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Ruth Wilson

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A STUDY OF GIBBERELLIN RESPONSE IN ARABIDOPSIS THALIANA
BY

Ruth N gaio Wilson

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Genetics Program
1993

ABSTRACT
A STUDY OF GIBBERELLIN RESPONSE IN ARABIDOPSIS 171ALIANA
By

Ruth Ngaio Wilson

In an effort to learn more about how the hormone gibberellin triggers plant
responses, mutants of Arabidopsis thaliana have been analyzed. An existing mutant called
gibberellin-insensitive (gar) has been further characterized, and some new gibberellin
response mutants have been isolated.

Previously the gai mutant was known to be impaired in stem elongation, apical
dominance, and seed germination. It is shown that the gai mutation also affects two other
gibberellin responses. The gai mutation impairs gibberellin stimulated development of
fertile flowers in continuous light, and also delays the transition from vegetative growth
to flower initiation in short days. Flowering under short day growth conditions is shown
to be completely blocked in a severely gibberellin-deficient mutant (gal ~3).

New mutants have been isolated in two different ways. A search for suppressors
of the gai phenotype has turned up gibberellin responder (gar) mutants at relatively high
frequencies in mutagenized populations. These mutants fall into at least two different
classes; Members of the most abundant class, exemplified by the mutant garI-I, show
semidominance for suppression of the gibberellin-insensitive phenotype. The garI-I
mutant has been found to be very closely linked to the gai mutation. The gar2-I mutant,
which is completely dominant for suppression and is unlinked to the gai locus, clearly

defines a new gene involved in gibberellin signalling.

Another new mutant called gas], which may show gibberellin hypersensitivity,
was found by screening for plants resistant to an inhibitor of gibberellin biosynthesis
(paclobutrazol). The gas] mutant flowers early in short days. i

The gai mutation was mapped relative to four linked DNA markers in a wide
cross, using a novel technique to score the gai phenotype. Also described in this
dissertation is the use of a new Arabidopsis strain for testing the technique of pollen

mutagenesis.

ACKNOWLEDGEMENTS

I would like to thank everyone who helped me with this work. I am indebted to
John Heckman for performing microscopy. Laura Weislo helped substantially with the
RFLP mapping, and Sherry Hill assisted with a number of experiments. I thank Chris
Somerville for allowing me to work on this project for so long and for providing me with
useful advice and discussions, funding, and now and then enthusiasm. Jan Zeevaart first
kindled my interest in gibberellin, and has provided me with a' wealth of GA literature
and experience as well as help when Ine‘eded it. I thank Lenny Robbins for many;
informative discussions, calculations, and much encouragement. I am grateful to Rebecca
Grumet reading this work so carefully, and providing helpful criticisms. Lastly I thank

my husband Edward Hanley for his patience, support, and good cooking.

iv

TABLE OF CONTENTS

LIST OF TABLES .................................... ,, vii
LIST OF FIGURES .................................... viii
LIST OF ABBREVIATIONS AND SYMBOLS ..................... x
ARABIDOPSIS GENES AND GENOTYPES ...................... xi
CHAPTER 1
A BRIEF HISTORY .............................. ; ...... 1
REFERENCES ............ 5
CHAPTER 2
SUPPRESSION OF THE GIBBERELLIN INSENSITIVE PHENOTYPE IN
ARABIDOPSIS ......................................... 8
INTRODUCTION .......................... ' ........ 8
MATERIALS AND METHODS .......................... 9
RESULTS ...................................... 16
DISCUSSION .................................... 40
REFERENCES ................................... 44
CHAPTER 3
GIBBERELLIN IS REQUIRED FOR FLOWERING IN ARABIDOPSIS' THALIANA
UNDER SHORT DAYS . , ................................. 47
INTRODUCTION ................................. 48
MATERIALS AND METHODS ......................... 49
RESULTS ...................................... 52
DISCUSSION............, ....................... 62
REFERENCES ................................... 64
CHAPTER 4
MAPPING OF THE GIBBERELLIN-INSENSITIVE MUTATION RELATIVE TO DNA
POLYMORPHISMS .................................... 67
INTRODUCTION ................................. 67
_ MATERIALS AND METHODS ......................... 68
RESULTS ...................................... 69
DISCUSSION .................................... 79
REFERENCES ................................... 79

CHAPTER 5

ISOLATION OF A PACLOBUTRAZOL RESISTANT MUTANT ......... 80
INTRODUCTION .......... ‘ ....................... 80
MATERIALS AND METHODS ......................... 81
RESULTS ......................... * ............. 83

‘ DISCUSSION .................................... 88
REFERENCES ................................... 91

CHAPTER 6

POLLEN MUTAGENESIS ................................ 92
INTRODUCTION ................................. 92
MATERIALS AND METHODS ......................... 93
RESULTS ...................................... 94
DISCUSSION .................................... 96
REFERENCES ................................... 96

CONCLUSION ..................... _ .................. 97

vi

LIST OF TABLES

Table 2-1. Frequenciesof Suppressor Mutants of gai ................. , 21
Table 2-1B. Suppressor Mutants of gai. ........................ 22
Table 2-2. Comparison of Flowering Times in Short Days. ............ 26
Table 2-3. Seed Germination. ........................... , . . 28
Table 2-4. Plant Height on Various Media. ...................... 29
Table 2-5. Dominance of Suppressor Mutants of gai. ................ 34
Table 2-6. Complementation Tests Among Suppressor Mutants ........... 36
Table 2-7. Mapping of the garI-I Mutation Relative to gai. ............ 39
Table 2-8. Variations of the "Two Gene" Model: Comparison of Predictions to
Observations. ............................. . ....... 43
Table 3-1. Flowering Time in Short Days. ...................... 53
Table 3-2. Flowering Time and Leaf Number in Continuous Light. ....... 53
Table 4-1. Mapping of gai Relative to DNA Markers. ............... 77
Table 6-1. Generation of adh Null Mutants by Pollen Mutagenesis. ....... 95

vii _

LIST OF FIGURES

Figure 2-1. Flower Morphology. ............................ 17
Figure 2-2. The Effect of the gai Mutation on Flower Fertility Restoration. . . 18

Figure 2—3. A Primary ”Revertant" Among Gibberellin Insensitive Dwarfs (gai,

an). .......................................... 20
Figure 2-4. Plant Height Comparison of gai and Suppressor Mutants. ...... 24
Figure 2-5. Phenotypes of Mutants and WT. ..................... 25
Figure 2-6. The garI-I Mutation is Semidominant. ................. 31
Figure -2-7. The garZ-I mutation is Dominant. .................... 32
Figure 2-8. Comparison of Plant Heights of Various Genotypes. ......... 33
Figure 24’. Testing for Linkage of a Suppressor Mutant to gai. .' ........ '. 38
Figure 2-10. Model to Explain Suppression of Gibberellin Insensitivity. ..... 42

Figure 3-1. The effect of exogenous GA3 on flowering time in Short days. . . . 54

Figure 3-2. Phenotypes of Arabidopsis mutants in SD. ............... 56
Figure 3-3. Total leaf number in short days. ..................... 57
Figure 3-4. Mutant gal-3 after 113 days of growth in SD at 21-23°C. ..... 58
Figure 3-5. Median sections of Shoot apices. ...................... 61
Figure 4-1. Phenotypes of the Strain LA—O on Various Media. ........... 71

Figure 4-2. Phenotypes of the gai Mutant and the Strain ND-O on Various

Figure 4-3. Autoradiogram of a Typical Genomic Blot to Score a Restriction
Fragment Difference ................................. 76
Figure 4-4. Genetic Map of a Portion of Chromosome 1. .............. 78

Figure 5-1. The Effect of Paclobutrazol and GA on Seed Germination and

SeedlingGrowth. 82
Figure 5-2. The gas] Mutant Is Resistant to Paclobutrazol. ............. 85
Figure 5-3. Plant Height in the Presence of Paclobutrazol. ............. 86
Figure 5-4. Flowering in SD. Bars represent mean i SE (n=27-28). ...... 87

Figure 5-5. Early Steps in Gibberellin Biosynthesis That Can Be Inhibited by the

Growth Retardants Paclobutrazol and AMO-1618. .............. 9O

ix

LIST OF ABBREVIATIONS AND SYMBOLS

chi-square statistic

continuous light

centimeter

centiMorgan = 1% recombination

day(s)

gibberellin insensitive dwarf mutant of maize
ethylmethanesulfonate

first generation resulting from crossing

generation resulting from self-fertilization of an F 1 hybrid plant
generation resulting from self-fertilization of F2 individuals.
gram

gibberellin

gibberellin Ax

any gibberellin which has 19 carbon atoms

hour

KiloRad

first generation after mutagenesis

second generation resulting from self-fertilization of mutagenized plants
meter '
Molar

millimeter

micromolar

milliliter

photon flux density

dwarf mutant of wheat (shows reduced GA sensitivity)

second ‘

short days

vegetative mutant of pea

volume

adh
an
cer2
er
gal-I
gai
gar
gas]
glI
LA-O
. ms]
ND-O
P)’
W 1

ARABIDOPSIS GENES AND GENOTYPES

(Use of lower case letters indicates a mutant allele.)

alcohol dehydrogenase

angustofolia

eceriferum 2

erecta

gibberellin deficient (allele 1)
gibberellin insensitive

gibberellin responder (suppressor of gut)
gibberellin super-responder 1
glabrous 1

Landsberg erecta ecotype
male-sterile 1

Neiderzenz ecotype

thiamine auxotroph

Landsberg er, an, gII, cer2, py, ms]

wild type (Landsberg er)

xi

CHAPTER 1
A BRIEF HISTORY

Gibberellins (GAS) are diterpenoid molecules that trigger growth and
developmental changes in plants. GAs and structurally related plant hormones function
in angiosperms, gymnosperms, ferns, and possibly also in psilophytes and algae
(T akahashi et al., 1986; Takahashi et al., 1991). Although numerous (62 at least)
different gibberellin (GA) structures have been found in flowering plants, apparently only
a very few specific GAs actually act to stimulate plant responses (sponsel, 1987). Most
of the other GAs seem to consist of precursors and side products generated during the
biosynthesis of active hormone, 'or else molecules which have been modified in the plant
so as to destroy activity (Sponsel, 1987). In the plant species in which GA biosynthesis
has been studied the most thoroughly, such as Zea mays, Pisum sativum, and Arabidopsis ‘
thaliana, only GA, and a few very similar structures such as GA3 and, perhaps 6A4 seem
to be intrinsically active (Spray et al., 1984; Ingram, 1984; Zeevaart and Talon 1991).
For simplicity the term gibberellin (GA) will henceforth be used specifically to refer to
these active molecules.

At present it is not understood how GA acts in plants and no receptor has been

identified since GA was first discovered more than 50 years ago (MacMillan,1987).

- _ Photoaffinity labelling has recently revealed a soluble protein which specifically binds

active GA4, but it is not yet clear whether this polypeptide will prove to be a receptor or

2
a GA metabolic enzyme (Hooley et al., 1992). Because purely biochemical approaches

have not yet succeeded, genetic methods are being used to study the problem of GA
action.

Mutants that show defects in GA biosynthesis have revealed that this hormone
normally plays several roles in plants. Across a broad spectrum of flowering plant
species, including representatives of the grass, legume, solanaceous, composite and
crucifer families, GA deficient mutants are generally short (Reid, 1986; Waycott and
Taiz, 1991). Application of GA can reverse this dwarfness by causing cell elongation and
division in stem intemodes to increase (Reid et al., 1983). GA deficient dwarfs often
show additional defects, but the spectrum of symptoms varies somewhat depending on the
severity of GA depletion and on the species of plant. For example, in maize, GA
auxotrophs may have anthers in the ears due to bisexual development of the normally
female cob (Harberd and Freeling, 1989), while an analogous mutant in tomato shows
sterility due to the failure of both male and female meiosis (Nester and Zeevaart, 1988).
In Arabidopsis thaliana, GA deficient mutants have been isolated at five loci (CAI-GAS).
The most severe of these mutants have revealed that gibberellin is required in Arabidopsis
for normal seed germination, stem and leaf elongation, apical dominance, development
of complete (male-fertile) flowers (Koomneef and van der Veen, 1980), and as shown
here, GA is also required for flower initiation under short day growth conditions.

