when 2 mg/l 2,4-D was included in the culture medium.
Under the latter growth factor regime callus was softer and more
uniform in texture as well. The inclusion of 1000 mg/l glutamine and
0.3 mM adenine sulfate further enhanced growth rate. The standard
medium on which 'Superior' callus was routinely subcultured consisted
of Murashige and Skoog inorganic salts (68), Nitsch and Nitsch
vitamins (69), 30 g/l sucrose, 10 mg/l NAA, 2 mg/l 2,4-D, and 1000
mg/l glutamine. This medium was designated N10 D2 + GlnlOOO' Gluta—
mine was frequently reduced to 500 mg/l during culture maintenance

to decrease subculture frequency. Cultures were routinely maintained
at ambient room temperature and illuminated with 40 - 80 foot-candles

of fluorescent light.

Growth Assay

 

Accurate assessment of growth is essential to understanding the
mechanisms by which growth processes are affected. Without a univer-
sally recognized method of reporting growth in a form which is

compatible with all experimental conditions, comparisons of the
effects of different growth promotors or inhibitors among unrelated
tissues cultured under dissimilar conditions is not possible.
Unfortunately, almost all reports in the plant literature which
describe the effects of growth promotors or inhibitors on cell
cultures are meaningful only within the rigid confines of the

specific experimental conditions defined by the investigator. This
dilemma is due to the common practice of reporting growth promotion or
inhibition as percent increase or reduction of control growth at an

arbitrary point in time following initiation of the experiment. The

23

flaw in this approach is to have compared two points from an exponen-
tial growth curve in linear terms without having first transformed to
linearity. With this approach an apparent 50% growth reduction attri-
buted to a particular concentration of inhibitor can just as easily be
reduced to an apparent 20% growth reduction by shortening the
duration of the experiment. Alternatively, it can be increased to an
apparent 90% growth reduction by extending the duration of the experi-
ment. Figure 2, a plot of fresh weight increase over time as
affected by various concentrations of ethionine, illustrates this
situation. The apparent percent reduction in fresh weight increase

- attributable to any level of ethionine is wholly dependent upon the
time at which the comparison is made. Data reported in this manner is
highly subject to manipulation and lacks real meaning.

Among the few investigators who legitimately report their results
on a linearized basis such as change in growth rate (doublings per
unit of time) or change in generation time, almost all assume constant
growth velocity from initiation to completion of an experiment.
However, when growth is monitored over frequent time intervals, growth
rate can often be shown to vary over the course of an experiment.
Figure 3, a linear transformation of Figure 2, demonstrates that
growth rate was in fact not constant for all treatments throughout the
experimental growth period. The initially steep slope of all treat—
ments receiving ethionine diminished as ethionine toxicity appeared.
Following an equilibration period of approximately five days, the
growth rate became constant and accurately reflected the level of

inhibitor present. For other systems an initial lag phase followed

24

Figure 2. Time course of the effect of ethionine concentration on

fresh weight increase in 'W-973' potato cell cultures.

fresh
weight
increase

(mg)

700

600

500

400

300

200

100

 

 

 

 

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0.01
0.03
0.10
0.30
1.0
3.0
10.0

23233223

10 15

days of culture

//
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20

25

26

Figure 3. Time course of cell doubling in 'W-973' potato cell cultures

as affected by ethionine concentration.

4.0
3.0
cell
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2.0
1.0

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0.3
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25

28

by a period of constant exponential growth and a later growth rate
decrease may be noted. For some of the inhibitors investigated in this
project, high concentrations induced an initial growth increase
followed by an apparent negative growth rate as cells died and released
their contents into the medium. Such negative growth was later
followed by growth stasis. From these examples it is apparent that an
accurate growth rate can be determined only within a time frame during
which growth proceeds at a constant rate.
To assess growth rate in potato cells a nondestructive method
was devised in which growth of all cells within an individual callus
could be followed without disturbing the orientation of cells within
‘the callus or of cells at the callus-culture medium interface. This
was accomplished by culturing cells on a nitrocellulose filter which
could be aseptically weighed at convenient time intervals. Nitro-
cellulose filters are rigid enough to be easily transported to a
sterile petri dish for weighing. Because they absorb a fixed amount
of liquid from the agar medium, the weight of filter plus liquid,
recorded prior to transfer of cells to the filter, can be subtracted
from the measurement of callus plus filter weight.
Cultures were routinely prepared by transferring approximately

40 mg of cells to a filter which had previously been saturated with
liquid, lightly blotted, and weighed. These cultures were then
weighed in a tared petri dish on an analytical balance in a laminar
flow hood. Subsequent weighings were performed at carefully
monitored time intervals. When weights were recorded to the nearest
tenth milligram, the method proved to be extremely sensitive to very

slight growth increments. Because of this, a growth mate as low as

29

two orders of magnitude below that of uninhibited cells could be
detected within a 20 - 30 day assay period.

Cell growth proceeding at a constant rate can be accurately
described as y = a2” where y is the fresh weight increase from time
7‘: = 0, a is the initial fresh weight, and IV is the number of mass
doublings per unit of time. Growth rates for individual callus
cultures in all experiments were calculated by a least squares
exponential regression procedure. The regression equation was

2 t-Zm y - -E¥£—EJELJL
b a. m Zn 2

2.2 - 1723
m

 

where growth rate, b, equals N/t, the number of doublings per unit of
time and m is the number of time points sampled within the exponential
phase of growth. The unit of time was standardized to days; b is
therefore reported as doublings per day.

In preliminary experiments designed to characterize normal and
inhibited growth of W—973 cells, frequent time intervals of 4 or 5
days were sampled throughout a period of up to six weeks. These
experiments indicated that constant growth rates occurred within the
period of approximately eight to thirty-five or forty days following
culture initiation. Therefore culture weighings were routinely
performed between days 10 and 30. A minimum of three sampling points
was used for all regressions calculated. The coefficient of determi-
nation served as a useful check that growth rate was indeed constant.
Rarely did this indicator fall below a value of 0.98. Experimental

treatments were typically replicated at least four times while control

30

treatments were replicated a minimum of eight times.

The physical properties of cells used in growth assays were
critical to the accuracy of growth determinations. Only exponential
phase callus which was soft, finely dispersive, and brightly cream—
colored was used. To overcome difficulties due to somaclonal
variation, callus for an entire experiment or for an entire replica-
tion was thoroughly mixed to assure uniform response.

Preparation of culture medium was accomplished by autoclaving a
pH adjusted,concentrated version of the final medium devoid of all
amino acids and/or growth inhibitors. These were prepared as pH
adjusted, concentrated stocks and were filter-sterilized before their
addition to the basal medium. All cultures on which growth assays
were performed were placed in a dark incubator maintained at 240 -

25° 0.

Effects of Aspartate Family Amino Acids on Growth of Potato Cultures

 

Methionine

A selection strategy dependent upon methionine accumulation
should be designed so as to permit growth of variant clones which
synthesize product at levels that do not adversely affect normal
growth processes. This requires an estimate of methionine levels
which are toxic to cell growth. While growth inhibition by exoge-
nously supplied methionine can provide a minimal estimate of the
concentration at which toxicity occurs, information provided by a
methionine growth inhibition curve is useful in studies designed to
determine whether methionine can overcome growth inhibition due to

competition with methionine analogs or to feedback-induced methionine

31

deficiency.

The effect of methionine on growth rate in 'W~973' cells was
determined by culturing cells on D3 + Gln1000 medium containing
methionine at concentrations ranging from 0 to 30 mM. Cultures were
prepared, maintained, and assayed as described under 'Growth Assay'.

Figure 4 depicts the response of potato cell cultures to
supplemental methionine. Concentrations between 0.01 and 0.1 could
not be distinguished from the control (<1= 0.05) while higher concen-
trations resulted in growth depression.

Since the cells were not assayed for methionine content, the
degree to which they concentrated methionine from the culture medium
cannot be discerned. Other amino acids have been shown to be trans-
ported by energy dependent, saturable permease systems which function
at amino acid concentrations of up to approximately 5 mM (9, 63).
These systems can concentrate an amino acid from 10 to nearly 100
fold, depending upon the uptake phase of the permease. At still
higher amino acid concentrations an energy independent, unsaturable
uptake system is noted; this system may be primarily diffusional.
Therefore at very high levels of supplemental methionine, the cell's
endogenous methionine level may more closely relate to the concentra-
tion provided to the culture medium.

A lower limit estimate of the highest intracellular methionine
concentration which fails to evoke toxicity can be inferred from free
pool concentrations in cells in which methionine occurs in greatest
abundance, i.e. tuber cells. Assuming potato tubers to be 80% water,
the free methionine pool in 'W-973' tubers can be estimated at 1.14 mM

(see Appendix B). A comparison of this estimate with the highest

32

Figure 4. Effect of methionine concentration on growth rate in 'W—973'

potato cell cultures.

33

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34

extracellular level of methionine which does not elicit toxicity
leads to the conclusion that potato cells are capable of concentra-
ting methionine by a factor at least ten times that provided in the
culture medium. This estimate, of course, is valid only for methio-
nine levels for which transport is permease dependent. As will
later become apparent, the limit of potato cell tolerance to supple-

mental methionine is less than for lysine, threonine, or cysteine.

Lysine

Lysine-induced growth inhibition was examined by assaying growth in
'W-973' callus cultured as described under 'Growth Assay' on D3 i;
Gln1000 medium supplemented with lysine from 0 to 100 mM. Inhibition
curves relativized against control growth rates appear in Figure 5.
It is clearly evident that glutamine relieves lysine-induced growth
inhibition, particularly at weakly toxic lysine levels. A 50% growth
rate reduction was noted at 25 mM in glutamine—free medium while in
the presence of glutamine, this level of growth rate depression
occurred at 60 mM. The phenomenon of glutamine-conferred protection
against growth inhibition will later be discussed in relation to
other amino acids and analogs. While the basis for protection is
unknown, glutamine is known to competitively affect uptake of other
amino acids in cells cultured in its presence (18). It is therefore
conceivable that in cells cultured in the presence of glutamine, the
internal pool of competing amino acid is effectively reduced. Never-
theless, glutamine was included in most culture media because of its
effect in accelerating growth rate. While not apparent in Figure 5,

control growth rate in medium containing glutamine was 20% greater

35

Figure 5. Lysine-induced growth inhibition in 'W-973' potato cell

cultures.