An Arabidopsis mutant which closely resembles a GA auxotroph in appearance,
but which is much less sensitive to application of GA has also been discovered
(Koornneef et al., 1985). This GA insensitive mutant (gai) exhibits impairment in every

GA response known in Arabidopsis. The plant elongates much less than wild type

3
Landsberg erecta (WT), and can produce more axillary shoots. The gai mutation also

greatly reduces the ability of a GA deficiency mutant (gal -1) to respond to exogenous
GA by growing taller. Seed batches of the gai mutant may also show dramatically lower
germination rates than comparable WT batches, especially if the WS are treated with
GA and kept in darkness, but the extent of germination difference observed depends on
the dormancy state of the seeds (Koornneef et al., 1985). Only one mutant allele of the
gai locus has been found (Koornneef, 1982), and the mutation is semidominant. Wl‘lgai
plants have an intermediate semidwarf phenotype (Koornneef et a1. 1985).

I have found that the gai mutation also retards initiation of flowering in short days
and interferes with the ability of applied GA to restore normal flower fertility to the gaI-
1 mutant. Far from displaying any reduction in GA synthesis, gai mutant plants have
been found to have increased levels of all c,9-GAs except GA29, and to accumulate 27
times more GA, than WT (Talon et al., 1990), perhaps due to some sort of fwdback
regulation. Phenotypically this mutant appears analogous to the D8 mutant of maize and
the Rht_3 mutant of wheat, which are also GA-insensitive (Harberd and Freeling, 1989;
Reid, 1990). It seems likely that the gai mutation impairs a GA receptor or some other
primary element in the GA response pathway.

An alternative possibility might be that the gai mutant is defective in GA
transport, and somehow fails to move GA to the cells or compartments where it is active.
However it has'been proposed that due to its partially hydrophobic, weakly acidic
chemical nature, GA can cross cell membranes by diffusion without requiring a
transporter (Hooley et al., 1992). GA synthesis has generally been found to occur in the

shoot'apical region, in roots, and in developing seeds (Sponsel, 1987), but unfortunately

4

little is known about sites of hormone action. One tissue in which GA is known to ‘
function directly is the seed aleurone of barley, and in this case GA may act at the outer
surface of the cell membrane (Hooley et al., 1992).

A much different type of GA mutant shows hyperactive or constitutive responses
to GA. Examples of such mutants are the slender mutants of barley and pea, both of
which have lower than normal GA levels (Lanahan and Ho, 1988; Potts et al., 1985).
Slender mutants behave like WT plants which have been treated with saturating quantities
of exogenous GA (Reid, 1990), but actually have lower than normal endogenous GA
levels (Scott, 1990). These plants are not readily dwarfed by chemical inhibitors of GA
synthesis (Lanahan and Ho, 1988; Potts et al.,1985). Slender pea plants still grow
completely tall even in a GA-deficient mutant background (Potts et al., 1985).

The slender mutant of pea shows unusually long internodes, rapid growth,
abnormal flower development, and production of some parthenocarpic (seedless) pods
(Potts et al., 1985). This phenotype is caused by a combination of two recessive
mutations at different loci (1a and cry“), which may be duplicate genes (Potts et a1.
,1985).

In barley, a single recessive mutation causes a slender phenotype. Slender barley
plants develop long intemodes and- leaves, aerial roots, and sterile flowers (Lanahan and
Ho, 1988). Seed aleurone tissue of the barley slender mutant produces a-amylase
constitutively, whereas the synthesis of this enzyme is normally GA inducible (Chandler,
1988).

In this dissertation genetic and phenotypic characterization which adds to our

knowledge of the gai mutant is described, as well as the isolation and characterization of

5

novel GA response mutants. These new mutants were obtained in two different screens.
One productive approach was to look for mutations which could suppress the gai dwarf
phenotype, while another strategy was to search for mutant plants which showed
resistance to a high concentration of paclobutrazol (an inhibitor of GA biosynthesis). The
hope was that new mutants would identify more genes 8 involved in mediating GA
responses and that these would provide new insights into the nature of the gai mutation.

The overall aim of the work described here was to learn more about how GA
functions in plants.

REFERENCES

Chandler P.M. (1988). Hormonal regulation of gene expression in the "slender" mutant .
of barley (Hordeum vulgare L.). Planta 175,115-120

Harberd N. P. and Freeling M. (1989). Genetics of dominant gibberellin- -insensitive
dwarfism 1n maize. Genetics 121, 827- 838.

Hooley R., Beale M.H., Smith SJ ., Walker R.P., Rushton P.J., Whitford P.N. and
Lazarus C.M. (1992). Gibberellin perception and the Avena fatua aleurone: do our
molecular keys fit the correct locks? Biochemical Society Transactions 20(1), 85-89

Ingram T.J., Reid J.B., Murfet I.C., Gastin P., Willis C.L., MacMillan J. (1984).
Intemode length in Pisum. The Le gene controls the 3B—hydroxylation of gibberellin A20
to gibberellin A,. Planta 160: 455-463.

Koornneef M. and van der Veen J. H. (1980). Induction and analyses of gibberellin
'. sensitive mutants in Arabidopsis thaliana (L. ) Heynh. Theor. Appl. Genet. 58, 257-263.

Koornneef M., Dellaert L.W.M., van der Veen J .H. (1982). EMS and radiation
induced mutation frequencies at individual loci in Arabidopsis thaliana (L. ) Heynh.
Mutation Res. 93, 109-123.

Koornneef M., Elgersma A., Hanhart C.J., van Loenen-Martinet E.P., van Riin L.,
and Zeevaart J .A.D. (1985). A gibberellin insensitive mutant of Arabidopsis thaliana.
Physiol. Plant. 65, 33-39.

Lanahan M.B., and Ho T.J.D. (1988). Slender barley: a constitutive gibberellin -
response mutant. Planta 175, 107-114.

6

MacMillan J. (1987). Gibberellin—deficient mutants of maize and pea and the molecular
action of gibberellins. In Hormone Action in Plant Development-A Critical Appraisal,
G. V. Hoad et al. eds (London: Butterworth & Co. Ltd).

Nester J .E. and Zeevaart J .A.D. (1988). Flower development in normal tomato and a
gibberellin deficient (ga;_2_) mutant. Amer. J. Bot. 75, 45-55.

Potts W.C., Reid J .B.,Murfet LC. (1985). Intemode length in Pisum. Gibberellins and
the slender phenotype. Physiol.Plant. 63, 357-364

 

Reid J.B., Murfet I.C., Potts W.C. (1983). Internode length in Pisum 11. Additional
information on the relationship-and action of loci Le,La,Cty,Na and Lm. J. Exper. Bot.
34 (140), 349-364

Reid J .B. (1986). Gibberellin mutants.ln A Genetic Approach to Plant Biochemistry,
A.D. Blonstein and P.J. King eds (Vienna: Springer-Verlag), pp.1-33.

Reid J. B. (1990). Phytohormone mutants in plant research. J. Plant Growth Regul. 9,
97-111

Scott I.M. (1990). Plant hormone response mutants. Physiol.Plant. 78, 147-152
Sponsel V.M. (1987). Gibberellin biosynthesis and metabolism. In Plant Hormones and

Their Role in Plant Growth and Development, P.J. Davies ed (Dordrecht: Martinus
Nijhoff), pp.43-75.

 

Spray C., Phinney B.O., Gaskin P., Gilmour SJ., MacMillan J. (1984). Intemode
length in Zea mays L. The dwarf-1 mutation controls the 3B-hydroxylation of gibberellin
A20 to gibberellin A,. Planta 160, 464-468.

Takahashi N., Yamaguchi 1., and Yamane H. (1986). Discovery of gibberellins and
their occurence in nature. In Chemistry of Plant Hormones, N .Takahashi ed (Boca Raton,
Florida: C.R.C. Press) pp.58-68.

Takahashi N., Phinney B.O., and MacMillan J. (1991). Gibberellins.(New York:
Springer-Verlag).

Talon M., Koornneef M. and Zeevaart J.A.D. (1990). Accumulation of C,,,-
gibberellins in the gibberellin-insensitive dwarf mutant gai of Arabidopsis thaliana (L. )
Heynh. Planta 182: 501-505

Waycott W. and Taiz L. (1991). Phenotypic characterization of lettuce dwarf mutants
and their response to applied gibberellins. Plant Physiol. 95, 1162-1168.

Zeevaart J .A.D.and Talon M. (1991). Gibberellin mutants in Arabidopsis thaliana. In

7

Plant Growth Regulation 1991, C.M. Karssen, L.C. Van Loon and D. Vreugdenhil
eds.(Dordrecht: Kluwer) pp.34-42.

 

 

 

8 .
CHAPTER 2

SUPPRESSION OF THE GIBBERELLIN INSENSITIVE PHENOTYPE IN

ARABIDOPSIS

ABSTRACT
The gibberellin insensitive mutant (gal) of Arabidopsis thaliana shows impairment in
multiple responses to the plant hormone gibberellin, including seed germination, stem
elongation, apical dominance, and rapid flowering in short days. Here it is shown that
the semidominant gai mutation also interferes with development of fertile flowers in
continuous light. A total of 17 independent mutants were isolated in which the
gibberellin-insensitive phenotype is suppressed. These suppressor mutants appeared at
relatively high frequencies following mutagenesis, and fall into at least two classes.
Members of the most abundant class, represented by the garI-I mutant, show
semidominance for suppression. The garI-I mutation maps very close to the GA] gene.
A model is proposed to explain thesefindings. The garZ-I suppressor mutation, which
is completely dominant and unlinked to gai, clearly defines a new gene in the GA
response pathway.
INTRODUCTION

Although the hormone gibberellin (GA) stimulates dramatic growth and
developmental changes in a wide variety of plants, almost nothing is known about how
it acts (MacMillan, 1987). In an attempt to learn more about the mode of action of GA,

I have analyzed the gibberellin insensitive (gai) mutant of Arabidopsis thaliana

 

 

9

(Koornneef et al., 1985). This mutant seems to be impaired in some primary step of GA
perception or response. Superficially, a gai mutant plant resembles a somewhat "leaky"
GA-deficient dwarf. It shows reduced stature, apical dominance, and seed germination
(Koornneef et al., 1985), as well as delayed flowering in short days (Wilson et al., 1992;
this dissertation chapter 3). Unlike mutants defective in GA biosynthesis, the’gai mutant
fails to substantially elongate in response to exogenous GA treatment (Koornneef et al.,
1985), and the mutant accumulates much higher than normal levels of active GAs (Talon
et al., 1990).

The existence of the gai mutation raises a number of questions. Is the mutant
impaired in all GA responses, or just some of them? How does the GA] gene product I
function in GA signalling? Are there other early steps in a GA response pathway? Why
is the gai mutation semidominant? Are such mutants rare?

In order to learn more about the nature of this mutation, I undertook further
characterization of the gai mutant, and also initiated a search for new mutations which
suppress the gai phenotype. Results presented here support a possible model in which two
or more homologous GAI genes encode subunits of a multimeric protein.

MATERIALS AND METHODS
Arabidopsis Strains. The mutants of Arabidopsis thaliana (L.) Heynh Characterized here
were all originally derived from the Landsberg (erecta) line which we call WT. The
”gibberellin-insensitive mutant (gai), the severely GA deficient mutant (gal-3), and the
gaI-I, gai double mutant have been described (Koornneef et al., 1983; Koomneef et al.,
1985; Wilson et al.,1992), as has the W1 (an, py, gII, cer2, msI) strain (Koornneef and

Stam, 1992). The nomenclature for Arabidopsis mutants has recently been changed

 

10

(Koornneef and Stam, 1992). The semidominant gai mutation was originally designated
as Gai, which now refers to the normal phenotype. The gal -I and gal -3 mutants were
originally called lines N65 and 31.89 respectively (Sun et al., 1992).

The gai, an strain was constructed by crossing gai x W1 and subsequently

isolating F2 segregants which were dwarf and also showed the angustofolia phenotype of
narrow leaves and twisted siliques (Koornneef et al., 1982). The gai and an mutations
are linked 21.8 map units apart on chromosome I (Koornneef et al., 1985b). Progeny
testing revealed one plant which was homozygous for both mutations and failed to
segregate other visible markers. WT (GAI), an plants were obtained as segregants from
a WT x gai, an cross. GA responder mutants (gar) were isolated from second generation
(M2) mutagenized gai,an seeds as ”revertants". .
Growth Conditions. Mutagenized populations of M, seed were planted on plastic flats.
(900 cm2 x 8 cm deep) which had been filled with potting soil (BACTO), soaked with
distilled water, and topped with 3 mm fine vermiculite. M2 seed was planted on 13-15
cm diameter round clay pots filled with Arabidopsis mixture (equal parts by volume of
perlite, vermiculite, and either potting soil or Sphagnum moss). The pots were wetted
with mineral nutrient solution (Somerville and Ogren, 1982) and topped with 4 mm fine
‘ vermiculite. Small square plastic pots (4 cm wide) prepared in the same way were used
to grow individual plants for physiological comparisons. Within an experiment the same
batch of mix and nutrient solution was generally used for all pots.

Seeds were allowed to dry in paper envelopes for at least one week before
planting. Generally, seeds were mixed with a suspension of 0.1% agar in water or

mineral nutrient solution and chilled for 7 d at 4°C, before dispersing on the surface of

 

 

ll
pots, either with a 10 ml pipet, or a 250 ml separatory funnel (for large volumes). Pots

were covered with plastic wrap or sheeting for several days to ensure sufficient humidity
for seed germination.

In the greenhouse, flats were covered with cloth sheets to screen out intense
sunlight during the first weeks of growth. Plastic coverings were usually slit with a raZor
blade 1-2 d before removal in order to allow the seedlings to gradually acclimatize to dry
air. Pots were generally kept in plastic trays and bottom-watered, while flats were misted
or watered from the top. Each flat was fertilized once with 0.5-1 liter mineral nutrient
solution 3-4 weeks after planting.

Several different approaches'were taken to achieve the goal of growing one plant
per small pot. One approach was to plant 2-10 seeds per pot, and subsequently to thin out
seedlings (4-7 (1 after planting). Another technique was to plant only one seed per pot,
using a small pipet tip. A third approach was to germinate seeds on agar plates for 4-7
days, and then to gently transplant the seedlings to pots using a small curved forceps.
Transplants were covered with plastic wrap for the first 2-3 (1 of acclimatization to soil
and growth chamber conditions. Germination on plates was preferred for scarce seeds
such as the F, products of crosses.

Potted plants developed. in growth chambers unless otherwise indicated. Standard
growth chamber conditions were a temperature of 22 i 1°C and continuous light of 110
-_l-_ 10 umol m'2 s‘1 photon flux-density (PFD) emitted by cool white fluorescent bulbs.
SD conditions consisted of 8 h light followed by 16 h darkness. Plants were grown in
greenhouses only during thewinter and early spring. FluOrescent lights or sodium lamps

extended daylength to 16 h. Greenhouse temperatures ranged from l7-25°C.

 

 

l2
Mutagenesis by Ethylmethanesulfonate. Seeds were pretreated to break dormancy

before mutagenesis. Roughly 20—40,000 seed of the gai, an genotype were imbibed in
0.1 % agar and then spread on filter paper discs in petri dishes at a density of roughly 65
seeds/cmz. The dishes were sealed with parafilm and placed at 4°C for 7 days. The discs
were then lifted out and placed on a sheet of blotting paper in a sterile hood and left to
dry. The seed were collected and treated with 50 ml of 0.3% (v/v) EMS solution for 15
h at 21°C. Then they were rinsed with distilled water 15X, transferred to a clean 500 ml
container, and rinsed 15X more (200 ml /rinse). All EMS waste was collected and
inactivated by reaction with NaOH. After dispersal in 0.1 % agar the seed were planted
on flats (900 cm2 x 8 cm deep) at a density of 1-2 seeds/cm2 in the greenhouse. M2 seed

was collected in 7 separate pools.

Mutagenesis by Gamma Radiation. 'After pretreatment to break dormancy,

approximately 55,000 dry md of the gai,an genotype in a glass vial were treated with

radiation from a °°Co source at the M.S.U Chemistry Department. A dose of 83 Krads

i 20% was applied at a rate of approximately 1.8 KRad/ minute. The mds were planted

at a density of 5-6/cm2 on flats. Mature M, plants were bulked in groups of 500-1500 to

yield separate batches of M2 seed.

Mutagenesis by Fast Neutrons. Dry gai, an seeds were treated with 6 KRads (60 Gy)

i 4.2% fast neutrons in a nuclear reactor (at the Plant Breeding Unit, FAO/IAEA I
Program, Seibersdorf, Austria). The dose of fast neutrons was applied at a rate of 3

Rads/second j; 1 (mean energy= 1.5 Mev; maximum energy =15 Mev). Contamination

by gamma rays and thermal neutrons was low enough as to be considered biologically

negligible (gamma contamination< 2%, thermal neutrons< 0.01%). Seed was either

13
imbibed shortly after irradiation (within 1-2 weeks) or else stored dry at -80°C for up to

6 months. Seeds were imbibed in 0.1% agar suspension containing 100 11M GA3 at 5:1;
1 X 103 seed/ml. After chilling for 7 d at 4°C, the seeds were diluted into 0.1 % agar to
a final concentration of 38 i 8 seed /ml and planted at 3—4 seed/cm2 on flats in a
greenhouse or growth chamber. Mature M, plants were harvested in pools of 500-1500.
In all mutagenesis experiments, survival was estimated by counting the number of living
plants in a small sample of representative flats (2-14% of total) 3-4 weeks after
germination.
Mutant Screening. After drying for at least 7 days and chilling in 0.1 % agar to break
dormancy, 500-1000 M2 seeds from each M, bulking were planted. Tall ”revertant"
plants with large leaves were identified by eye. The fact that these big plants carried the
an marker was apparent due to narrow leaf shape, twisted siliques and other symptoms
of this morphological mutation (Koornneef et al., 1982). Seed from putative mutants was
collected and planted to test heritability and perform backcrosses, generally to the parental
gai, an strain, but occasionally to the unmarked gai strain.
Growth of Sterile Plants. Seeds were surface sterilized in plastic microfuge tubes (up
to 1000 seed/tube). The seed were washed twice with 1 ml 70% ethanol and then twice
with 1 ml of a solution containing 1.6% hypochlorite (v/v) and 0.02% Triton X-100. For
each wash, the seeds were vortexed in the solution for 30 seconds, before the supernatant
was removed by aspiration. In a sterile hood the final bleach rinse was removed, and then
the seeds were washed three times with sterile Arabidopsis mineral nutrient solution.
After sterilization, the seeds were usually suspended in 0.1-1 ml of a 0.1 % agar

suspension and chilled at 4°C for 7 days. Sterilized seed of the gal and gal ,gai mutants

 

14

was treated with GA3 to promote germination. GA treatment consisted of soaking in 100
uM GA, in sterile water for 2-7 days at 4°C followed by 30-36 hr incubation at 20°C
under fluorescent lights (SO-100 limo] m'2 s’1 PFD).

Agar medium consisted of Arabidopsis mineral solution (Somerville and Ogren,
1982) solidified with 7 g per liter agar (SIGMA). For experiments in which plants were
grown for longer than one week under sterile conditions, the medium was supplemented
with 10 g/L sucrose. For some experiments GA3 and/or paclobutrazol were added to
autoclaved medium after it had cooled to 55°C. Agar plates for seed germination
measured 9 cm in diameter by 1.3 cm deep. For long term growth plants were grown on
9 cm diameter by 1.7—2.0 cm deep petri plates or in individual test tubes (18 X 150 mm)
loosely capped with translucent polypropylene 25mm caps (Bellco Kaputs). Each deep -
petri plate received approximately 50 ml medium; each test tube received 5 ml.

Dry, nonsterile seeds were distributed on plates with flat toothpicks. Sterilized
seeds were planted on the agar surface with pasteur pipets. Generally 50 seeds were
arranged in a grid pattern on each deep plate and 3 mds were placed in each test tube.
- Seedlings in tubes were thinned to one 4-6 days after planting. All sterile plants were
grown in continuous fluorescent light (Sylvania ”grow lux" bulbs 50 -100 limo] m“2 s‘l
PFD) at 20 i 1 °C in a still growth room.

GA and Growth Retardants. A stock solution of GA3 (SIGMA) was prepared at 0.1 M
in dimethylforrnamide. A stock solution of paclobutrazol (ICI) was prepared at 10‘3 M
in acetone. Both solutions were stored at —20°C in tubes sealed with parafilm.

Measurements. For flower fertility experiments, plants were grown in test tubes until the

majority of siliques had dried on the fertile plants (2-3 months). Each plant was then

15

carefully removed from the tube with a forceps and threshed. The sides of the test tube
and surface of the agar were inspected for stray seed. A plant was scored as fertile if it
yielded at least one seed.

Plant height was measured either by holding a ruler up to an intact plant or by
cutting off the plant at the shoot base and laying it out on graph paper. Either way, height
was defined as the distance from the surface of the medium to the top (apex) of the plant.
Test tube plants were generally measured before they contacted the lid.

Flowering time was defined as the number of days from placement in the growth
chamber until flower buds became visible to the naked eye.

Seed germination experiments were performed on batches of seed which had
matured simultaneously in the greenhouse in spring or summer. The seeds were dried in
envelopes for 5-7 d before testing. Dry seeds were placed directly on agar plates
containing 100 pM GA3 at 36 seeds/plate. These were wrapped in 2-3 layers of aluminum
foil in order to keep them dark during chilling (7 days) and germination (5-7 days). To
determine seed viability, some seeds from each batch were germinated in the light on
minimal medium. 1
Genetic Mapping. Sterilized F2 seeds from a WT x garI-I, gai cross were planted on
deep agar plates supplemented with 0.1 uM paclobutrazol and 1 11M GA3. This medium
had been previouly found to help accentuate the growth difference between WT and
gai/WT heterozygotes. The plants were scored for height, leaf size and green color when
WT control plants had "bolted" and pushed up the plate lid (28-35d). Putative dwarfs
were progeny tested. A similar experiment was performed with the garZ-I mutant,

although progeny testing was not performed in this case.

1 6
RESULTS

Effect of the gai Mutation on Flower Development. Severely GA deficient mutants of
Arabidopsis produce defective flowers which are male-sterile and have shortened petals
and stamens, while weaker GA synthesis mutants, which are only partially dwarfed, may
show normal flower morphology and fertility (Koornneef and van der Veen, 1980).
Apparently, less endogenous GA is needed to trigger proper flower development than is
required to fully stimulate stem and leaf growth in the plant.

The gai mutant produces fertile flowers (Figure 2-1) under
standard growth conditions (Koornneef et al., 1985). Initially it was uncertain whether
the gai mutant has normal flowers because the GA] gene plays no role in flower
development, or because this mutant is somewhat "leaky". Even if Gai function is
necessary for petal and stamen growth, the small amount of GA sensitivity shown by the
gai mutant might be enough to cause normal flower development, because extraordinarily
high levels of active GAs can accumulate in this genotype (Talon et al., 1990).

A gai,gaI-I double mutant, which is deficient in GA synthesis as well as
responsiveness, produces defective male sterile flowers (Figure 2-1). This mutant requires
a substantially larger amount of applied GA than does the gal-3 single gene mutant to

stimulate development of fertile flowers (Figure 2-2).

 

 

gai

gai, ga1

 

Figure 2-1. Flower Morphology. The fertile flowers of the gai mutant appear similar
to those of WT, while the flowers of the double mutant gai, gal (gal-I) which is
impaired both in GA response and in GA synthesis, show defects similar to those of a
severe gaI mutant. The plants were photographed at comparable stages of development
(gai at 34 d and gai,gaI-I at 53 d post-planting).The white bar equals 2mm.

18

Stimulation of Flower Development by GA In Arabidopsls Mutants
100 J. 4,-0—

 

—O'- 931
+ gaigal

I

 

% Fertile Plants

 

 

 

 

 

Figure 2-2. The Effect of the gai Mutation on Flower Fertility Restoration. GA
concentration refers to the initial concentration of GA3 present in the sterile media on
which seeds were planted. Plants were classified as fertile if they produced any detectable
seed. Each point represents the mean of 3 trials. For each trial, n=7-10 plants. Bars
represent SE. The single gene mutant shown here carries a slightly more severe allele of
gal (gal-3) than does the double mutant gai, gal (gal-I).

19

In this experiment, without GA treatment gai,gaI-l plants were found to grow

somewhat taller than gal-3 mutant plants [mean height of gai,gaI—I = 12.2 :1; 2.6 mm;
mean height of gal-3 = 6.4 i 1.1 mm (n=19)]. This finding suggests that fairly
substantial levels of active GAs may exist in the double mutant due to ”leakiness” of the
gal-1 allele and enhancement of GA accumulation by the gai mutation. The gal-3
mutation is the most severe lesion known in the important GA biosynthesis gene called
GA] (Zeevaart and Talon 1992). The gai mutation increases the total amount of active
GA necessary to stimulate flower fertility even more dramatically than is indicated by the
graph (Figure 2-2), but some of this hormone is provided endogenously.
Isolation of Suppressor Mutants of gai. A total of 17 mutants in which the gibberellin-
insensitive phenotype is suppressed were obtained from independent mutagenized M,
populations of a homozygous mutant gai line (Table 2-1, 2-1B). This line also carried the
angustofolia (an) mutation as a visible marker. Suppressor of gai, or GA responder (gar),
mutants were readily recognized by their large size and light green color (Figure 2-3).
All primary gar mutants manifested the angustofolia phenotype of narrow leaves and
twisted siliques, so it was apparent that they were not WT contaminants.