36

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37

than that in glutamine-free medium.

Threonine

The influence of threonine on growth rate in 'W—973' potato cell
cultures was examined over a range of 0 to 100 mM on D3‘:_Gln1000
medium. Growth rates relativized against glutamine-supplemented or
glutamine-free controls are shown in Figure 6. As with lysine,
glutamine conferred a nearly 100 fold protection against threonine—
induced toxicity. The 50% level of growth inhibition in the absence
of glutamine was 4 mM while in its presence, 40 mM threonine was

required. Glutamine enhanced control growth rate by 19%.

Cysteine

Cysteine-induced growth depression in 'W-973' cells was examined
in cultures grown on D3 iGln1000 medium. Over the range of 0.15 to
100 mM cysteine, provided as cysteine HC1, toxicity was most prominent
in glutamine-free medium (Figure 7). At approximately 6 mM cysteine,
glutamine rapidly lost its capacity to protect against toxicity. In
glutamine-free medium 50% growth inhibition occurred at 0.8 mM;
in glutamine-supplemented medium the half maximal growth rate occurred
at 10 mM. Cell stasis or death was elicited by 4 and 15 mM cysteine
in D3 and D3 + Gln1000 medium, respectively.

Consistent growth responses were not always observed when
cysteine was included in the medium. Unanticipated differences in
toxicity between cysteine HC1 and the free base were noted; the latter
was much more toxic. Since a time dependent pH decrease in cysteine-

supplemented medium was suspected, medium prepared with the free base

38

Figure 6. Threonine—induced growth inhibition in 'W-973' potato cell

cultures.

39

00H

AH\mmHozEv :oflumuucmocoo mcflcomnnu

mm ow mm ma 0H

 

mcwfiouoam 9593?» o In.

oGHEmn—SHU cums all.

mgw

o.¢ m.N

 

m

H o.H

mo.

ow.

mm.

mH.

ov
om
on much
:u3oum
or Houucou
mo
unmouoa
om
om

ooa

40

Figure 7. Effect of cysteine on growth rate in 'W-973' potato cell

cultures.

41

00H

 

mm

ov

mm

ma

OH

AZEV cowumuucoocou mcflmummo

m.m

o.v m.N

m.H

 

o.H mo. ow. mm.

ma.

ocfieousam usonufi3 o

mcflfiouaam muss

¢

 

out
on:
o
mush
on nu3onm
Houucoo
ov mo
unmoumm
om
cm

OOH

42

form of cysteine was buffered with MES at concentrations ranging from
0 to 20 mM. After adjusting the pH of filter sterilized cysteine and
MES to 5.7, D3 + Gln1000 medium was prepared and the pH monitored over
several days. In unbuffered medium the pH had spontaneously dropped
to 3.95 by 115 hours while at 20 mM MES the pH had only decreased to
5.42. The inclusion of 20 mM MES in culture medium supplemented with
cysteine only slightly reduced cysteine toxicity. Despite this weak
relief MES was provided in several experiments involving cysteine-
induced growth inhibition.

A further difficulty encountered with cysteine was the development
of a dense precipitate at high cysteine concentrations. Cysteine is
known to be catalytically oxidized by ferrous ion; indeed, deletion of
iron and microelements from the culture medium diminished the rate at
which precipitation occurred. As a consequence of cysteine precipita-
tion, the quantity of cysteine that was available to the cells could

not be accurately known.

Selective Systems Based upon Methionine Analogs

 

Preliminary Considerations

An understanding of the regulatory controls discussed in the pre-
ceeding chapter allows one to construct selection schemes in which the
overproduction of methionine might confer growth advantage. One
useful approach is to provide structural analogs of methionine to
a cell population. Presumably the analogs will be transported into the
cell where they will be perceived as methionine. As the analogs

interact with the cell's metabolic machinery, they may inhibit

43

certain metabolic processes and/or they may be used to produce defec-
tive products, often with the expenditure of considerable metabolic
energy. With regard to analogs, any of a number of mutations which
might increase the quantity of intracellular free methionine,and thereby
lead to effective methionine-analog competition, could be expected to
enhance cell proliferation of a variant clone. Alternatively, a
scheme based upon artificially induced methionine starvation through
concerted feedback inhibition might result in selection of a mutant
with relaxed feedback control. Resumption of synthesis of the
precursors required for methionine production could permit such a
cell to grow while wild type cells would die. Selection based upon
relaxed feedback inhibition would be expected to involve fewer
mutational classes than selection for analog resistance because
desirable mutations downstream from the target feedback genes would
be unable to be expressed.

Although one can develop selective schemes which favor growth
of methionine overproducers, other mutational classes might be repre-
sented among variant cell lines capable of resisting the selection
pressure. Permease mutants in which the uptake of feedback inhibitors
or methionine analogs is impaired could escape growth inhibition.
Improved enzyme specificities capable of distinguishing between
methionine and its analog(s) would also confer resistance. Increased
relative specificity of methionyl—tRNA synthetase for methionine would
result in reduced analog incorporation into protein. Similarly,
improved specificity of methionine adenosyltransferase or other methyl

transferases would prevent an analog such as ethionine from propagating

44

its alkylating effects to other metabolic pathways. Finally, an
epigenetic (or even possibly stable) resistance might be conferred by
the acquisition of a mode in which synthesis of inhibited enzymes is
elevated or by induction of a feedback insensitive isozyme not
normally produced by the cell type under selection. Assumption of a
dormancy mechanism in which overall metabolism during the period of
selection is reduced could also result in an apparently resistant

variant.

Selenomethionine

Selenomethionine serves as an analog of methionine by virtue of a
selenium substitution for sulfur. Its incorporation into protein has
been examined in order to determine whether translational processes
are affected. In Vigma radiata, a plant species sensitive to selenium,
selenomethionine is efficiently charged onto methionyl-tRNA,and
ribosome binding is normal (26,27). Peptide formation, however, is
reduced. This reduction is especially pronounced when selenomethionine
is charged to methionyl-tRNA-l, the polypeptide initiator species,
with the result that polypeptide initiation is frequently aborted.
Despite this effect, selenomethionine is freely incorporated into
protein. E. caZi proteins in which selenomethionine has largely
replaced methionine function at near normal levels. In hepatoma
cells selenomethionine increases the heat lability of enzymes into
which it has been incorporated (52). Methionine adenosyltransferase
utilizes selenomethionine to produce Se-adenosylselenomethionine.
Methyl transferases can transfer the methyl group of this species to

methyl acceptors.

45

Resistance to selenoamino acids has been described for tobacco
cultures (28). Two mutants resistant to selenocystine alone and one
mutant resistant to selenocystine and selenomethionine in combination
were isolated from cultures treated with both analogs. The mechanism
of resistance in these mutants has not been determined.

To assess conditions under which cell selection for resistance
to selenomethionine might be performed, the toxicity of this compound
to 'W-973' cells was determined. Cultures which had been prepared

as described under 'Growth Assay‘ were transferred to D3 + GlnIOOO

medium supplemented with selenomethionine at concentrations ranging
from 0 to 10 mM. Growth rates expressed as a percentage of control
growth rate are presented in Figure 8. The selenomethionine inhibi-
tion curve displays an unusually sharp transition between innocuous
and highly toxic concentrations. A response of this nature leads to
difficulty in accurately assessing the degree to which growth rate is
reduced within the transition portion of the curve. Fifty percent
growth rate inhibition occurs near 0.3mM selenomethionine while

ninety percent depression is caused by approximately 0.7 mM analog.

Ethionine

Ethionine is a structural analog of methionine in which an ethyl
group is substituted for the methyl moity of methionine. This analog
is effectively incorporated into protein, but little is known concer-
ning its effects on enzyme activity (30). Perhaps the most pervasive
effects attributable to ethionine derive from its activity as an
ethyl donor in reactions which normally accept methyl groups.

S-adenosylethionine is readily synthesized by yeast and Caprinus

46

Figure 8. Selenomethionine toxicity in 'W—973' potato cell cultures.

47

OOH

O.H

AH\mmHOZEO wchoHsuoEocmem

H.O

H0.0

HO0.0

 

O
ON
ow
cums
on £u30Hm
unmoumm
0>HuoHoH
Om

OOH

48

methionine adenosyltransferase, thereby setting the stage for exten-
sive ethylation. Ethyl groups originating from S-adenosylethionine
have been identified in choline and in rat liver DNA and RNA.
Ethionine is recognized as a mutagen and displays carcinogenic
activity as well.

Resistance to ethionine can occur by a number of mechanisms (30).
A permease mutant which excludes ethionine and concurrently exhibits
resistance to a phenylalanine analog has been described in
Saccharamyces. A Neuraspara mutant is resistant by virtue of
methionine overproduction. Enhanced discrimination in methionyl-tRNA
charging has been reported in a Cbprimus mutant. Other mutants also
exist for which the resistance mechanism is not understood.

As a guide to identifying a concentration of ethionine appropriate
for variant selection, an ethionine growth inhibition curve was
established. The response of 'W—973' cell cultures prepared as
described under 'Growth Assay' was examined over the range of 0 to
10 mM ethionine. Fresh weight increase, cumulative cell doublings, and
percent growth rate relative to a control are presented in Figures 2,
3, and 9, respectively. In Figure 9 the concentration producing 50%
growth inhibition appears as 0.01 mM; 90% growth rate depression
occurred near 0.1 mM ethionine.

It is noteworthy that during the course of this experiment an
occasional callus sector appeared which grew distinctively more rapid-
ly than background cells on medium containing 0.1 mM ethionine. One
such variant which continued to express resistance was propagated and
designated clone El. These observations indicated that selection in

the vicinity of 0.1 mM ethionine might allow relatively rapid growth of

49

Figure 9. Growth rate inhibition in 'W—973' potato cell cultures as

a function of ethionine concentration.