Independent suppressor mutants appeared at relatively high frequencies following
mutagenesis, although not before (Table 2-1). Approximately 1/1,500 M, plants that had
been mutagenized with EMS yielded GA responsive mutants in the next generation. This
frequency is comparable to the incidence of heritable loss of function mutations found in
an average gene in heavily EMS mutagenized M, populations of Arabidopsis. One survey

of 15 loci revealed that on average, approximately l/2,000 EMS mutagenized M,

20

 

Figure 2-3. A Primary "Revertant" Among Gibberellin Insensitive Dwarfs (gai, an).
Plants were grown under SD conditions. Its long narrow leaves show that, although the
gai mutation has been suppressed, the big mutant plant still carries the angustololia
marker. The white bar equals 1cm.

21

 

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22

 

Table 2-1B. Suppressor Mutants of gai.

 

 

Mutagen Mutant (Old Name) Mutant (New Name)‘
ethylmethanesulfonate ems-A gar3
ems-C gar4
ems-D gar5
ems-G gar6
gamma rays gamma-1B gar7
gamma-2B gar8
gamma-2C . garZ-I
fast neutrons ' fn—15 gar9
fn-25 garIO
fn-41 garII
fn-46 gar] -1
fn-51 garIZ
fn—65 gar13
fn-67 gar14
fn-70 gar15
fn-83 gar16
fn-99 gar] 7

 

' The mutants gar-I-I and gar2-l define the GAR] and GARZ genes respectively.
Many of the othermutants may be allelic to garI-I, although the semidominant
nature of the mutations has precluded meaningful complementation tests.

 

23

plants carried a mutation at an individual locus (Koornneef et al., 1982).

Phenotypic Characterization. All gar mutants were backcrossed to the parental gai, an
strain and reisolated at least one time prior to characterization. The mutants were tested
for height, flowering time in SD, seed germination, and response to applied GA. These
studies focused mostly on the garI-I and garZ-I mutants, which represent two different
4 classes of gai ”revertants".

The most obvious effect of the gai mutation on Arabidopsis is moderately severe
dwarfing. Mutant gar, gai, an plants grew substantially taller than gai, an plants (Figure
2-4). In an angustofolia background garI-I mutant plants were found to reach the same
height as normal GAI plants (Figures 2-5). In one experiment, the mean height of GA],
an plants was 18.4 i 1.9 cm and the mean height of garl-I, gai, an plants was 20.0 i
3.0 cm (n=15). '

When grown under SD conditions, homozygous gai mutant plants flower later
than WT plants (Wilson et al., 1992). Several .of the gar mutants were tested for
flowering time in SD, and all were found to flower earlier than comparable gai or gai,
an plants (Table 2-2). These gar, gai mutants flowered at approximately the same time

as WT (Table 2-2).

24

 

 

mean height (cm)

 

5““MVWQNQQQNNMVl-nmk
"Kkkkkkksv—shhhhh
“FNNCBN‘BNNQkastKg
“kuuumuuummmmmmmm
gg muuuuuuu

Figure 2-4. Plant Height Comparison of gai and Suppressor Mutants. Each bar
represents the mean of measurements on 5-17 plants :1; SE. Height was recorded 31 days

after planting. All genotypes pictured were homozygous for the an trait and also the gai
trait (unless mutation had occured at that locus).

25

 

Figure 2-5. Phenotypes of Mutants and WT. Pictured are left to right: WT; gai; garI-I ,
gai; and gar2-1, gai. All plants pictured were homozygous for the an trait. Plants were
photographed 29 d after planting. The white bar equals 2cm.

26

 

Table 2-2. Comparison of Flowering Times in Short Days

Experiment . Genotype‘ . Days to Flowering
#1 WT‘ 40.7 i 3.3
gai 63.0 :t 4.5
gar3, gai, an i 45.6 i 2.9
#2 WT 48.6 i 2.9
gai, an 67.8 i 5.9
garll, gai, an 50.3 j; 2.6
garI-I, gai, an 52.4 i 3.1
#3 WT 63.9 i 3.1
gai, an 85.4 i 3.6
garll, gai, an 64.8 i 3.2
garZ-I, gai 68.8 i 5.1

(segregating an)

The mean and SE were calculated from observations on 7-16 plants per trial.
Differences in light quality and intensity probably account for most of the variation
between experiments.

' Any possible affect of the an mutation on flowering has not been explored.

 

27

Depending on a number of factors, such as light conditions during seed
development and length of the after-ripening period, seeds of the gai mutant can
sometimes show unusually strong dormancy (Koornneef et al., 1985). The gai mutant
tends to germinate especially poorly in darkness, even in the presence of large enough
quantities of GA to fully stimulate germination in WT. Dormant seed batches of WT and
mutants which had developed and been stored in parallel were tested (Table 2-3). Both
gai and gai, an mutant seed batches failed to germinate in darkness in the presence of
GA3, but germinated well in the light (Table 2-3). In contrast, seeds of the gar, gai
mutants tested showed normal germination both in darkness and light (Table 2-3).

Paclobutrazol is an inhibitor of GA biosynthesis which interferes with the
oxidation of ent-kaurene, and to a lesser extent with sterol synthesis and other reactions
(Davis and Curry, 1991). WT Arabidopsis norrnally shows little response to applied GA,
probably because endogenous levels of the hormone are already close to saturating
(Koornneef et al., 1985). Paclobutrazol was used to inhibit GA production in plants so
that differences in growth response to applied GA could be readily perceived. In the
presence of this chemical, WT plants show a large growth response to application ’of
GA3, while gai plants show only a small change (Table 2-4). The garI-I, gai mutant
responded substantially to exogenous GA3 in the presence of paclobutrazol. Much like

WT, this mutant elongated very little on paclobutrazol alone (Fable 2-4).

28

 

Table 2-3. Seed Germination

Experiment Genotype % Germination in ‘ % Germination in
Darkness + GA3 Light
' 1 WT 99 100
gai, an . 0 100
garII, gai, an 100 ' 100
garI-I, gai, an 100 100 I
2 WT 100 100
gai 0 97
gar3, gai, an I 96 i 100

 

In experiment #1, n= 108. In experiment #2, n=71-72 in darkness and n=36
in light.

 

29

 

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30
The garZ-I mutant grew taller than expected on paclobutrazol (Table 2-4). It

seems possible that this suppressor mutant constitutively performs processes normally
induced by GA. In this respect it may resemble the slender mutants of pea and barley
(Lanahan and Ho,“ 1988).
Genetic Characterization. Analyses of (gar, gai x gai) F, progeny has shown that every
gar mutants except one is semidominant for suppression of the homozygous gai trait
(Table 2-5). The exceptional garZ-I mutant is completely dominant for suppression of the
gai dwarf phenotype (Table 2-5). In a homozygous gai mutant background, plants which
are heterozygous for the gar] -I trait grow to a height intermediate between that of the
GAR] and garI-I homozygotes (Figure 2-6). In contrast, plants heterozygous for the
garZ-I mutation grow just as tall as garZ-I homozygotes (Figure 2-7). These plants are
somewhat taller that gai/GA] semidwarfs (Figure 2-8). Because the gar2-I, gai mutant
does not grow as tall as WT, it appears that the garZ-I mutation can only partially
reverse the dwarf phenotype, even though the mutation is completely dominant genetically
(Figure 2-7, 2-8). 1

All gar, gai mutants tested (15) yielded tall F, progeny upon crossing to WT
(T able 2-5). For these mutants suppression is completely dominant in a heterozygous
GAI/gai background. However, heterozygous gar2-1/GAR2 mutant plants which are also

heterozygous for the gai mutation do not reach fully WT height (Figure 2-8).

31

 

 

10-

mean height (cm)

 

 

 

 

 

 

 

 

GARi/GARi,gal/gal
GAR1/gar1,gai/gal
gar1/gar1,gaI/gal

Figure 2-6. The garI-I Mutation is Semidominant. Plants were measured 26 d after
transplanting. Each bar represents the mean of measurements on 5-9 plants 1 SE. All
genotypes were homozygous for the angustofolia trait.

.32

 

 

 

mean height(cm)

 

 

 

 

 

 

 

 

 

 

 

GA HZ/GA 32, GA l/GAI (wr)
GA 32/ GA R2, ya I/ GA I
gar2/ger2,gaI/gal
GA RZ/gar2,gaI/gai

Figure 2-7. The gar2-1 mutation is Dominant. Plants were measured 26 d after
transplanting. Each bar represents the mean of measurements on 2-7 plants :1; SE. All.
genotypes manifested the normal An phenotype.

33

3O

 

 

__'

 

 

 

mean height(cm)

d
O
I

 

 

 

 

 

 

 

 

 

 

 

GA RZ/GA R2, gal/GA!
GA Rz/garz, gal/gal
, garz/GA Bagel/GA!

GA RZ/GA 32, GA l/GAI (WT)

Figure 2-8. Comparison of Plant Heights of Various Genotypes. Plants were measured
26 d after transplanting. Each bar represents the mean of measurements on 2-17 plants
:1: SE. All of these plants manifested the normal An phenotype.

34

 

Table 2-5. Dominance of Suppressor Mutants of gai.
(n=number of F, progeny)

 

Mutant‘ (gai X s)°F, (n) (s X gai)°F, (WT X s) F, (s 'X M) F,
gar3 semidwarf (10) semidwarf (10) tall (1) tall (1)
gar4 semidwarf (3) semidwarf (1)

gar5 semidwarf (5)

gar6 semidwarf (3) .

gar7 semidwarf (11) semidwarf (3) tall (3) tall (1)
gar8 semidwarf (1) semidwarf (5) tall (1)
garZ-I tall (27) tall (6) tall (3) tall (3)
gar9 semidwarf (3) semidwarf (2) tall (1)

gar10 semidwarf (1) tall (3)

gar] I semidwarf (9) semidwarf (4) tall (5) tall (2)
garI-I semidwarf (15) semidwarf (4) tall (14)

gar12 semidwarf (5) semidwarf (4) tall (6)
gar13 semidwarf (17) tall (6)
gar14 semidwarf (3) tall (3)
gar15 semidwarf (4) tall (6)
garI6 semidwarf (6) tall (6)
gar17 semidwarf (18) tall (3)
WT semidwarf semidwarf tall tall

gai dwarf dwarf semidwarf semidwarf

 

‘Primary "revertants" were generally backcrossed to the gai, an strain once
before testing. Data from primary and backcrossed suppressor mutants is
pooled. All suppressor mutants were homozygous for gai (unless mutation had
occurred at that locus). ‘
bBoth parents carried the angustofolia (an) trait in most of these crosses.

 

35

Pairwise complementation tests were performed among homozygous gar mutants,
even though the interpretation of such tests is difficult in cases of semidominance (Table
2-6). If two semidominant mutants produce F, progeny which show the complete mutant
phenotype, One cannot be sure whether the phenotype is caused by two mutant alleles of
the same gene, or an additive affect of mutations in two different genes. All pairs of gar

mutants crossed so far have yielded tall (mutant) progeny (Table 2-6).

36

 

Table 2-6. Complementation Tests Among Suppressor Mutants.
A H symbol signifies that F, plants showed the tall phenotype. All
mutants were homozygous for the an mutation, and also (presumably)

for the gai mutation. (n=number of F, progeny)

 

 

 

 

 

 

 

 

 

 

 

Male Parent:

Female Parent garI-I gar-2 gar-4 gar-6 gar2-I '
garI-I - (6) - (2)b
gar3 - (9) - (9)"
gar4 - (2)‘I
gar5 - (6)
gar6 ' (2) - (10)
gar7 - (2)
gar8 " (2) - (1)' ' (6F
gar12 - (3)
gar16 - (1)
garI7 - (2)

 

 

 

 

 

 

'One or both parents in this cross was a primary mutant.
”The F, plants from this cross appeared slighty dwarfed (garI-Ilgai <

F, < garI-I)

‘37

Precise reversion of the original X-ray induced mutation (Koornneef et a1. , 1985)
in the GA! gene would probably be a very rare event. Because GA responder mutants
were frequent, the vast majority are not expected to be true revertants at the gai locus.
Since the gai mutation is semidominant however, it seemed possible that any random
inactivating mutation in this gene might somehow free the plant to grow normally again.
To test this theory, suppressor mutations were tested for linkage to the gai locus as
outlined in Figure 2-9.