SO

much
:u3oum

unmouom

o>HMMHmu

AH\mmHozev :oHumuucoocoo mchoHnuo

OH O.m O.H m.O H.O M0.0 H0.0 O

O
OH. ON.
ON 0‘.
on OO.
OO OO.
om OH.
OO

NH.
on

«H.
OO

OH.
OO

OH.
OOH

ON.

moo
mom
mmcaanaou
:H
mums
£u3oum

51

resistant cells which could later be analyzed under more stringent
selection pressure.

The kinetics of methionine-ethionine antagonism were assessed to
determine whether these compounds act in a competitive or noncompeti-
tive manner. Even though the biochemical process most disturbed by
ethionine is not known, the effect of ethionine on any rate limiting
step should be reflected in cellular growth rate. Therefore the
extent of antagonism can be measured. The nature of methionine—
ethionine antagonism is important because a noncompetitive inter—
action will not necessarily permit selection for resistance based
upon methionine overproduction. It was also of interest to determine
the level of supplemental methionine required to achieve a particular
level of relief from inhibition at various ethionine concentrations.
Since the extent to which ethionine and methionine are transported
and concentrated within the cell was not known, the influence of the
endogenous free methionine pool (0.24 mM in 'W-973' cells) upon
antagonism could not be determined.

From Table 1 it is evident that methionine can negate ethionine-
induced growth inhibition. An analysis of variance performed on
growth rates corrected for methionine-induced toxicity indicated
strong interaction between methionine and ethionine (F‘<0.0005). That
the nature of interaction is competitive is demonstrated by the
double reciprocal plot in Figure 10. As required of competitive
interactions, all curves converge at the reciprocal value of the unin-
hibited growth rate. Growth depression was relieved to the extent of
fifty percent for 0.01, 0.03, and 0.1 mM ethionine by 0.023, 0.046, and

0.057 mM methionine, respectively (Figure 11).

52

 

Table 1. Relief of ethionine-induced growth inhibition by methionine
in 'W4973' callus cultures.z
ethionine methionine concentration (mM)
concentration
(mM) 0.01 0.03 0.1 0.3 1.0
0 100.0 100.0 100.0 100.0 100.0 100.0
0.003 90.2 86.4 91.5 96.1 95.1
0.01 28.4 38.0 54.4 73.2 95.3 100.4
0.03 8.8 18.3 39.6 63.5 95.4 95.6
0.1 -1.8 1.8 30.7 64.5 98.2 98.3

 

2- growth rates are expressed as percentage of growth rate in ethionine-
free controls and are corrected for inhibition due to methionine.

The experiment-wise standard error is 2.6 percent.

53

Figure 10. Competitive interactions between ethionine and methionine

in 'W—973' potato cell cultures.

54

AcoHuouucmocoo mchoHnumE EEO m\H

 

HI
OOH mm 9H m.m.H
o c H OH
.\\
0
ON
. Q
on “wasp :H
oEHu
coHumnmcmmv
ov >
\H
ocacoH um 28 .O o
n. . n moo om

mchoHnuo SE aH0.0 o
9.3023 as. 85 a

2.335.... as 35 fl 8

55

Figure 11. Reversal of ethionine—induced growth inhibition in 'W-973'

potato cell cultures.

56

AZEO coHuoHucoocoo mchoHsvoE

 

 

m.O H.O m0.0 H0.0 MO0.0 O
.
D Ill‘t
ochoHnuo :5 H.O .§ .
9:533 as 85 o .N
ochoHnuo SE H0.0 .1
ochoHsuo :5 mO0.0 o
23033 as o a
o .v
moon
Susana
Houucoo
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a mo
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. 111‘:
o
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l
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ml .IIII LI .1 ml a. 4ft .3

 

(‘1

In

E. I

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57

Glutamyl-Y -Methyl Ester

Glutamyl—‘r-methyl ester (GluOMe) is an amino acid analog known
to antagonize glutamate through its activity with glutamyl-tRNA
synthetase in E. caZi. In the course of screening E. caZi for
resistance to GluOMe, mutants were isolated which mapped to genes
associated with the methionine pathway (53). These mutants also
exhibited resistance to other methionine analogs though not to
analogs of other amino acids. The cross resistance patterns to other
methionine analogs proved compatible with those of mutants known to
reside in the methionine biochemical pathway. Since GluOMe had not
previously been recognized as an analog of methionine, it was of
interest to determine whether GluOMe might exhibit methionine analog
activity in a higher plant.

An inhibition curve for GluOMe within the range of 0 - 100 mM
was determined both in the presence and absence of supplemental
glutamine. Reversal of GluOMe by methionine was also examined. As is
evident in Figure 12, the inclusion of glutamine in the culture medium
confers resistance to the analog. Whether this indicates that GluOMe
also acts as an analog of either glutamine or an immediate product of
glutamine is not clear. It is possible that activity as a dual analog
may not even be cause for great concern. Another methionine analog,
methionine sulfoximine, is also rescued by methionine in E. 001i and
has been successfully used for the selection of methionine overpro-
ducers in tobacco (16), yet this analog also acts as an analog of
glutamine.

Figure 12 shows that GluOMe toxicity is partially reversed by

58

Figure 12. Inhibition of growth rate in 'W—973' potato cell cultures

by glutamyl-~y—methyl ester.

59

OOH

   
       

om

AH\mmH02EO umumm HanumethloHoo UHEmuon

O.m

O.H

m.O

H.O M0.0

wchoHnuoE
ochoHnuoE
ocHEousHm

mchonuoE
ochoHcme
ocHEousHm

H0.0

2e O.H + v
SE H.O + iv

zeo.H+ Av
:5 H.O + *v

 

ON
Ow
moon
0
OO nusou
ucoouom
o>HuoHou
Om
OOH

60

nontoxic levels of methionine. This observation suggests that GluOMe
may well be useful for selecting methionine overproducers in plant
cells. Selections might most effectively be performed in the absence
of added glutamine since glutamine-free medium provided a greater
growth differential between GluOMe toxicity and methionine rescue.
Selection on glutamine—free medium might also identify variants which

need not produce self-inhibitory quantities of methionine.

Selection Strategies Utilizing Feedback Inhibition
Preliminary Considerations

The extensive system of feedback controls operating within the
aspartate family of amino acids has led to the development of selection
schemes capable of favoring growth in cells expressing a reduced
sensitivity to feedback regulation. Selection for concomitant
resistance to lysine and threonine has been performed in maize and
barley with the objective of isolating mutants in which lysine content
may be enhanced. Selection in partially organized maize tissue
cultures has revealed a mutant with diminished sensitivity to the
lysine sensitive isozyme of aspartate kinase (40). This mutant
expresses an approximately 1.3 fold increase in free lysine and a
four fold increase in free methionine in homozygous seed but is most
remarkable for a 75 fold increase of free threonine (C.E. Green,
personal communication). It is very likely that threonine enhance-
ment in this mutant is due to stimulation of threonine synthase
activity by elevated levels of S-adenosylmethionine ( refer to Figure
1). In experiments similar to those with maize, selections performed

with M2 embryos from mutagenized barley seeds resulted in the

tic

CY

(10

rn

61

identification of adominant gene responsible for increased concentra-

tions of lysine, threonine, and methionine (10)..

Cysteine-Threonine Selection

Reports concerning selections based upon the combined effects of
cysteine and threonine do not exist. However, feedback regulatory
controls described for homoserine dehydrogenase suggest that cysteine-
threonine selection might be useful in identifying variants with
deregulated carbon flow to homoserine. Since elevated methionine
levels might overcome cysteine-threonine inhibition, the effect of
cysteine and threonine on growth of potato cell cultures was investi-
gated.

Cysteine-threonine studies were performed with 'W-973' cultures.
Selection of cysteine and threonine levels was based upon previous
determinations of growth inhibition individually induced by these
amino acids. Cysteine (free base) levels of 3, 11,21, and 38 mM and
threonine concentrations of 1, 2.1, 3.3, and 7.2 mM had previously
been determined to result in 0, 10, 25, and 50 percent growth rate
depression, respectively. These levels of inhibition were selected
so as to provide evidence for synergism. Cysteine-threonine combina-
tions containing cysteine at 11 mM or less reduced growth rate no
more than 43% while at cysteine levels of 21 mM or more all cultures
were killed.

A subsequent experiment was performed to identify cysteine-
threonine combinations which might result in growth rate reductions
greater than the moderate levels observed yet less stringent than the

most severe. In this second experiment cysteine HCl was substituted

u.

I85

by

re

C)

(9

{is

UT}

ge

in

IE!

aaj

62

for the free base. Table 2 reveals a very different pattern of
response than that observed in the preliminary experiment. The
pattern of growth inhibition closely paralleled the response elicited
by threonine alone with little effect attributable to cysteine. This
result indicates that selection for resistance to cysteine HC1-
threonine may be of no more value than selecting for resistance to
threonine alone.

The basis for discrepancy in these two experiments is not known.
While it is tempting to attribute differences to the form or batch
of cysteine used, the results might just as easily be attributed to
differences in inherent cysteine sensitivities of 'wild type' cells.
It is well known that cell cultures maintained for long periods of
time can be separated into subclones which are distinguishable
from the parental population (56). The potato cultures designated
as 'wild type' were under continuous selection for characters
associated with good culturability. It is very possible that
subclones with altered sensitivity to amino acid inhibition were
inadvertantly selected. Even in the absence of selection for cultura-
bility characteristics, unselected differences in amino acid sensiti-
vity could occur. This possibility is supported by the frequent
observation that apparently identical subclones isolated from
apparently homogeneous regions from a single callus mass and propagated
under identical conditions for a period as brief as four or five cell
generations can respond very dissimilarly when treated as replicates
in the same experiment. Such instability in wild type populations
required frequent standardization of subclone response to individual

amino acids and analogs. Of all the amino acids and analogs

63

Table 2. Influence of various concentrations of cysteine and

threonine on growth rate in 'W-973' potato cell cultures.2

threonine (mM)
cysteine (mM)

 

 

0 3 6 15 50
0 100 62 51 37 25

100 78 62 45 34
8 86 73 48 39 33
10 74 68 50 38 35
12 82 68 57 47 35
20 81 44 42 36 29

 

z- values are growth rate means relativized against the
growth rate of cells cultured on medium devoid of
cysteine and threonine.

which have been restandardized over time, wild type cell sensitivity

to cysteincshifted most dramatically.

Lysine-Threonine Selection

End product regulation of aspartate kinase activity by lysine and
threonine results in growth inhibition due to methionine starvation.
Mutations which desensitize an aspartate kinase isozyme to feedback
inhibition should result in the resumption of carbon flow through the
aspartate pathway and thereby relieve methione deficiency. In the
absence of artificially maintained levels of lysine and threonine,
cells with altered aspartate kinase feedback sensitivity should
synthesize aspartate aminoacidsat rates greater than in regulated
cells. Selection for resistance to the joint effects of lysine and
threonine should therefore provide a means for enhancing free

methionine levels.

To determine the effects of lysine and threonine on growth of

64

potato cell cultures various combinations of these amino acids were
added to D3 + Gln1000 medium. 'W-973'cultures were prepared as
described under 'Growth Assay'. Figure 13 depicts the response of
potato cells to lysine-threonine induced growth inhibition.
Unexpectedly, the surface topography is very irregular and displays
frequent departures from what might be anticipated in a model based
upon concerted feedback inhibition. Nevertheless, the establishment
of a topographical feature by several contiguous points was deemed
as evidence for a real effect. Of particular interest is the relief
of lysine inhibition in the 85 to 140 mM range conferred by low levels
of threonine. From this observation it might be inferred that lysine
sensitive aspartate kinase represents the bulk of the total aspartate
kinase activity. Its inhibition by excess lysine would be expected to
lead to cell deprivation of both threonine and methionine. Threonine
supplementation of lysine inhibited cells may then restore growth rate
to the level observed in cells inhibited by threonine alone. At high
levels of both lysine and threonine, cell death occurs.

It has recently been reported that high concentrations of lysine
antagonize arginine dependent processes (17). In this context, growth
inhibition observed in lysine-threonine treated cells may not be

attributed wholly to methionine starvation.

65

Figure 13. Response surface topography of growth inhibition in 'W-973'

potato cell cultures induced by lysine plus threonine.

67

Interactions AmongiLysine, Threonine, Cysteine,iand Methionine

The effects of lysine plus threonine and cysteine plus threonine
on growth of potato cell cultures have already been described. It was
of interest as well to determine whether lysine, threonine, and
cysteine might depress growth rate to an even greater extent when
acting in concert. Additionally, it was necessary to determine the
degree to which supplemental methionine could alleviate growth
inhibition due to lysine, threonine, and cysteine individually and
in various combinations. A factorial experiment was designed to
assess these interactions.

'W-973' potato cultures were transferred to D3 + Gln1000 medium
containing ZJSmM MES (pH 5.7) and various combinations of lysine,
threonine, cysteine, and methionine. Distinct growth inhibition was
observed over the range of each individual amino acid except cysteine
(refer to Table 3). Methionine relieved growth inhibition due to
threonine but was not effective in reversing lysine inhibition.

Growth depression due to lysine plus threonine was not consistently
observed, nor was it regularly relieved by methionine. Cysteine
generally exerted little effect on lysine-induced growth inhibition.
However, cysteine partially reversed growth reduction due to threonine.
Interactions among lysine, threonine and cysteine were weak. Had
arginine been included in the culture medium, methionine reversal
of lysine—threonine inhibition may well have been more pronounced

(17).

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68

 

 

 

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69

_ReIeIsal_of_Analog_and_Eeedback_Inhihitorzlnduced_firomth_Rate
W_

Specific reversal of end product induced growth depression by
methionine precursors synthesized downstream of the target block
in methionine synthesis indicates that,(1)no other enzymes occurring
downstream of the feedback inhibited enzyme are also subject to
feedback inhibition by end product(s) effective against the target
enzyme and,(2)a variant which overcomes end product growth inhibition
by virtue of relaxed feedback regulation will likely synthesize
methionine at elevated rates. Similarly, reversal of growth
inhibition due to methionine analogs by methionine and methionine
precursors indicates,(1)the capacity of methionine to overcome
analog-induced growth depression and,(2)the noninterference of
analog with upstream processes in methionine synthesis. Each of
these conditions is required for analog selection of a methionine
overproducer.

To test these criteria, methionine and the methionine precursors,
homoserine and homocysteine, were challenged with methionine analogs
and with proposed feedback inhibitor selection systems. Table 4
demonstrates complete reversal of ethionine-induced growth inhibition
by a two fold excess of methionine. Very weak relief of growth
inhibition was conferred by homocysteine at ten fold molar excess.
Equimblar concentrations of either homoserine or homocysteine were
without effect. Nevertheless,higher concentrations of these compounds
might be expected to overcome ethionine inhibition. It was

unanticipated that neither methionine nor its precursors would reverse

O
7 II.
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mums a“: ammo mm 2: mm 3 am a: VH2: mmSLBEES
ass; as no aso; as H.O aso; as no 23%
mmmHHonmmmE
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71

growth inhibition due to selenomethionine. This suggests that seleno-
methionine may act noncompetitively with methionine, that toxicity
may reach into pathways outside those directly related to methionine
synthesis, or that the levels of methionine and its precursors tested
were inadequate to overcome growth inhibition. In any event, seleno-
methionine is decidedly more toxic to the cell than ethionine.

Relief of growth inhibition induced by glutamyl-YL-methyl ester
was not examined in this experiment since methionine at concentrations
well below those of the inhibitor were shown to provide substantial
reversal of growth inhibition.

End product growth depression due to 80 mM lysine plus 121m4
threonine was clearly relieved by 1 mM methionine. Homoserine and
homocysteine, however, appeared to have no statistically discernible
effect on growth.

Medium containing 2 mM threonine and 21 mM cysteine (provided as
free base) was highly toxic and could not be reversed by methionine
or methionine precursors.

The choice of an appropriate selection system useful in
identifying methionine accumulators is somewhat simplified by the
information provided in Table 4. Cysteine-threonine selection is not
tenable, primarily because of the failure of methionine to rescue
inhibited cells. With selenomethionine rescue by methionine was not
demonstrated; however very high methionine levels relative to
selenomethionine have negated toxicity in tobacco cells (28). Seleno-
methionine may still be useful, although less stringent selective
conditions may be necessary. Selections based upon lysine-threonine

growth inhibition may well prove useful in revealing methionine

72

overproducers, particularly if arginine supplementation can be shown
to increase the ability of methionine to promote growth. In this
vein it is particularly noteworthy that methionine failed to relieve
lysine-threonine inhibition in callus cultures of Arabidbpsis and
barley ( but not in barley seedlings) until arginine was supplied to
the culture medium (17).

Ethionine selection may also prove useful in identifying
methionine accumulators. Growth inhibition is strongly relieved by
methionine and less strongly so by homocysteine, and mutations at
sites in addition to the gene for aspartate kinase can result in
oversynthesis. Variants of alfalfa (71) and carrot (104,105) in
which free methionine pools appear to be elevated have been revealed
by ethionine selection.

The usefulness of glutamyl-‘Y-methyl ester as a selective agent
in plant cells also appears promising. Successful reversal of
growth inhibition by relatively low methionine supplements suggests
that mutants in which methionine need be only moderately elevated

can be selected.

IV. SELECTIONS AND RESISTANCE PROPERTIES OF RECOVERED VARIANT CELL
LINES

Introduction

 

Cell selections were performed with three of the selection
strategies described earlier. Of these strategies ethionine and
selenomethionine were anticipated to produce useful variant cell
lines. Selection for resistance to cysteine-threonine was deemed
less promising but nevertheless was performed to see what types of

variants might appear.
Ethionine Selection and Variant Characterization

A spontaneous ethionine resistant variant from 'W—973' identi-
fied among callus cultures growing on 0.1 mM ethionine has already
been mentioned. This variant, clone E1, appeared as one of several
healthy proliferating nodules on a background of deteriorating cells.
Further variants were sought using a more intense selective pressure
of 0.5 mM ethionine.

Suspension cultures of clone 'W-973' were prepared by adding
six grams (5.4 X 107 cells) of friable agar—cultured callus to 30 ml
of liquid D3 + Gln1000 medium in 125 ml erlenmeyer flasks. These
suspension cultures were placed on a gyrotory shaker operating at
125 rpm at ambient room temperature in darkness. Following six days

of acclimatization, filter sterilized ethionine was added to provide

73

74

a final dilution of 0.5 mM. Seventy days later the cultures were
decanted. The cells were spread onto fine mesh nylon microfilament
discs and transferred to nonselective solid medium for a three week
growth period. A second cycle of selection was then initiated by
transferring the nylon screens to solid medium containing 0.1 mM
ethionine.

Among 15 original ethionine-treated suspension cultures, 13
produced from three to several hundred colonies on nonselective
medium. Resistance was retained in colonies from 10 flasks following
a second cycle of selection. From each of the latter 10 flasks
a single, healthy colony was isolated; these were designated clones
E3 — E12. Thereafter these variant clones were maintained on
D3 + GlnlOOO medium. The frequency of resistance was calculated to
be 9.4 X 10’10 on the basis of the number of flasks retaining ethionine
resistance after two cycles of selection.

Although ethionine resistant variants E3 - E12 successfully sur-
vived two cycles of ethionine selection, it was of interest to
determine whether these clones might retain their resistance following
long term growth in the absence of selection pressure. Accordingly,
wild type potato cell clone 'W-973' and ethionine selected clones E3 -
E12 were cultured on D3 + GlnIOOO medium containing 0, 0.01, and 0.1
mM ethionine following 260 days of continuous growth on ethionine-free
medium.

Table 5 demonstrates that all variants expressed high level
resistance to 0.01 mM ethionine after greater than eight months'
unchallenged growth. At 0.1 mM ethionine, 'W—973' and E9 were totally

inhibited while the remaining variants continued to grow. The

75

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76

resistant clones fluctuated widely in their inherent, unchallenged
growth rates. Clones E5 and E10 exhibited the strongest growth rates
on ethionine-free medium and also proliferated to the greatest extent
on 0.1 mM ethionine. Further characterization of these two clones
revealed a complete absence of cross resistance to 1 mM seleno-
methionine. When cultured on medium containing 160 mM lysine and

24 mM threonine, E5, E10, and 'W-973' grew at 20.2 i_2, 35.6 :_1,

and 22.2 :_3 percent of their normal growth rates.