Analyses of (WT x gar] -1) F2 progeny showed that the garI-I mutation is in fact
located very close to the gai gene (Table 2-7). Mapping of the gar2-I mutation gave a
much different result. F2 seed from the cross WT x garZ-I yielded a substantial number
of dwarf plants (37/475 = 7.8%). A fraction of 30/475 =1/16 dwarfs would be expected
if garZ-l and gai are unlinked. Semidwarfs were not actively searched for in this
population. Assuming that most or all of the dwarfs observed were gai homozygotes, the

gar2 gene is unlinked to gai.

38
Linkage

Is a mutation that suppresses the gal phenotype
located in the gai gene itself 7

To try to answer this question,cross:

gai "revertant" X wild type

 

if very closely
if unlinked: linked:
g , __S__ (F1) gal S
+ + - + +
self fertilize $le fertilize
(F2)
("1- g— 9 :— Dwarf No Dwarfs
16 gal or semidwarfs
and
(3..) g , :1. semidwarf
8 +
(L) E _s_ semidwarf
8 gal +

Figure 2-9. Testing for Linkage of a Suppressor Mutant to gai. S represents a
suppressor mutation. If there is absolute linkage, the F2 will not contain any dwarf or
semidwarf offspring. If there is independent assortment, the F2 will contain 1/16 dwarfs
and 1/4 semidwarfs. If the two genes are loosely linked, the F2 generation will contain
an intermediate fraction of dwarfs and semidwarfs. '

39

 

Table 2-7. Mapping of the gar] -1 Mutation Relative to gai.

 

Genotype Number Number Dwarf Total Map
Tall or Semidwarf Plants Distanceb
(WT x 463 0‘ 463 dsl.3 cM
garI-I,gai,an) F2 (p=0.95)
(gai, an x WT) F2 18 31 49
WT 45 0 45
gai, an 0 50 50

 

' Although 13 plants from the garI-I cross initially appeared short, all
selfed to yield 90-100% tall progeny (n=47-50).

" Because significantly fewer than the expected 75% of plants in the control
(gai, an x WT) F2 population appeared moderately to severely dwarfed
(63%), it seemed likely that some semidwarfs were missed in this
experiment. It was conservatively assumed that half of all semidwarfs were
detectable.

 

40
DISCUSSION

In summary, a number of facts are now known about gibberellin insensitivity in
Arabidopsis:
1) The gai mutation is semidominant and affects every GA response known in
Arabidopsis (Koornneef et al.,1985). Comparison of the gai mutant to a gai, gal—3
double mutant has shown that the semidominant gai mutation interferes with the ability
of applied GA to stimulate normal flower development (Figure 2-2). Clearly the GA]
gene product must play a fundamental role in the mechanism of action of gibberellin.
2) Suppressor mutations of gai occur relatively frequently in mutagenized Arabidopsis
populations. The frequency of independent GA responder mutants induced by EMS
mutagenesis seems to be comparable to the frequency of loss of function mutations
expected for a single moderately sized gene.
3) Phenotypically, homozygous gar, gai mutants resemble WT with respect to seed
germination, stem elongation growth, and flowering time in SD.
4) Analyses of F, heterozygotes show that the majority (16/17) of the suppressor
mutations obtained act semidominantly to suppress the homozygous gai phenotype. Most
gar/GAR, gai/GA] heterozygous plants grow completely tall.
5) No case of complementation was observed in tests involving 8 different semidominant
mutants.
6) At least one of these suppressor mutants maps very near to the gai gene itself (< 1.3
map units), and no recombinants have yet been found.

One possible explanation for these results is proposed here. If the normal function

of the Gai protein is to induce GA responses in Arabidopsis, we might imagine that the

 

41

gai mutation is semidominant because it'causes an altered polypeptide to form, that can
aggregate to form a multimer, but that prevents the resulting protein from functioning.
A second mutation in the gai gene, which destroys the ability of the dysfunctional
polypeptide to aggregate with other subunits or eliminates the ”poisonous" gai gene
product altogether, would allow the plant to grow normally again, provided that sufficient
normal GAI protein can be manufactured by another gene.

This, idea is illustrated in Figure 2-10, where open circles represent normal
polypeptide subunits, and striped circles represent mutant subunits. For simplicity, only
one copy of each of the two proposed GAI genes has been illustrated, although a diploid
would naturally contain two copies of each.

The model can readily account for the relatively high frequency of suppressor
mutants obtained. A specific alteration within a gene is not required to restore GA
response. Instead, any one of a number of possible mutations which inactivates the gai
gene product would suffice.

If the suppressor mutant is assumed to contain a null allele of GAI, this model can
also explain why a "suppressor” mutant of gai would produce semidwarf F, progeny in
a backcross to the gai homozygote, and tall F, progeny in a cross to WT (Table 2-8),
AsSuming that the two GAI genes encode functionally equivalent polypeptide subunits and
are expressed at equal levels, and that one "poisonous" subunit is sufficient to kill protein
activity, one can calculate the fraction and amount of functional protein expected in,
different genotypes. These calculations have been performed assuming that various

numbers of homologous subunits composes the Gal protein (T able 2-8).

42

Model to Explain Suppression of Gibberellin lnsensitivity

wild type :1 12:1
gene 1 gene 2

o <5

Response
active multimer j
g_a_i mutant 2': 1:1
9010 1 gene 2
lmpalrod

——> GA
Response

inactive multimer

Suppressor .
mutant of gai 5.2.: =3

"’1" ”'1” _ #
‘96. 00 -+ 22......

no functional gene product

Figure 2-10.

 

43

Table 2-8. Variations of the ”Two Gene" Model: Comparison of
Predictions to Observations.

 

 

 

 

Genotype gai/gai gai/GAI s/s s/gai s/GAI
Observed dwarf semidwarf tall semidwarf tall
Phenotype‘
got—TIM"
Fraction of Protein that is Active.
Number of
Subunits
n (1/2)n I (3/4)n 1 (2/3)n l
2 0.250 0.562 1 0.444 1
4 0.062 0.316 1 ’ 0.197 1
6 0.016 0.178 1 0.088 1
8 0.004 0.100 1 0.039 1
10 0.001 0.056 1 0.017 1
I! E . . n°
Amount of Active Protein (Relative to WT Amount).
Number of
Subunits
n (1/2)n (3/4)n 1/2 3/4(2/3)n 3/4
2 0.250 0.562 0.5 0.333 0.75
4 0.062 0.316 0.5 0.148 0.75
6 0.016 0.178 0.5 0.066 0.75
8 0.004 0.100 0.5 0.029 0.75
10 0.001 0.056 0.5 0.013 0.75

 

‘These observations apply to 14/ 15 independent suppressors tested.

l’If competition is presumed to occur, the ratio of functional to disfunctional
protein molecules is the major determinant of phenotype.

cIn this case, only the absolute amount of functional Gai protein is

- important. The threshold of active protein needed for the tall (WT)
phenotype is assumed to be 50% of normal.

 

44

If the Gai protein is assumed to be a dimer, and competition occurs between active
and inactive protein molecules, the ratio of active to inactive dimers would determine
phenotype. This model can explain all of the observed phenotypes.

If inactive protein does not compete with the active form, only the concentration
of active protein is relevant. Such models can also explain the data, but only if the
multimer has at least 4 subunits.

The garZ-I mutation, which is dominant rather than semidominant and does not
map near the GA] gene, is clearly different from the other suppressor mutations. This
mutation does not cause simple loss of function of GAI. The garZ-I mutation may cause
some specific change in the hypothetical second GAI gene, perhaps increasing Gai activity
or amount enough to compensate for inactivation by "poisonous" gai polypeptides.
Another possibility is that the gar2-I mutation might affect some other step in a GA
response pathway so as to override the need for Gai function or somehow compensate for
the gai defect.

. Molecular isolation of the GAI gene and its products will probably be necessary
before it can be determined if the proposed model really presents an accurate picture of
what happens in Arabidopsis. Because many of the suppressor of gai mutants described
here were induced by high energy radiation, we hope that some of the mutants contain
genetic deletions which will make gene cloning possible via the technique of genomic

subtraction (Straus and Ausubel, 1990; Sun et a1, 1992).

REFERENCES

Davis T.D. and Curry E.A. (1991). Chemical regulation of vegetative growth. Crit.
Revs. in Plant Sci. 10(2), 151-188

45

Koornneef M. and van der Veen J. H. (1980). Induction and analyses of gibberellin
sensitive mutants in Arabidopsis thaliana (L. ) Heynh. Theor. Appl. Genet. 58, 257-263.

Koornneef M., Dellaert L.W.M., van der Veen J .H. (1982). EMS and radiation
induced mutation frequencies at individual loci in Arabidopsis thaliana (L. ) Heynh.
Mutation Res. 93, 109-123.

Koornneef M., van Eden J ., Hanhart C.J., De Jongh A.M.M. (1983). Genetic fine
structure of the GA—I locus in the higher plant Arabidopsis thaliana (L. ) Heynh. Genet.
Res. Camb. 41, 57-68.

Koornneef M., Elgersma A., Hanhart C.J., van Loenen-Martinet E.P., van Riin L.,
and Zeevaart J .A.D. (1985). A gibberellin insensitive mutant of Arabidopsis thaliana.
Plant Phys. 65, 33-39.

Koornneef M., Elgersma A., Hanhart C.J., van Loenen-Martinet E.P. (1985b).
New linkage data of chromosome 1 with a very close linkage of three genes affecting
plant height. Arabidopsis Info. Serv. 22,43-48

Koornneef M. and Stam P. (1992). Genetic analyses.In Methods In Arabidopsis
Research. K.Csaba et al., eds (Singapore: World Scientific).

Lanahan M.B., and Ho T.J.D. (1988). Slender barley: a constitutive gibberellin -
response mutant. Planta 175, 107-114.

MacMiIlan J. (1987). Gibberellin-deficient mutants of maize and pea and the molecular
action of gibberellins.ln Hormone Action in Plant Development-A Critical Appraisal,
G.V.1Hoad et al. eds (London: Butterworth & Co. Ltd). ‘

Somerville C.R. and Ogren W.L. (1982). Isolation of photorespiration mutants in
Arabidopsis thaliana.In Methods in Chloroplast Molecular Biology. M. Edelman et al.
eds (New York: Elsevier)

Straus D. and Ausubel F .M. (1990). Genomic subtraction for cloning DNA
corresponding to deletion mutations. Proc. Natl. Acad. Sci. USA 87, 1889-1893. _

Sun T.P, Goodman H.M., Ausubel F.M. (1992). Cloning the Arabidopsis GAI locus
by genomic subtraction. Plant Cell 4: 119-128.

Talon M., Koornneef M. and Zeevaart J.A.D. (1990). Accumulation of C,,,-
gibberellins in the gibberellin-insensitive dwarf mutant ga_i of Arabidopsis thaliana (L. )
Heynh. Planta 182: 501-505

Wilson R.N., Heckman J.W., Somerville C.R. (1992). Gibberellin is required for

46
flowering in Arabidopsis thaliana under short days. Plant Physiol. 100, 403-408

Zeevaart J .A.D. and Talon M. (1991). Gibberellin mutants in Arabidopsis thaliana. In
Plant Growth Regulation 1991, C.M. Karssen, L.C. Van Loon and D. Vreugdenhil
eds.(Dordrecht: Kluwer) pp.34-42.

47
CHAPTER 3

GIBBERELLIN IS REQUIRED FOR FLOWERING IN ARABIDOPSIS THALIANA

UNDER SHORT DAYS

ABSTRACT
Mutants of Arabidopsis thaliana deficient in gibberellin synthesis (gal -3 and gal —6), and
a gibberellin insensitive mutant (got) were compared to the wild type Landsberg erecta
line for flowering time and leaf number when grown in either short days or continuous
light. The gal -3 mutant, which is severely defective in ent—kaurene synthesis because it
lacks most of the GA] gene, never flowered in» short days unless treated with exogenous
gibberellin. After a prolonged period of vegetative growth, this mutant eventually
underwent senescence, without having produced flower buds. The gai mutant and the
”leaky" gal -6 mutant did flower in short days, but took somewhat longer than wild type.
All the mutants flowered readily in continuous light, although the gal -3 mutant showed
some delay. Unlike wild type and gal -3, the gai mutant failed to respond to gibberellin
treatment by accelerating flowering in short days. A cold treatment promoted flowering
in the wild type and gai, but failed toinduce flowering in gal -3. In light of these results,

it appears that gibberellin normally plays a role in initiating flowering of Arabidopsis.