Further characterization of clonal stability to ethionine
resistance was performed with clone E1, the earliest isolated ethionine
resistant potato clone. Shortly following its isolation clone E1

was divided into two subclones. One subclone, El-O was continuously
subcultured on D3 + Glnl000 medium plus 0.1 mM ethionine while its
sister subclone, E1-1, was maintained on nonselective D3 + GlnlOOO
medium. At various time intervals secondary subclones from E1-0 were
transferred to ethionine-free medium.

The resistance of these subclones to ethionine challenge
following various periods of time in the absence of ethionine is
depicted in Figure 14. Since the unstressed growth rate of each
subclone varied, the resistance of each subclone is expressed as a
percent growth rate relative to growth on ethionine-free medium.
Growth rates of wild type cells were reduced to 16 and 4 percent on
0.05 and 0.2 mM ethionine, respectively. From 0 to 40 days following
culture on ethionine—free medium, relative growth rates of
rechallenged cells were not substantially altered. In contrast,
subclone E1-1,which had been maintained free of selection pressure

for 450 days,exhibited a sharp growth reduction when again challenged

77

Figure 14. Stability of resistance to ethionine among clone E1 derivatives
as a function of duration and intensity of selection.

78

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79

with ethionine. Despite this growth reduction its phenotype was
still resistant in comparison to wild type cells.

That long term culture in the absence of selective pressure
should be associated with partial loss of ethionine resistance might
be explained in several ways. Had resistance been due to the main—
tenance of multiple copies of a major resistance gene by multiplica-
tion of the chromosome hearing such a gene, then loss of one or more
of these chromosomes umder nonselective conditions would have reduced
the level of resistance expressed upon resumption of selective
conditions. Alternatively, the partial resistance expressed by
subclone El—l might indicate an epigenetic component to the high level
state of resistance expressed by subclone E1-0. Loss of the epi-
genetic component could be expected to lead to moderate level
resistance.

In a parallel experiment subclone El-l was cultured without ethionine
andrechallenged 11 cell generations before growth rates were measured.
This was done to determine whether El-l was composed of two pheno-
typically, and perhaps genetically, distinct subpopulations, one
highly resistant to ethionine and the other moderately resistant. The
prechallenge period was sufficiently long to permit a subpopulation of
highly resistant cells occuring at a frequency of 2% to accrue to
greater than 30% of the total population at the time of growth assess-
ment. When growth rates of prechallenged and nonprechallenged El-l
cells on ethionine medium were measured, no substantial differences
were noted. This indicates that the cells comprising El-l are

relatively homogeneous with respect to their tolerance to ethionine.

80

Selenomethionine Selection and Variant Characterization

 

Selection for resistance to selenomethionine was performed with
'Superior' callus at an analog concentration capable of inducing
growth stasis in 'W-973' cultures. Photosynthetic 'Superior' callus
cultured under 40 foot-candles of fluorescent light was plated thinly
on fine mesh nylon microfilament discs at a rate of 2 g (1.8 X 107
cells) per 30 ml of N10 D2 + GlulOOO medium containing 1 mM seleno-
methionine. Thirty plates were selected in the light; fifteen were
cultured in darkness. After 80 days of culture the cells were
transferred to fresh selection medium. Following two additional
45 day passages on selective medium, four slowly growing resistant
clones were isolated and transferred to N10 D2 + Glnlooo medium.
Three of these clones, SEMI, SEMZ, and SEM3, originated from cultures
selected in darkness. SEM4 was isolated under light selection. The
overall frequency of resistant variants was calculated directly as
2 x 10'3.

Selenomethionine resistant variants were subsequently cultured
for 160 days on selenomahionine-free medium. They were then retested
for resistance to growth inhibition by the seleno-analog as well as
to ethionine and lysine plus threonine. A feature common to all
selenomethionine selections was their sharply reduced unstressed
growth rates relative to wild type 'Superior' callus. This may
indicate a greater than necessary stringency of the original
selection procedures since clones with diminished metabolic rates
would tend to be favored in an inhibitor-laden environment. The

wild type clone proved sensitive to selenomethionine while SEMI

81

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82

and SEM4 exhibited strong and moderate resistance, respectively (see
Table 6). Clones SEM2 and SEM3 had lost all their earlier expressed
resistance. Cross resistance to a first time exposure to 0.1 mM
ethionine was noted for SEMI and SEM3. SEM2 and SEM4 proved
sensitive to ethionine. One clone, SEM4, was examined for tolerance
to lysine plus threonine, and like wild type callus, proved
sensitive.

Among the four selenomethionine selected variants all possible
combinations of resistance and sensitivity to selenomethionine and
ethionine appeared. The dual resistance expressed by SEMI may
conceivably be due to analog competition due to enhanced methionine
production or to mutual discrimination by an altered permease.
Selenomethionine resistance in SEM4 appeared to be due to a mechanism
specific to this analog since ethionine completely suppressed growth.
SEM3 is an anomaly. This variant proved highly resistant to 0.1 mM
ethionine but lost all earlier expressed resistance to selenomethionine
during growth away from this analog. SEM2 was also no longer resistant

to selenomethionine and proved sensitive to ethionine as well.
. O

Cysteine—Threonine Selection

 

Selection for concurrent resistance to cysteine and threonine was
performed as a test to determine whether such a selection might result
in methionine overproduction. On the basis of the regulatory controls
operating in the aspartate amino acid family, this selection would
appear to be directed toward homoserine dehydrogenase activity.
Desensitization of homoserine dehydrogenase might be expected to

increase carbon flow through the pathway. The inability of

83

supplemental methionine to reverse cysteine-threonine induced growth
rate depression was described earlier. Nevertheless it was considered
of interest to isolate resistant variants since such selections had
not previously been described.

For selection six grams of 'W-973' cells were dispersed per 125
ml erlenmeyer flask containing 35 ml D3 + Gln1000 liquid medium
with 21 mM cysteine (free base) and 2 mM threonine. Flasks were dark
incubated on gyrotory shakers rotating 125 rpm. Selection continued
over a 90 day period during which the culture medium was once decanted
and replenished. Following selection only three of thirty-two flasks
contained viable cells. One isolate from each was maintained on
D3 + Gln1000 medium. The frequency with which resistant variants
appeared was calculated as 3.9 X 10'11.

The three surviving clones, designated CTI, CT2, and CT3, were

maintained for 360 days in the absence of selection. Upon retesting
these clones for resistance to 21 mM cysteine and 2 mM threonine, all

were killed. Resistance in these clones may therefore have been

epigenetic.

V. AMINO ACID ANALYSIS OF WILD TYPE AND VARIANT CLONAL SELECTIONS

Methionine-Specific Assays

To assess whether endogenous methionine levels in variant clonal
selections correlate with the degree of resistance to methionine
analogs, it was necessary to select a methionine assay procedure
sufficiently sensitive to discriminate between real and trivial
differences in both free and protein-bound methionine levels.

Turbidometric microbial bioassays utilizing Streptococcus
zymagemes or Leucamastoc mesemteraides have frequently been used to
estimate methionine levels in biological material including potato
(57). Such tests detect an available fraction of the total methionine
present.

Several assays dependent upon specific chemical properties of
methionine have been described. Gehrke (33) devised an automated
method for determining methionine levels in hydrolyzed seed meals.
This assay is based upon a methionine-nitroprusside color reaction.

A second assay depends upon the selective oxidation of methionine by
chloramine T (98). Due to its simplicity and promised specificity,
considerable effort was invested to adapt this procedure for use with
potato tissue.

The chloramine T assay originated from the observation that under

mildly alkaline conditions methionine alone among the common amino

84

85

acids is oxidized (81). Upon oxidation of methionine to methionine
sulfoxide with known excess of chloramine T, the remaining chloramine
T can be assayed ina second reaction. Unreacted chloramine T oxidizes
the highly colored reagent, 2-nitro-5-thiobenzoic acid (NTB) to a
colorless species, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The
quantity of chloramine T remaining after the methionine oxidation
step can be determined spectrophotometrically at 412 nm. Absorbance
at 412 nm is therefore directly related to the quantity of methionine
originally present. Positive interference due to sulfhydryl or amino
groups can be removed by prior acylation with diethylpyrocarbonate
(DEPC). Although DEPC also reacts with chloramine T, small quantitiesof
DEPC hydrolyze to 002 and ethanol within several hours.

In a preliminary examination of the chloramine T method of
methionine estimation, the procedures suggested by Trout (98) were
tested. By this method the relation between methionine concentration
and absorbance at 412 nm was linear for methionine within the range of
0.12 and 1.88 pg/ml.

To more closely simulate true sampling conditions the effective—
ness of DEPC as a remedy for amino and sulfhydryl interferences was
examined. Methionine standards were assayed in the presence of gluta-
mine and cysteine at levels anticipated to exceed those which might
interfere in potato tuber samples. DEPC was provided at the level
recommended by Trout, and acylation accompanied by DEPC decay pro-
ceeded for 24 hours.

Positive interferences proved not to be completely suppressed.

In subsequent tests DEPC levels up to 10 fold that recommended by

Trout were used, and the decay period was extended to 48 hours. At

86

the ten fold DEPC level a very pronounced negative interference was
observed due to NTB oxidation. Reducing the DEPC to six fold the
recommended level eliminated both DEPC-induced negative interference
and the positive interference contributed by amino and sulfhydryl
groups. A test could now be performed on potato tissue samples.

A whole, sound tuber of the cultivar 'Superior' was cubed,
frozen in liquid nitrogen, lyophilized, and milled to pass a 60 mesh
sieve. Free amino acids were extracted by agitation in distilled
water or 80% ethanol for periods of 0.5 to 24 hours. Samples
clarified by two centrifugations at 12000 X g were than assayed.
Samples extracted in 80% ethanol appeared to yield twice the free
methionine level that had previously been reported for 'Superior'
tubers (42). The apparent methionine levels measured in aqueous
extracts were approximately four times greater than the published
value. The association of unduly high apparent methionine levels with
the development of phenolic substances in these extracts suggested
that enzyme activities might well be causing positive interference in
the assay. Accordingly, DEPC, which also acts as a potent enzyme
inactivator, was included during the extraction step. This adjustment
resulted in apparent free methionine levels more comparable to
published results. The concentration of DEPC required to reduce
positive interferences was so high that residual, undecomposed DEPC
interfered with subsequent steps in the assay. Methods to accelerate
DEPC decomposition were therefore investigated.

To eliminate DEPC interference,a compound capable of reducing
DEPC as well as being easily removed from the assay system was sought.

Sodium borohydride is a very potent reducing agent which is reported to

87

be very reactive with methanol and labile to heat (77). Post extrac-
tion reaction of NaBH4 with DEPC eliminated negative interference
attributable to DEPC; however, excess NaBH4 could not in turn be
purged from the system by heating in the presence of methanol. There-
fore attempts fo measure methionine with the chloramine T technique

were discontinued in favor of ion exchange chromatography.

Ion Exchange Chromatography

High level resolution and quantitative determination of amino
acids is readily provided by ion exchange chromotography (8). With
reference to selection systems for methionine overproduction which
might be expected to alter synthetic rates of amino acids other than
methionine, ion exchange chromatography is useful in comparing amino
acid profiles among variant and wild type clones. Recognition of
altered amino acid spectra coupled with the regulatory phenomena
associated with amino acid synthesis can reveal the genetic lesion
underlying a novel variant phenotype. For this reason alone the
additional effort and expense involved higenerating the complete
amino acid profile for a variant clone is justified.

One of the more critical steps in automated amino acid analysis
relates to sample preparation. For carbohydrate-containing samples,
the conditions under which acid hydrolysis is performed become
especially crucial (78). The sulfur amino acids as well as tryptophan,
serine, threonine, and tyrosine are highly susceptible to degradation
and require special treatment (51, 62, 76, 87). Performic acid oxida-
tion of cysteine to cysteic acid and methionine to methionine sulfone

(41, 66) or derivatization of cysteine to acid stable compounds

88

(31, 76) prior to hydrolysis permit quantitative determination of the
sulfur amino acids. Alternatively, providing mercaptoacetic acid or
mercaptoethanol during hydrolysis enhances methionine recovery

(62, 87). Tyrosine and serine recoveries are improved by addition of
phenol (51, 76). Preparation of free amino acid samples requires that
all free amino acids are extracted under conditions that do not
degrade polypeptides. Furthermore, soluble proteins must be excluded
from the sample (49).

Amino acid analyses were performed on potato tissues representing
variable levels of morphogenetic organization, physiological state, and
resistance to in vitro selection procedures. Callus cultures of
wild type and variant 'W-973' clones were dark grown at 24° C on
D3 + Gln1000 medium for 26 days (late exponential phase) before
harvest. Wild type callus cultured on medium lacking glutamine was
included as a control to assess the effects of supplemental glutamine
on the resulting amino acid profile. Callus cultures of wild type and
variant 'Superior' clones were raised at 24° C under 40 foot-candles of
fluorescent light. Cells were harvested following 16 days of culture
(mid-exponential phase) on N10 D2 + Gln500 medium. Young, fully
expanded leaves were collected from greenhouse grown plants. Field
grown tubers from 'Superior' were stored two months at 40 C prior to
sampling. Tubers from 'W—973' were freshly harvested from greenhouse
grown plants. All tissues were frozen in liquid nitrogen, lyophilized,
milled to pass a 60 mesh screen, and stored dessicated and frozen at
-20° C before use.

For free amino acid extractions a carefully weighed tissue sample

of approximately 50 mg was vortexed in 1 ml of 1% sulfosalicylic acid

89

containing 1 umol norleucine and 10 umols dithiothreitol. This
mixture was then agitated for one hour on a gertory shaker adjusted
to 125 rpm. The sample was twice centrifuged for 15 minutes at
12000 X g, the pH was adjusted to 2.2 with crystalline lithium
citrate, and the volume was doubled with lithium citrate buffer. A
white flocculent, precipitate was removed by a final centrifugation,
and the sample was filtered through a 0.2 pm nitrocellulose filter
before ion exchange chromatography.

For total amino acid digests tissue samples of approximately 6
mg were very carefully weighed in pretared 40 ml ignition tubes. To
each tube was delivered 6 ml of 6 N HCl containing 0.5% phenol,

0.67 umols norleucine, and 300 umols dithiothreitol. After degassing
the samples the tubes were flushed with nitrogen and tightly sealed
with teflon lined caps. Acid hydrolysis proceeded for 16 hours in an
autoclave at 121° C. Digested samples were filtered, ether extracted,
and evaporated under vacuum on a rotory evaporator at 55° C. After
two cycles of dissolving the residue in 10 ml water followed by
rotary evaporation, the final residue was taken up in 1 ml of lithium
citrate buffer plus 1 ml of 0.01 N HCl, and the sample was twice
centrifuged for 15 minutes at 12000 X g. Dithiothreitol was added to
provide a 50 mM concentration and the sample was then stored in
darkness at 350 C for 72 hours to reduce methionine sulfoxide to
methionine (43). After filtration through a 0.2 um nitrocellulose
filter the sample was analyzed. In all sample preparations great care
was taken to assure cleanliness of glassware and reagent purity.
Double-distilled deionized water was used for all reagent stock

solutions and sample dilutions.

90

Ion exchange chromatography was performed on a 27 cm X 3.2 cm
single column instrument utilizing Dionex DC-4 resin in conjunction
with a Pico IV lithium buffer and ninhydrin detection system. The
flow rate was 0.1 ml per minute. Chromatography proceeded at 37° C
to glycine elution whereupon the temperature was increased at a rate
of 1° per minute to 60° C. A single amino acid profile was
determined for each tissue sample. Traces were hand integrated for
all amino acids, and actual concentrations were determined by
relation to a norleucine internal standard after compensation for
absorbance factors specific to each amino acid.

The capabilities of this system in large measure determined the
methods of sample preparation. Since protected sulfur amino acid
derivatives such as methionine sulfone and cysteic acid are not well
resolved from other amino acids in this system, it was necessary that
unoxidized methionine and cysteine be measured. These compounds are
especially subject to degradation so a new means for their protection
was assessed. The use of mercaptoacetic acid and mercaptoethanol as
methionine protectants had been reported ( 62, 87), yet because of
their noxious nature dithiothreitol was used instead. It was found
that 50 mM dithiothreitol during a 16 hour hydrolysis period allowed
39% greater recovery of methionine compared to a control lacking
dithiothreitol. Post hydrolysis treatment with 50 mM dithiothreitol
of a sample hydrolyzed in the absence of dithiothreitol resulted in
16% greater recovery of methionine. As a result of these observations
dithiothreitol was routinely used both to recover methionine from
methionine sulfoxide and to prevent methionine destruction during

acid hydrolysis.

91

Cysteine is also recognized as being labile to oxidation and
degradation (8). Its oxidation was investigated with dithiothreitol
protected and nonprotected digests of insulin. In the absence of
dithiothreitol cysteine was completely converted to cystine. Cysteine
recovered from samples hydrolysed in the presence of dithiothreitol
occurred wholly in the reduced state. In potato tissue cysteine
occurs at very low concentrations. Its precise measurement is
therefore difficult and subject to error.

Additional limitations imposed by the available chromatography
system relate to its inability to resolve several amino acids.
Asparagine in free amino acid extracts was not resolved from glutamate.
Cystathionine cochromatographed with isoleucine. Homoserine eluted in
the tail of glutamate. Glutathione was confounded with the aspartate
peak. Cystathionine, homoserine, and glutathione were of particular
interest as intermediates and end products of sulfur metabolism in
plant cells. Although cystathionine occurs in very low quantities in
higher plants, glutathione can be present at greater than 10 fold the
level of methionine (20). In this regard it is remarkable that levels
of a compound interpreted as cystathionine in wild type and ethionine
resistant alfalfa clones ranged from two to ten times the level of

recovered methionine (71).

Total Amino Acid Profiles

 

The primary objective of this project has been to devise in vitro
selection strategies capable of revealing cell lines whose synthetic
capacities for methionine have been enhanced. Realization of this

objective requires that stable mutational events leading to an

92

overproducer phenotype be distinguishable from transitory events which
might lead to an apparent overproduction only. It has become a tenet
in plant cell culture work that the process of growing cells in culture
generates variability. The greater the temporal displacement of two
cell lines from their progenitor, the more likely their phenotypes

will have diverged. Cell culture conditions tend not to select against
aberrant cytogenetic changes which would quickly be suppressed in a
whole plant growing in a natural environment. The resulting collagecfi?
different euploid, aneuploid, and chromosomally rearranged cell types,
with their attendant perturbations in gene dosage, cell cycle time,

and gene regulation, provides a highly heterogeneous background

against which a phenotype which represents a truly novel genetic
element must be distinguished from a phenotype which is merely the
consequence of a new pattern of expression for essentially unaltered
genetic material. For this reason the patterns of amino acid varia-
bility expressed in different variant clones must be compared in
different ways and on different bases.

The amino acid profiles presented in Appendices A and B are
cataloged so as to allow direct comparisons of individual and total
amino acid distributions within and among clones on a reference index
related to wild type callus cultures. All data presented are on a
molar basis. The 'umoles per gram' category provides the molar amino
acid concentration per gram dry weight as determined by ion exchange
chromatography. The 'molar %' category indicates the distribution of
any amino acid as a percent of the total amino acid concentration.
'PDI' or percent distribution index is a relative measure comparing

the molar % distribution of an amino acid with the molar % distribution

93

of that amino acid in wild type callus tissue. The 'AAR' or amino
acid ratio measures the total amino acid content of a tissue relative
to the content of all amino acids in wild type callus. Tables 7 and 8
summarize these indices for methionine alone and also introduce a
'methionine ratio' for comparisons of relative methionine levels

among variant lines.

Upon examination of Appendices A and B it quickly becomes
apparent that substantial variation in amino acid expression exists
among the tissue samples analyzed. Tubers of 'W-973' and 'Superior'
possess a much greater total free amino acid pool than wild type
callus, yet total amino acids from hydrolysates (AAR) are sharply
depressed, indicating much lower relative levels of tuber protein.