48
INTRODUCTION

Exogenous gibberellin has been shown to promote the switch from vegetative
growth to flowering in a variety of plants. Most species in which applied GA can induce
flowering are long-day or cold-requiring plants, and many of these normally grow as
rosettes under noninductive conditions (17). Exogenous GA fails to stimulate flowering
in many other angiosperms (11). It is still unclear what role, if any, endogenous GA
plays in floral induction. In a very few cases, such as Samolus parviflorus, an inhibitor
of GA biosynthesis has been shown to prevent flowering, in a GA reversible manner
(17). In other species, however, the application of GA synthesis inhibitors failed to block
flowering (8). For these plants, it remains uncertain whether GA is normally involved in
the induction of flowering.

Mutants which are specifically impaired in GA production have been obtained in .
a number of species. The GA deficient-mutants of rice, maize, Arabidopsis, pea and
tomato all flower readily under normal growth conditions (11), although these flowers
may show various structural defects, depending on the species (3,10). A GA deficient
mutant of Brassica rapa takes somewhat longer to flower than normal (16), as does a GA
deficient mutant of Thlaspi arvense (9). A mutant of red clover never flowers without
exogenous GA (2), but it is not clear whether this variant will prove to be defective
primarily in GA metabolism.

It is possible that all the GA biosynthesis mutants which have been examined to
date are "leaky" to some degree, and produce small amounts of active GAs, sufficient to
induce flowering. In order to determine whether GA is necessary for flowering, it is

essential to study a mutant which contains very little or no active GA. Therefore, I

49

examined an extremely GA-deficient mutant of the quantitative long-day plantArabidopsis
thaliana, in which a major portion of the GAI gene is deleted (14). This gene is thought
to encode a product necessary for carrying out the first committed step in GA
biosynthesis, the formation of ent-kaurene (18). The availability of this apparently non-
leaky mutant provided the opportunity for a definitive test of the role of GA in flowering
of Arabidopsis. In order to gain a better understanding of GA action in flowering, I have
also characterized the effect on flowering of the gai mutation, which impairs GA
responsiveness. The results presented here indicate that under short photoperiods, GA is
required for flowering in Arabidopsis.
MATERIALS AND METHODS

Plant Genotypes. The mutants of Arabidopsis thaliana (L.) Heynh. characterized here
were derived from the line Landsberg (erecta) which we refer to as wild type. The
severely GA deficient mutant, gal-3, was induced by fast neutrons (4), and contains a
deletion of a major portion of the GA] gene (14). The "leaky” mutant allele of the GA]
locus, gal -6, was obtained by ethylmethanesulfonate mutagenesis (4), and the gibberellin
insensitive mutant (gar) was induced by X-rays (5). The gal-3 and gal-6 alleles were
originally designated as isolates 31.89 and d352 respectively, but have recently been
renamed (14).

Growth Conditions. Seeds of the gal-3 mutant generally require exogenous GA to
germinate. In order to minimize the effects of residual hormone on plant development,
the seeds were treated for the minimum time necessary to induce germination. Dry seeds
of all genotypes were soaked in 0.1 mM GA3 (approximately 1 ml per 100 seed) in a

loosely capped centrifuge tube for 2 to 7 days at 4°C in darkness, and then for 30 to 32

50
_h at 20°C under fluorescent lights (100 limol m‘zs’l photon flux density (PFD). At this

point the seeds had not yet visibly germinated. The seeds were rinsed with four 10 ml
changes of water and then resuspended in 0.1% agar and pipetted into plastic pots, 7cm
diameter (round) or 4 cm wide (square). These pots contained a mixture composed of
equal parts by volume of potting soil (BACTO), perlite, and vermiculite,which had been
soaked with a mineral nutrient solution (13) and topped with approximately 4 mm of fine
vermiculite. The pots were covered with plastic wrap and moved to growth chambers.
After 4 days the wrap was removed, and the seedlings were thinned to 1 per pot. The
plants were sub-irrigated with distilled water and, after 2 months, drenched with fresh
nutrient solution.

Plants were grown in chambers illuminated by cool white fluorescent bulbs (120
i 10 limo] rn'zs'l PFD. The spectrum of irradiation emitted by such bulbs has been
described (7). The plants either received SD (8 hours of light and 16 hours of darkness)
or CL. The growth temperature was 21 °C except where indicated otherwise. For
experiments in which a chilling treatment to promote vernalization was tested, seeds were
treated with GA and rinsed as described above, and then planted in a 5°C SD chamber
and incubated for 45 days. The seeds germinated and were thinned out during this period.
The seedlings were then shifted to the 21°C SD chamber.
Measurements. Nine to 15 plants were assayed for each treatment.ln leaf counting
experiments, true leaves in the main rosette were counted once a week. Each time, the
youngest leaf was marked with a small dot of pink nail polish (Maybelline).This made
it possible to keep track of all the leaves on a plant despite loss of older leaves, due to

senescence, during the coilrse of the experiment.

51

Flowering time was defined as the number of days from placement in the growth
chamber until flower buds became visible with the aid of a hand-held magnifying glass
(2X). Plants were checked for flower buds every 2 to 3 days. Because leaf number and
flowering time were measured in separate experiments, it is possible that some
parameters, such as light quality, may have varied slightly from one experiment to
another. A
Hormone Application. Beginning 17 days after planting in SD, GA-treated plants were
sprayed generously once a week with 0.1 mM GA3 (Sigma) and 0.02% (vol/vol) Tween-
20. Control plants were sprayed with a solution containing only the Tween-20 and 0.1%
(vol/vol) dimethylformamide (the solvent for the GA3 stock solution).

Microscopy and Photography. This work was performed by John Heckman. The shoot
apical region was excised from a representative plant of each of the following types: the
gal-3 mutant after 81 days growth in SD, wild-type after 45 days growth in SD, the gal -
3 mutant after 25 d growth in CL. The apical regions were fixed in CRAF III fixative
(1), under vacuum, for at least 24 hrs. The tissue was then serially dehydrated in an
ethanol tert-butanol series prior to infiltmtion in paraffin. Serial longitudinal sections were
cut at 10 microns, mounted on slides and stained ’with hematoxylin (1). Median
longitudinal sections were selected from these series and photographed under a Zeiss light

microscope.

52
RESULTS

Effect of daylength on flowering time and leaf number. In SD, the extremely GA
deficient mutant, gal -3, never flowered during any of four independent experiments,
unless treated with exogenous GA3 (Table 3-1; Figure 3-1). However, in CL the mutant.
flowered readily, although later than wild type (Table 3-2). Thus, extreme GA deficiency
converts Arabidopsis from a facultative long-day plant to an obligate long-day plant. After
a growth period of 5-6 months in SD, all untreated gal-3 mutant plants eventually died
without flowering. In three separate experiments the mean time to senescence of the gal -
3 mutant in so was found to be 190 i 9 days, 178 i 13 days, and 168 i 17 days (i
standard error, n=7-12).

. The "leaky" GA deficient mutant, gal-6, did flower in SD, although it took
somewhatlonger to do so than wild type (Table 3—1; Figure 3-1). Like gal-6, the GA
insensitive (gai) mutant also flowered somewhat late in SD (Table 3-1; Figure 3—1,3-2).
This result seems consistent with a general picture of gai as a mutant in which GA
response is greatly reduced, but not abolished (5). In SD at 25°C, all genotypes flowered
more rapidly than at 21 °C, but at both temperatures the gai and gal -6 mutantsflowered
later than wild type. In contrast, in CL, both the gai mutant and the gal-6 mutant

flowered at the same time as wild type (Table 3-2).

53

 

Table 3-1. Flowering Time in Short Days. The mean and SE were
calculated from observations on 1014 plants per trial.

 

Genotype . Temperature (°C) Days to Flowering

WT 21 57 i 2
gai 21 77 i 12

gal-6 21 ‘ 70 i 7

gal-3 21 never flowered (>117 d)
WT 25 40 i 3
gai 25 62 i 4

gal-6 25 55 i 2

gal-3 25 never flowered (>117 d) ‘

 

 

Table 3-2. Flowering Time and Leaf Number in Continuous Light. The
mean and standard error were calculated from observations on 9-15 plants
per trial. The growth temperature was 21 °C. Flowering time and leaf
number were obtained in separate experiments.

 

Genotype Days to Flowering Leaf number
WT 18 i 0 8.2 :l: 0.7
gai 18 i 0 ' 11.6 i 0.7

gal-6 18 i 0

gal-3 29 i 2 11.7 _+_, 0.7

 

54

 

 

 

 

 

 

 

 

 

 

 

120
-WT
-goi
100 ' :lgoi-CS ‘
OJ
.5 so . .
1v
3
_°.
11. 50 .. .
.8
(I)
g; 40 - Ti— .
20 - -
O

 

SD SD + GA

Figure 3-1 The effect of exogenous GA, on flowering time in short days. Each bar
represents the mean of observations on 7 to 11 plants :1: standard errors. GA treated
plants were sprayed weekly with the hormone as described. Untreated gal -3 mutant
plants failed to flower, but the plants began to senesce at the indicated time.

55

Like many annual plants, when Arabidopsis begins to produce flowers, it stops
making true leaves. Thus, generally, a plant which fails to flower, but continues to
produce new nodes at a normal rate, will accumulate more leaves than one which flowers
early (7). In SD, the gal—3 mutant plants produced more than twice as many leaves as
wild type on the main stem rosette, and appeared to produce additional leaves (uncounted)
on lateral rosettes (Figure 3-3, 3-4). Leaf production slowed in these plants eventually,
although they never switched to making flowers. In SD, gai plants produced about twice
as many leaves as wild type (Figure 3—3). Flowering time showed more variability than
leaf number for the gai mutant (Table 3-1; Figure 3-1, 3-3), perhaps reflecting variability
in the growth rates of individual plants.

In CL, gai and gal-3 plants produced somewhat larger numbers of leaves than
wild type plants (Table 3-2). The gal-3 mutant showed retarded flowering in terms of
absolute time as well as leaf number. The gai mutant produced a larger number of leaves
than wild type in CL in one experiment, while flowering as rapidly as wild type in
another (Table 3-2). Since the mutant does not seem to exhibit an unusually rapid rate
of leaf production (gai actually appears to develop more slowly than wild type), this
discrepancy may have been caused by a slightlvan'ation in some parameter between the
two experiments. '

In contrast to wild type, the gai mutant failed to respond to applied GA3 by
accelerating flowering or decreasing leaf number. The gal-3 mutant responded
dramatically to regular spraying with GA3 by producing many fewer leaves and flowering

almost as promptly as GA3 treated wild type (Figure 3-1, 3-2, 3-3).

56

 

Figure 3-2 Phenotypes of Arabidopsis mutants in SD. The top three panels show WT,
gai, and g0] (gaI-3) after 60 days of growth under SD conditions at 21 °C. The lower
panels show 45 day old plants of the above genotypes which were grown under the same
conditions, but which received GA3 treatments. The white bar equals 1 cm.

57

 

Loo

 

 

 

tmo-mwmnu . +
WE- \
.e a - 2

 

 

 

 

 

 

 

m0 + o> .mo + 005

U)
U

Enid ab HOS— 58. 5:52 5 «so: :36. m8: SEE: 8:383: =5 38: em

0:82:33 o: o 8 8 358 H «3:3:— onoa. goo—.35 S»: 83:38: m. :5
can 3. 29.815 we: :53» £30: :5 mofioa. Canaan: :5: 85-:88: mauw E38
:92 aesaaoa. 8 8:535 8:55: o: 596 32.8 :3: 8:658 83838 89:48.
005 :88: :53: :88 958 a 9n «egg—Sm «Ema 3 won «.9. 3 :8.“ :32. m6
:12 8 :5 as: on :5 9601.33.

58

 

,...._. .....

Figure 3-4 Mutant gal-3 after 113 days of growth in SD at 21-23°C. The scale is in
cm.

59

Effect of cold treatment. Because a chilling treatment has been shown to accelerate
flowering in some Arabidopsis genotypes (7), we tested whether low temperature would
induce or promote flowering of, GA mutants. A prolonged cold treatment (45 days) in
SD, prior to growth at 21°C succeeded in reducing leaf number and accelerating
flowering in the gai mutant, but it also reduced leaf production by wild type plants
(Figure. 3-3). The net result was that gai still produced roughly 2-fold more leaves than
wild type. Cold treatment failed to stimulate the gal-3 mutant to flower, and likewise
failed to cause this mutant to produce fewer leaves (Figure 3-3).