In contrast leaf tissue contains a much depressed total free amino
acid level relative to wild type callus while amino nitrogen in

leaf hydrolysates is enhanced. It is noteworthy that total amino
nitrogen in leaf tissues is two to three times that observed in the
tuber. In tubers of both 'W—973' and 'Superior' the free amino acid
pool represented 40% of the total amino nitrogen. Since one third of
the methionine in 'W—973' and one half of the methionine in 'Superior'
were found in free form, it becomes clearly apparent that methionine
accumulation in tubers is not dependent upon its incorporation into
protein. It is also notable that free methionine in the tuber
represents a much higher proportion of total free amino acids than
does free methionine in cell cultures. A small increase in
methionine synthetic capacity might therefore translate to substantial
quantities in the tuber. These observations strongly support the thesis

that potato cell clones selected for enhanced methionine production

94

might well express this phenotype in the tuber.

Supplemental glutamine increased total free amino acids by more
than 50%. Despite this increase the proportion of methionine in
glutamine-supplemented cells was half that in cells cultured without
glutamine. This observation suggests that the inclusion of glutamine
in selective medium may actually increase the stringency of selection.

As an aid in determining whether the various indices of methionine
content calculated for each cell line are associated with either total
amino acid levels or growth kinetics of the cell lines, correlation
coefficients among these variables were computed for ethionine-
resistant variants of clone 'W-973' (Table 7). Of critical interest is
the association of ethionine resistance with methionine concentration

in variant tissue samples. In neither the free pool nor in total

digests is such a correlation apparent. This observation suggests that
in the majority of ethionine-resistant variants the mechanism of
resistance may not be via methionine overproduction.

A paradoxical but explainable correlation is the observation that
as the proportion of methionine in the free pool increases, the level
of resistance to ethionine decreases. This apparent anomaly can be
resolved by noting the high correlation of ethionine resistance with
total free pool amino acids. Ethionine resistance then is more likely
to be associated with cell lines in which the synthetic capacity for
amino acids other than methionine is generally enhanced. An inspection
of the PDI values for all free pool amino acids of ethionine-selected
cell-lines exhibiting a total AAR greater than 1.00 reveals that while
the proportion of methionine generally decreases, the relative contri-

bution of aspartate to the free pool increases. Glutamine and/or

95

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96

glutamate tend to increase as well. This suggests that these amino
acids somehow interfere with ethionine.

The indices of methionine concentration presented in Tables 8 and
9 allow an assessment of whether methionine has in fact been increased
in any variant cell lines. Among variant derivatives of clone 'W—973'
few exhibit elevated methionine concentrations. Clone E3 contains
approximately 20% more methionine in both the free pool and in total
digests. This clone is also characterized by an exceptionally large
total free amino acid pool. Clone E5, a selection which is highly
resistant to ethionine, is similarly distinguished by a large free
amino acid pool. Its methionine ratio in total hydrolysates is 1.44.
Since its amino acid ratio for the total hydrolysate is not greatly
increased compared to 'W-973', methionine may be accumulated by
incorporation into protein(s) which are overexpressed relative to
other proteins. A very high free methionine pool exists in clone E11;
however, the high concentration of the free pool is not reflected in
the levels found in total tissue digests.

Among selenomethionine-selected clones SEMI and SEM3 are clearly
distinguished from wild type in their methionine content. SEMI was
earlier shown to be highly resistant to selenomethionine and ethionine
while SEM3 is resistant to ethionine alone. For SEMI free methionine
is elevated 2.29 times that of wild type cells. This increase is the
result of both a 30% increase in total free amino acids and a 76%
greater proportion methionine in the free pool. SEM3 also
expresses a.greater proportion of its total free‘amino acid pool as
methionine yet accumulates free amino acids to a lesser extent than

wild type cells. The increase in free methionine concentration in

97

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98

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.m mmsmm

99

SEM3 was 61% greater than that of wild type tissue. In total cell
digests methionine increased 38%.

In both these variants the enhancement of methionine levels can be
attributed to free methionine rather than to protein bound methionine.
Since the tuber contains more than eleven times the free methionine
contained in callus tissue, it is reasonable to expect that tuber
methionine levels will be sharply enhanced in these variants. If the
percent increase in methionine concentration observed in callus cultures
of SEMI and SEM3 prevail in the tuber as well, the potato will have
become a wholly adequate source of dietary methionine. This prediction,

however, must await regeneration of these variants to intact plants.

VI. SHOOT AND ROOT MORPHOGENESIS IN POTATO TISSUE CULTURES

Until such time that za mutant population of in vitra selected
cells can be regenerated into an entire, healthy, plant capable of
expressing its newly acquired character at the whole plant level,
the value of such a mutant will be extremely limited. For this
reason the process of shoot regeneration in potato cultures was
examined.

Initial experiments were designed to define conditions pro-
motive of shoot regeneration in primary explants, i.e., tuber,
petiole, stem, and lamina segments. For cultivar 'Superior',
conditions previously defined by Jarret (47) and Lam (54) proved
satisfactory. Cool temperatures (18° C) and a light intensity of
500 foot-candles substantially enhanced regeneration frequency and
plant quality. Clone 'W-973' and twelve commercially important
cultivars produced shoots on media modified from Behnke (7).

Secondary callus, i.e., callus no longer in association with
primary explants was also examined for shoot regenerability. A
number of modifications of the conditions suggested by Behnke (5),
Binding (7), Lam (55), and Shepard (85) were examined as a possible
means for regenerating shoots from long term callus of 'W-973',
'Superior', and 'Russet Burbank'. No treatment resulted in shoot
regeneration. It is noteworthy that among the several protocols

published for potato shoot regeneration, very dissimilar culture

100

101

media and growth conditions are employed. Inorganic salts, vitamins,
carbon sources, osmotic stabilizers, organic supplements, and hormones
are highly variable. Protocols effective with some cultivars are
frequently ineffective with others. Typically, culture age is a
critical factor in shoot regeneration from unorganized tissues. As
cultures age, their resistance to shoot induction increases. Since
selection for altered cell phenotypes is a long term process which
cannot easily be hastened, the selection process impedes the final
objective of regenerating whole plants.

In the course of identifying media which promote growth of
potato callus, several were found which induced root regeneration.
Specifically, combinations of benzylaminopurine from 0.1 to 1.0 mg/l
and naphthalene acetic acid from 0.1 to 3.0 mg/l promoted root
formation while 2,4—D suppressed the process. Shoot regeneration
from primary explants appears to involve the vascular regions of
these explants. Since roots also possess well developed vascular
tissues, it was inferred that regenerated roots might serve as a
transitional organ from which shoots might later be regenerated. Very
few attempts to regenerate plants from root tissues have been
described (95). However, observations that certain solanaceous plants
infrequently produce shoots from true roots under field conditions
suggested that in vitro attempts to regenerate shoots from roots in
potato might be well worthwhile.

Aseptic shoot cultures from the cultivars 'Superior' and
'Michibonne"were from. disinfested tuber buds and were maintained

as lighted shaker cultures in 125 ml erlenmeyer flasks supplied with

102

30 ml of hormone-free Murashige and Skoog basal medium. Healthy, true
roots proliferated and were harvested for subsequent culture on solid
medium. Explants consisting of 5 to 8 mm root segments were placed on
medium composed of Murashige and Skoog FeEDTA and macro salts with the
exception of NH4N03 which was replaced by 8.3 mM NH4CI, half strength
micro salts, Nitsch and Nitsch vitamins, 500 gm/l low salt acid hydro-
lysed casein, 0.3 mM adenine sulfate, 0.2 M mannitol, and 5 g/l
sucrose at pH 5.7. This basal medium was modified from Shepard (82).
Plant hormones included zeatin, the phenylalanine conjugate of indole
acetic acid (IAA-phe), abscisic acid, and gibberellin A3. These were
examined in various combinations over a range of concentrations. All
cultures were maintained at ambient room temperature and were illumi-
nated with 40 foot-candles of fluorescent light.

Of all the hormones examined,zeatin proved most critical for
shoot initiation. For 'Superior' 0.1 to 0.3 pM zeatin elicited the
greatest response; for 'Michibonne' 0.3 to 1.0 uM zeatin was more
effective. In the absence of zeatin or at concentrations of 3 uM
or more, shoots never appeared. IAA-phe contributed very little to
shoot initiation. Responses at 0, 0.1, 1.0, and 5.0 uM IAA-phe were
all very similar. In later experiments auxin was entirely omitted
from the culture medium. Gibberellic acid at 0.65 or 1.3 pM
decisively promoted both the frequency of responsive roots and the
numbers of emergent shoots per root. Additionally,it reduced the
period to shoot emergence from three months to three weeks. In the
absence of gibberellin brachytic shoots or buds originated directly

from the distal surface of the root segment with no intervening

103

callus development. Proliferation of soft callus composed of shoots
and buds in various stages of development occurred when gibberellin
was present. By promoting shoot elongation gibberellin also led to
the formation of morphologically normal shoots which readily
produced roots upon transfer to hormone-free medium. Abscisic acid
had previously been reported to enhance shoot organization in proto-
plast derived cultures (82). Its influence at 0.3 pM on shoot
production from root segments was very modest and not always
reproducible. Higher levels suppressed shoot organization.

The physiological state of the root segment also influenced
shoot regenerability. Young white roots less than 10 days old were
more responsive than were older, thicker roots in which chlorophyll
had developed. Mother plant culture conditions also influenced
shoot initiation. Roots from plants cultured with 30 g/l sucrose
were several times more responsive than roots from plants cultured
with 5 g/l sucrose.

These results indicated that a transition from callus to root to
shoot might be useful in overcoming the recalcitrance of long term
callus cultures toward shoot regeneration. To date this concept has
not been confirmed because of newly arisen difficulties in regenera-
ting roots from cultures which a year earlier had produced roots
freely. Efforts to identify conditions under which rhizogenesis
might again occur are currently in progress. The success of this
approach will be critical to the determination of whether selected
genotypes in which methionine is modestly elevated in cell cultures

express this phenotype at the whole plant level as well.

APPENDICES

104

Appendix A

Patterns of amino acid distribution in wild type tubers, leaves,
and callus cultures and in variant callus cultures of tetraploid
S, tuberosum cv 'Superior'.