Dominance of the late flowering trait. In order to test whether the late-flowering
phenotype of the gai mutant line was due to a single mutation, the F2 progeny from a gai
x wild type cross were scored for late flowering in SD. The cross was scored at a time
when all of 10 wild type control plants had flowered, and all of 10 gai control plants
were vegetative. At this time, only 27 out of 100 F2 plants had visible. flower buds.
Thus, under the growth conditions used, late flowering appears to be inherited as a single
dominant nuclearmutation. (X2=0.213, p>0.5). Since the gai mutation shoWs partial
dominance for the dwarfrng phenotype, and visually dominant mutations are generally
rare, it seems likely that both the late-flowering and the dwarf phenotype are caused by ,

the same mutation.

6O .

Examination of Shoot Apices. In order try to determine whether the gal-3 mutant makes
any progress towards flowering in SD, such as formation of flower primordia, shoot
apices of the gal-3 mutant and wild type which had grown in SD or CL were examined
by light microscopy (Figure 3-5). In SD, no flower primordia or buds could be
discerned in the gal-3 mutant. Instead, the small, flattened apex appeared to be
surrounded by leaf primordia (Figure 3-5). In contrast, microscopic flower buds were
apparent in the mutant grown in continuous light, and also in the wild type grown in SD

(Figure 3-5).

61

gai/SD

  
 

ga1/CL

Figure 3-5 Median sections of shoot apices. The gal -3 mutant grown in SD was 81 days
old. The WT grown in SD was 45 days old. The gaI-3 mutant grown in CL was 25
days old. The black bar equals 0.1 mm.

62
DISCUSSION

Results presented here indicate that the Landsberg erecta line of Arabidopsis
thaliana requires the hormone GA in order to initiate flowers in SD. In spite of the fact
that it is missing a substantial portion of the GA] gene, the gal-3'mutant has been found
to produce greatly reduced but still detectable levels of several GAs (18). This may be
due to the action of a duplicate GAI gene which is poorly expressed in vegetative tissues
(18), or some alternative means of em-kaurene synthesis. Thus, one possible explanation
for the photoperiodism exhibited by the gal -3 mutant might be that in'this genotype GA
production drops to an even lower level in SD than in (CL, to a level below a critical
threshold needed for flowering. It is also possible that Arabidopsis may be more sensitive
to low levels of GA in CL than in SD. An alternative possibility for the photoperiod
effect could be that there are two different flowering pathways in Arabidopsis, a GA .
requiring pathway which operates in SD, and a GA independent pathway which functions
in CL. A true null mutant which lacks all GA would be nwded to test this hypothesis.
The observation that the gal -3 mutant flowered somewhat late even in CL would seem
to argue against the existence of a pathway which is completely GA independent (Table
3-2).

The gai mutant flowered promptly when grown in CL, but took longer to flower
than WT when grown in SD, whether or not it was sprayed with large quantities of GA3.
The gai mutant normally has greatly elevated endogenous levels of GAs (15), and is
defective in every known GA response of Arabidopsis, including: seed germination, stem
elongation, and apical dominance (5). We can now add rapid flowering in SD to the list.

The gai mutant appears to show impairment in a primary step in GA action. The

63

behavior (of this mutant suggests that some important change occurs in Arabidopsis when
photoperiod is lengthened, other than a rise in GA levels, and this unknown change
somehow accelerates flowering.

Like gai, the gal-6 mutant does flower in SD, although it shows some delay.
In spite of the fact that GA production is reduced enough to cause dwarfing in this mutant
(4), the plant can still synthesize enough GA to trigger SD flowering. Thus, it seems that
higher GA levels are needed by Arabidopsis for elongation growth than for flowering in
SD.

Apparently, "leaky” GA insensitive or deficient mutations exert a quantitative
affect on the time to flowering in Arabidopsis, and a moderate reduction in GA levels or
response delays, but fails to prevent flowering in SD. The reason for this delay is not
clear. It is possible that active GA must accumulate to a certain threshold level in order
for flowering to occur, and that mutants impaired in hormone synthesis take longer than
normal to accumulate sufficient GA in SD. Similarly, it might be proposed that the gai
mutant, which is not totally insensitive to GA, flowers rapidly in continuous light by
virtue of abnormally high levels of endogenous GA, but flowers slower under SD because
of a dramatic drop in GA production. However, this theory does not explain why
application of exogenous GA3 failed to noticeably accelerate flowering of the gai mutant
in SD. Perhaps, when either GA levels or the ability to perceive GA are low, the timing
of development somehow becomes perturbed. Many of the monogenic flowering mutants
which have been reported in Arabidopsis are late flowering types (6,7). By analogy with
the GA mutants, it seems possible that some of these might be leaky alleles of loci for

which only a trace of function is nwded to cause flowering.

64

A prolonged chilling treatment failed to substitute for GA in inducing flowering
of the Arabidopsis mutants. Since flowering was promoted in wild type but not in the
gal-3 mutant, it appears that the plants require some minimal level of GA as a
prerequisite for effective vernalization (Figure 3-3). Mutant gai plants responded to cold
treatment, although they failed to respond to exogenous GA. A simple explanation for
this result, in light of. the knowledge that the gai mutant is capable of accumulating very
high levels of endogenous Cw 6A3 (15), is that a vernalization treatment causes some
change, other than an increase in GA levels, which promotes flowering. Alternative
explanations are also possible however.

Flower formation is obviously not a necessary prerequisite for senescence, since
the gal-3 mutant plants eventually died without initiating any visible flower buds. This
is not the first known case of senescence in the absence of flowering. A similar response
was observed with the veg mutant of pea (12). Homozygous veg plants fail to initiate
flowers under any known conditions, but normally senesce after a maximum growth
period of 7 months. The shoots of veg plants could be kept alive indefinitely, however,
if they were periodically grafted onto new wild type roots (12). It is not known whether
the senescence of the Arabidopsis gal -3 mutant in SD might be preventable in a similar
1 fashion. In neither case is it clearly understood why nonflowering mutant plants
eventually cease production of new leaves.

We conclude that GA is crucial for flower initiation in Arabidopsis under certain
growth conditions.

REFERENCES

Berlyn GP, Mishke JP (1976) Botanical Microtechnique and Cytochemistry, Iowa State

65
University Press, Ames, 326 pp

Jones TA (1990) Use of a flowering mutant to investigate changes in carbohydrates
during floral transition in red clover. J Exp Bot 41:1013-1019

Koornneef M, van der Veen JH (1980) Induction and analyses of gibberellin sensitive
mutants in Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 58: 257-263

Koornneef M, van Eden J, Hanhart CJ, de J ongh, AM (1983) Genetic fine-structure
of the GA-I locus in the higher plant Arabidopsis thaliana (L.) Heynh. Genet Res 41: 57-
68

Koomneef M, Elgersma A, Hanhart CJ, van Loenen-Martinet EP, van mm L,
Zeevaart JAD (1985) A gibberellin insensitive mutant of Arabidopsis thaliana. Physiol
Plant 65: 33- 39

Koornneef M, Hanhart CJ, van der Veen JH (1990) The genetic analyses of flowering
time in Arabidopsis thaliana. Flowering Newsletter, Bernier G, Ed, Issue 10, Liége,
Belgium

Martinez-Zapater JM, Somerville CR (1990) Effect of light quality and vernalization
on late flowering mutants of Arabidopsis thaliana. Plant Physiol 92:770-776

Metzger JD (1987) Hormones and reproductive development. In PJ Davies, ed, Plant
Hormones and their Role 1n Plant Growth and Development, Martinus Nijhoff, Boston
pp 431-46210.

Metzger JD, Hassebrock AT (1990) Selection and characterization of a gibberellin-
deficient mutant of Thiaspi arvense (L.). Plant Physiol 94: 1655-1662

Nester JA, Zeevaart JAD' (1988) Flower development in normal tomato and a
gibberellin—deficient (ga-2) mutant. Amer J Bot 75: 45-55

Pharis RP, King RW (1985) Gibberellins and reproductive development in seed plants.
An Rev Plant Physiol 36: 517-568 '

Reid JB, Murfet IC (1984) Flowering in Pisum: A fifth locus, Veg. Annals of Bot 53:
369- 382

Somerville CR, Ogren WL (1982) Isolation of photorespiration mutants in Arabidopsis
thaliana. In M Edelman, RB Hallick, NH Chua, eds, Methods 1n Chloroplast Molecular
Biology, Elsevier, New York, pp 129-139

Sun TP, Goodnian HM, Ausubel FM (1992) Cloning the Arabidopsis GAI locus by
genomic subtraction. Plant Cell 4: 119-128

66

Talon M, Koornneef M, Zeevaart J AD (1990) Accumulation of C ,g-gibberellins in the
gibberellin-insensitive dwarf mutant gai of Arabidopsis thaliana (L.) Heynh. Planta
182:501-505

Zanewich KP, Rood SB, Williams PH (1990) Growth and development of Brassica
genotypes differing in endogenous gibberellin content. 1. Leaf and reproductive
development. Physiol Plant 79: 673-678

Zeevaart JAD (1983) Gibberellins and flowering. In A Crozier, ed, The Biochemistry
and Physiology of Gibberellins, Vol 2, Praeger, New York, pp.333-374'

Zeevaart JAD, Talon M ( 1991) Gibberellin Mutants in Arabidopsis thaliana. In CM
Karssen et al, eds, Plant Growth Regulation 1991, Kluwer, Dordrecht

67
CHAPTER 4

MAPPING ‘OF THE GIBBERELLIN-INSENSIT IVE MUTATION RELATIVE T0

DNA POLYMORPHISMS

ABSTRACT

The gibberellin-insensitive mutation (gal) of Arabidopsis thaliana was mapped
relative to four cloned DNA markers on chromosome 1 (219, 241, 333, and PhAral).
The closest of these, marker 219, maps 5 .1 CM away from the gai gene. All four of the
markers appear to be located on one side of the GAI locus. A novel method was used to
score the gai phenotype in the progeny of a wide cross.

INTRODUCTION

Cloned genomic DNA markers can serve as useful starting points for chromosome
walking if they are located near a gene of interest (Chang et al., 1988). Because the
gibberellin-insensitive (gal) mutation of Arabidopsis defines a gene which plays an
important role in gibberellin (GA) signalling, isolation of this locus would clearly be an
important step in learning how GA functions. To discover if walking to the GA] gene
might be feasible, 1 initiated a search for closely linked DNA markers.

The gai mutation was already known to be located on the left arm of chromosome
1 (Koornneef et al., 1985b), and had been positioned at 21.8 cM from the terminal
marker an on a genetic map of mutations that cause visible phenotypes (Koornneef et al.,
1985b). A map of cloned DNA markers which are polymorphic between different

Arabidopsis ecotypes was also available (Chang et al., 1988). Because the two maps had

68

only been aligned at one point on the left arm of chromosome 1 (an) and two different
pairs of divergent strains had been used in constructing the molecular map (Chang et al. ,
1988), it was unclear which cloned markers, if any, were located in the immediate
vicinity of the gai gene. Therefore several markers were picked and mapped directly
relative to the gai mutation.