The amino acid ratio, AAR, measures the total amino acid content
of a particular callus selection or tissue source relative to the total
amino acid content found in wild type 'Superior' callus. The PDI or
percent distribution index measures the molar percent frequency of any
amino acid in a callus selection or tissue source divided by the molar

percent frequency of that amino acid in wild type 'Superior' callus.

105

 

 

 

 

 

 

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«O.H OO.N ON.OO OO.N O0.0 OM.O
N0.0 OO.N OO.NO MN.O OO.N NO.m
ON.O Om.O ON.ON OM.O Om.q O0.0
N0.0 MH.O MN.OO OO.H OO.N OO.M
OH.O Om.mH Oq.HOH OM.O OO.NO OO.MOO
N0.0 NN.O NO.NN NO.N MO.N NN.O
O0.0 ON.O ON.OO MN.H NO.N ON.O
ON.O NO.NO OM.OOH OO.N OH.O OO.NH
N O mam N O mam
HOO maHoE OaH081 HOO mmHoe OaHoEQ.
aumONHomON: aamw

 

 

q 2mm

.AO.uCoov < xHOCaOO<

an

52
Hence

OOO
Om<

OHm
ONO

<m<0

asm
mNH
DOA
aHH
uaz
Ha>
OH<
3O
aHO
me
mOH

108

 

 

 

 

 

 

«O.H N0.0 O<<

O0.00H N0.00NO O0.00H M0.00 Hamoa
Nm.o mo.a OO.N. «H.O Ha.o aa.o ONO
NN.H O0.0 OM.OO O0.0 O0.0 NN.O Om<
ON.O MN.N ON.OM ON.O O0.0 Nq.O OH:
N0.0 NN.O M0.0~H HH.N M0.0 ON.O ONH
MN.O MN.O N0.0M HN.H NO.MN NN.ON <O<O
OM.H OO.N OH.NO O0.0 OO.H NN.O asm
«O.H OO.M OO.NO O0.0 O0.0 O0.0 mNH
ON.H ON.O ON.MNH Om.O O0.0 N0.0 CaH
NN.H OM.O ON.OO NN.O O0.0 MN.O aHH
ON.O MO.N N0.0M OO.N O0.0 O0.0 uaz
MO.H M0.0 MO.NOO OM.O ON.H HO.H Hm>
MO.H ON.O OO.MOH ON.O NN.M MO.N aH<
mo.a ON.N OO.NOa ON.O om.a so.a sac
O0.0 O0.0H ON.NOH H0.0 ON.NM O0.0M xHO
NN.O ON.O OO.NO MH.H NO.M OO.N mam
OO.H ON.O OO.NO ON.O NN.N HO.H mOH
OO.H N0.00 O0.00H O0.0 OO.NH OO.NO OO<

N OlmaO. N O mam» OHoa

HON maHoE OaH051 HON maHoe OaH081 OCHE<

amOONHomONn aamw

 

OOaH .monaOCO.

.AO.mCoov < xHOCaOO<

109

Appendix B

Patterns of amino acid distribution in wild type tubers, leaves,
and callus cultures and in variant callus cultures of dihaploid
S, tuberosum clone 'Wis AG-23l US-W973'.

The amino acid ratio, AAR, measures the total amino acid content
of a particular callus selection or tissue source relative to the total
amino acid content found in wild type 'W—973' callus. The PDI or
percent distribution index measures the molar percent frequency of any
amino acid in a callus selection or tissue source divided by the molar

percent frequency of that amino acid in wild type 'W-973' callus.

110

 

 

 

 

O0.0
OO.OOH OM.OOOH
~a.~ .quHII NN.Na
NO.H NO.N OO.NO
«O.H OH.N N0.0N
HO.H OO.N ON.OOH
.NH.H O0.0 MN.ONH
MO.H HO.M O0.00
OO.N OO.N OO.NM
ON.O NN.O ON.MNH
M0.0 ON.O H0.00
HN.O OO.N O0.0N
OH.O OM.N OO.MOH
N0.0 O0.0 O0.0MH
OO.H ON.O O0.00H
NN.O NH.OH NN.NqH
OO.H ON.O OH.ON
OO.H NM.O OO.HO
H0.0 MN.O ON.OMH
N w mag
HON mmHoE OaHoEQ
auaONHomONO

 

 

No.0
O0.00H wammw

om.a NN.N oa.s
ma.a ON.N ON.N
so.a ea.a mo._
NN.N ON.N CN.N
as.o mm.sa NO.NN
NN.O mm.a ON.N
NN.O NN.O NO.N
oc.a mo.~ ca.e
mm.a ON.O OO.N
os.a as.o ON.O
NN.O ao.s ON.O
wa.a No.Na om.NN
ON.N oo.q mo.o
No.a OO.ON sm.me
m~.a NN.N ON.O
om.a NN.N OO.M
MO.N om.~ mm.m

N w HON
Ham mNHOE OOHOEQ

NO.NM

 

CHO o\3 OamCuHCa OCHHaa.MNOI3.

O0.00H MO.NONH

 

 

 

 

O0.0 NN.OH
ON.O ON.NN
NO.N ON.NM
ON.O O0.0HH
OO.N O0.0MO
NO.M H0.00
OO.N OO.HO
OO.N O0.0MO
ON.O ON.NO
OO.N ON.ON
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O0.0H OH.NNH
ON.N OO.HMO
OO.MH OM.ONN
O0.0 OO.NO
OM.O ON.ON
OH.OH OO.MNH
N w maul
maHoE OaHoEQ
auOONHomON£

 

 

O0.00H O0.0MN
OM.N M0.0
OO.N OO.N
«O.H NO.N
ON.O OO.N
OO.HM ON.ON
NO.H NO.N
MO.H NO.N
OO.H NO.N
MO.N ON.N
ON.O OO.H
MO.N O0.0
MN.OH NM.OM
ON.N ON.O
OH.ON OO.NO
ON.N O0.0
ON.H ON.O
OM.O OO.M
N O mam
mmHoE OaHoi
aamw

 

OCHHao.MNOI3. aONH OHHB

aomu
anm
mNH
CaH
aHH
ma:
HO>
OH<
ONO
xHO
mam
mow
OO<

UHUQ
osms<

.O xHOCaOO<

111

 

 

 

 

 

mo.H mm.~
O0.00H Nm.ome oo.ooH a~.mcm
mm.m oq.H om.m~ Hm.o «H.~ Na.n
no.0 on.q mo.cm w~.~ N¢.N m¢.w
mm.o ow.~ Om.mm wN.o m~.o oo.~
~o.o -.o N~.mHH am.“ Hm.N «m.w
mN.H mm.o oo.mmfi qw.o mq.cm mN.Nm
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oo.H cq.m o~.mq Hc.~ wo.~ c~.o
Ha.o mm.c ¢H.w- mm.o we.~ w¢.m
on.o mm.q mm.mn No.0 mo.H Hw.m
o~.~ ma.H mm.¢m om.o mm.o mm.~
mo.o qm.o mN.NfiH NN.O m~.m «N.HH
mm.o NN.ON ~N.wwH ow.o o~.- ao.mq
NO.N qw.~ No.m<H MN.N wc.q mm.NH
Ho.H om.mH No.0qm mo.H mm.om qm.mofi
m~.H mm.e ON.NHH cc.~ cm.q mw.o~
mm.o mo.q om.qm oo.H mm.~ O¢.o~
NO.N nm.¢~ om.~mfi NN.N om.q mm.m~
N w awn N m Ham
Ham umHoE meoEQ Ham umHoE mmHoEQ
mumm%aouv%£ mmuw
m m

 

 

 

Hw.o

O0.00H ~N.¢NMH
OO.M ow.~ Hw.qm
mw.o qw.m w~.om
MN.N ¢~.N mm.fim
oo.~ mm.c Hq.ma
Hw.o Nfi.o HH.mw
mm.o mq.m qm.mq
m~.~ mo.m mo.mq
mm.o OH.N mm.~m
oa.o m~.m ~w.oN
mo.~ cw.H Hq.mm
mo.~ wa.© mm.oo
w©.o ON.N «$.Nm
NH.H oo.w oq.m-
NN.H «N.c~ om.om~
mo.H Hm.m q~.mN
o¢.o mH.q ow.mm
e¢.o NN.m Om.qm~
N w umm

Ham umHoE meoEQ

mummkaouv%£

 

 

 

no.0
oo.ooH qw.mmm

mm.~ mq.m ~¢.~H
ow.o 0N.H mm.m
NN.H mN.H mo.q
N~.~ N¢.H Hm.m
ON.O MN.NN NN.Om
mo.~ NH.~ mo.~
N¢.N «m.~ mn.m
mm.H mo.m mm.m
oq.H mm.fi mm.m
qw.o ~¢.o Nm.o
Nc.H mm.q om.¢
om.o mo.N «m.NH
mo.~ mm.m N0.0
N~.H gm.qm mm.n~
-.H HH.m NO.N
NN.N mm.m fim.w
NN.~ MN.N o~.o

N w\umm

Ham “macs mmHoEQ.
mmum

 

H

.Av.ucouv m xficcmmm<

m<<
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mag
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um:
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112

 

 

 

 

 

 

 

mo.~ ~o.~
O0.00H 00.5qmfi O0.00H mm.oqm
N0.H oo.H 00.5H H0.0 No.H m0.q
mo.H Om.q N0.0N mH.~ Hm.N 50.0
qo.o mw.H mm.mm mm.o MO.N qq.N
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q~.~ mm.w O0.~m~ NN.N mq.mm -.mw
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Na.o «0.0 om.NH~ ~0.o mm.~ m~.0
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m¢.~ ¢o.H~ om.mm~ @o.~ M0.0 mm.m~
0m.o ON.NN O0.0NN «N.o mm.o~ 00.00
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mm.o m~.q NN.NN mm.~ Hq.~ mm.m
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N M 3% N W umfl
Ham HmHoE mmaoen Ham umHoE mmHOEQ
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wq.~ $0.0 N~.0H ww.~ mq.q mm.mH
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mm.o mm.“ 00.0m N0.0 mw.o mo.m
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¢H.H 50.00 oq.mwm mm.H NH.Nm O0.mmH
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mummzaouumn mmum
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113

 

 

 

 

 

 

 

 

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