MATERIALS AND METHODS
Plant strains. The two strains Landsberg er (LA-O) and Neiderzenz (ND-O) have been
described (Chang et al., 1988). The gai strain contains the gibberellin-insensitive
mutation in a Iandsberg er background (Koornneef et al., 1985).
DNA clones. The cloned Arabidopsis markers 219, 241, 333, and PhAral were obtained
from E. Meyerowitz as lambda phage lysates. Phage DNA was isolated by several
different methods, the most successful being a large scale plate lyses procedure (personal
communication, Carrie Schneider). Phage DNA was gel-purified in low-melt agarose
prior to radioactive labelling.
Plant DNA. Each batch of DNA was prepared from one pot containing 50-500 plants
from the same F3 family or parental line, using a scaled up version of the method of
Dellaporta et a1. (1983). DNAs were prepared from a total of 50 selected F3 families: 32
families were homozygous GAI, 16 were segregating gai, and 2 were homozygous gai.
DNAs were digested in the presence of lmM spermidine. DNAs for blots probed with
the labeled clones 219, 241, and 333 were digested with the restriction enzyme Bgll.
DNAs for blots probed with the PhAral clone were digested with EcoRI.
Southern blots. The Southern hybridization procedure was performed according to the

method of Bematzky and Tanksley (1986). Probes were prepared by radioactively

69

labelling entire lambda clones using the method of random oligonucleotide priming
(Feinberg and Vogelstein, 1984). Each restriction pattern recorded was verified 2-3 times
on successive blots. '
Growth of plants on agar plates. Seeds were surface sterilized in plastic microfuge
tubes. The seed were washed twice with 1m] 70% ethanol and then twice with 1 m1 of
a solution containing 1.6% hypochlorite and 0.02% triton X-lOO. (For each wash, the
ms were vortexed in the solution for 30 seconds, before the supernatant was removed
by aspiration. In a sterile hood the final bleach rinse was removed,and then the seeds
were washed three times with sterile Arabidopsis mineral nutrient solution. -

After sterilization, the seeds were usually suspended in 0.1% agar suspension and
chilled at 4°C for 7 days. Very dormant batches of seed were then incubated for 30-36
hr at 20°C under fluorescent lights (50-100 PFD), but most seed batches were plated
directly. Sterile medium consisted of Arabidopsis mineral solution (SomerVille and Ogren,
1982) supplemented with 10 g/liter sucrose and solidified with 7g per liter agar
(SIGMA). Paclobutrazol (to a final concentration of 0.] 11M) and/or GA3 (to a
concentration of 1 11M) were added to autoclaved medium after it had cooled to 55°C.
Agar Plates measured 9 cm in diameter by 1.7-2.0 cm deep, and each received
approximately 50 ml medium. 25 seed were distributed in a grid pattern on each plate
with a pasteur pipet. A11 sterile plants were grown in continuous fluorescent light
(Sylvania "grow lux" bulbs 50 -100 umol m‘2 s'1 PFD) at 20 :1; 1 °C in a growth room.

RESULTS

Scoring the gai trait. The strain gai (LA-0) was crossed to the ND-O strain. A resulting

Fl plant grew as a semidwarf and was allowed to self-fertilize. Great variability was

70

observed in the F2 generation. The plants ranged widely in height, degree of leaf
expansion, shade of green color, and degree of apical dominance. It appeared that in this
wide cross several genes that interfere with dwarf morphology might be segregating. A
total of 136 F2 plants were allowed to self-fertilize, and their seed was collected as
individual F3 families.

Because visual inspection of height and other characteristics could not be relied
upon as a method for distinguishing between GA insensitive (gai/gai), partially GA
insensitive (gai/GAI), and GA sensitive (GAI/GAI) plants, a new method for assaying GA
responsiveness was invented. Other attempts to score the gai phenotype in wide crosses
'have failed (Brian Hauge, personal communication; Nicholas Harberd, personal
communication). The method used here was to plant seeds from each F3 family on each
of two different petri plates, one plate containing paclobutrazol, an inhibitor of GA
synthesis (Davis and Curry, 1991), and the other plate containing both paclobutrazol and
GA3 (Figures 4-1, 4-2). A GA biosynthesis inhibitor was used because Arabidopsis
normally shows little response to exogenously applied GA, unless endogenous GA
synthesis is first reduced (Figure 4-2) (Koornneef et al., 1985). In the presence of
paclobutrazol normal Arabidopsis of both the LA-O and the ND-O strains shows dramatic
increases in stem growth, leaf expansion, and often seed germination in response to
applied GA3 (Figures 4-1, 4-2). In contrast, the gai mutant shows very little response

(Figure 4-2).

71

 

72

Figure 4-1. Phenotypes of the Strain LA-O on Various Media. PAC=paclobutrazol(0.l
uM); GA=GA3 (luM)..

Figure 4-2. Phenotypes of the gai Mutant and the Strain ND-O on Various Media.
PAC=paclobutrazol (0.1uM); GA=GA3 (1 uM)

73

.. \if‘s»....flrf .35. t

9. .1
y b .
v.

Inward? ,

   

mm. + _u>0 + 0>

 

29.0 + 36,... o>

 

74

Comparison of the two petri plates enabled the amount of change shown by each
F3 family in response to a dramatic increase in GA concentration to be visibly assessed.
There are two potential problems in scoring homozygotes from a gai (LD-O) X ND-O
cross: 1)Extraneous segregating genes may cause gai/gai plants to grow tall.
2)Extraneous segregating genes may impair growth in GAI/GAI plants. Both of these
problems could be circumvented by the use of the 2 plate method. An F3 family was
scored as homozygous GA insensitive (gai/gar) if seed germination and plant growth
appeared very similar on the 2 media, even if some stem elongation was observed on both
plates. A family was scored as GA sensitive (GAI/GAI) if virtually all the plants showed
greatly increased leaf expansion on the GA containing plate, even if stem elongation was
poor.

F3 families which had come from potentially heterozygous F2 plants were the most
difficult to score. Because the gai mutation is semidominant, the family of a heterozygote
should consist of a ratio of 1:2: 1 of dwarf: semidwarf: tall plants. In practice, a family was
scored as segregating the gai gene only if very tall plants constituted approximately 1/4
of the total on the GA containing plate, and all plants appeared dwarf on paclobutrazol
alone. Otherwise the family was not used for mapping, since some other factor might be
causing variability in growth. The majority of F3 families. chosen for DNA analyses were
GAI/GAI homozygotes. All families used for mapping were scored for GA responsiveness

at least twice.

75
DNA analyses. Many (> 50) plants from each F3 family were bulked to yield a combined

DNA preparation which contained a chromosomal composition similar to that of the
parental F2 plant. Analyses of the various F3 DNAs on Southern blots allowed each to
be scored as having a diagnostic LA-O or ND-O pattern of bands, or some combination
of both patterns (Figure 4-3). Accordingly, the allelic composition of each F2 plant could
be inferred for each DNA marker used as a probe. 3

Mapping. A total of 50 F3 families were scored successfully for both the gai mutation
and 2 or more DNA markers. Only four DNA markers were tested. The gai mutation
was found to lie closest to DNA marker 219, and furthest from the marker PhAral (Table
4-1; Figure. 4-4). All the markers are thought to lie on the same side of the gai gene
because calculated genetic distances appear to fit this order (Figure. 4-4), and because all
four markers recombined away from gai in 5/100 chromosomes assayed. If two
recombination events were necessary to achieve this result, the rearrangement would be

expected to occur at a much lower frequency.

76

3: ‘; :‘g";h3332m3.6:l.;36

.
6

 

Figure 4-3. Autoradiogram of a Typical Genomic Blot to Score a Restriction Fragment
Difference. In this case DNAs were digested with Bgl II and probed with the
radioactively labeled clone #333. Left to right are LA-0, ND-O, and a series of F3
families.

77

 

Table 4-1. Mapping of gai Relative to DNA Markers.

Markers Recombinants Distance(cM)
. Total Chromosomes

gai—2 19 5/98 5.1

219-241 1/96 ' 1.0
' 241-333 0/96 0
333-phAral 1/80 1.25

 

‘ Analyses of 49 F3 families resulting from the cross
gai(Ld-O) X GAI(Nd-0) yielded these results.

78

 

 

 

 

 

— 251
1131 212 :11: mm
21.9. 16.8 15.8 14.5

- Figure 4-4. Genetic Map of a Portion of Chromosome 1. The numbers indicate distances
‘ from the left end of the chromosome in cM. The gai gene, which has previously been
located at 21.9 cM (Koornneef, 1985), serves as a reference point here.

 

79
DISCUSSION

It appears that chromosome walking to the gai gene will not become practicable
until closer DNA markers that flank the gene can be found.
REFERENCES

Bernatsky R. and Tanksley S.D. (1986). Methods for detection of single and low copy
sequences in tomato on Southem blots. Plant Mol. Biol. Rep. 4(1), 37—39

Chang C., Bowman J.L., Dejohn A.W., Lander E.S., Meyerowitz E.M. (1988).
Restriction fragment length polymorphism map for Arabidopsis thaliana. Proc. Natl.
Acad. Sci. USA 85, 6850-8860

Davis T.D. and Curry E.A. (1991). Chemical regulation of vegetative growth. Crit.
Revsrin Plant Sci. 10(2), 151-188

Dellaporta S.L., Wood J ., Hicks J .B. (1983). A plant DNA minipreparation: Version
1]. Plant Mol Biol. Rep. 1(4), 19—21

Feinberg A.P. and Vogelstein B. (1984). A technique for radiolabelling DNA
fragments to high specific activity. Analytical Biochem. 137, 266-267

Koornneef M., Elgersma A., Hanhart C.J., van Loenen-Martinet E.P., van Riin L.,
and Zeevaart J .A.D. (1985). A gibberellin insensitive mutant of Arabidopsis thaliana.
Plant Phys. 65 , 33-39 ' ’

Koornneef M., Elgersma A., Hanhart C.J., van Loenen-Martinet E.P. (1985b).
New linkage data of chromosome 1 with a very close linkage of three genes affecting
plant height. Arabidopsis Info. Serv.

Somerville C.R. and Ogren W.L. (1982). Isolation of photorespiration mutants in
Arabidopsis thaliana. In Methods in Chloroplast Molecular Biology. M. Edelman et al.
eds (New York: Elsevier)

80
CHAPTER 5

ISOLATION OF A PACLOBUTRAZOL RESISTANT MUTANT

ABSTRACT

A mutant of Arabidopsis thaliana has been isolated that shows resistance to the
growth retardant paclobutrazol. This mutant is suspected of showing somewhat elevated
sensitivity to the plant hormone gibberellin, and has been tentatively named gibberellin
super-responder (gasI). Mutant gas] plants grow taller than Landsberg erecta wild-type
plants on paclobutrazol containing media, and flower earlier in short days when grown
in the absence of paclobutrazol. Paclobutrazol resistant mutants are rare in Arabidopsis.

INTRODUCTION

Mutants which respond in a hyperactive or constitutive manner to the plant
hormone gibberellin (GA) have been found in barley, pea, and tomato (Scott, 1990). It
was hoped that similar mutants might be obtainable in Arabidopsis thaliana because this
plant has a small genome, short lifecycle, and other attributes which make it particularly
suitable for classical and molecular genetic studies (Bowman et al., 1988).

The slender mutantsof barley and pea which appear to show constitutive GA
signalling, contain lower than normal levels of active 6A5, and have been found to show
resistance to chemical inhibitors of GA synthesis (Lanahan and Ho, 1988;. Potts et al.,
1985; Scott, 1990). It was thought that slender-like Arabidopsis mutants would also be
likely to show growth retardant resistance, and that this phenotype might be used as the

basis of a screen for such mutants. Application of exogenous GA normally has little

81

visible affect on Arabidopsis plants, perhaps because endogenous GA levels are close to
saturating, so it was not clear if a constitutive GA response mutant would appear any
taller than a normal plant. It seemed likely that potential slender mutants would be most
readily recognizable if endogenous GA were lowered, as could be easily achieved by
growth retardant treatment. The chemical paclobutrazol which inhibits the oxidation of
ent-kaurene (Davis and Curry, 1991), was chosen for initial experiments
MATERIALS AND METHODS

Mutagenesis. Seeds of the Arabidopsis strains Landsberg erecta and Columbia were
treated with 0.3% ethylmethanesulfonate for 9-18 hours. Populations of approximately
40,000 surviving Columbia M, plants and 30,000 surviving M, Landsberg erecta plants
were grown in a cool (IS-20°C) greenhouse. Columbia M2 seeds were bulk harvested,
while the Landsberg M2 seed was collected in 19 separate pools of M, plants. To try to
preserve dormancy, dry seed was stored at -80 C. As an indication of the success of
mutagenesis, the approximate frequency of putative mutants at the APT] locus was
determined by selecting for resistance to 2,6 diaminopurine (Moffat and Somerville,
. 1988), and found to be 1/2500 among Columbia M2 seed and 1/6000 among Landsberg
M2 seed. _

Mutant Screening. ‘ Paclobutrazol (ICI) was incorporated into agar plates at a
concentration (1 11M) which severely inhibited md germination and growth of WT
plants. At this concentration, the affects of the chemical could be largely reversed by the
addition of 1 11M GA, to the plates (Figure. 5-1). Agar medium consisted of Arabidopsis
mineral nutrient solution (Somerville and Ogren, 1982) solidified with 7 g per liter agar

(SIGMA).

82

 

Figure 5-1. The Effect of Paclobutrazol and GA on Seed Germination and Seedling
Growth. The top panel shows WT on PAC (luM paclobutrazol), PAC + GA (+ luM
GA3), and on minimal (Arabidopsis nutrient agar). The lower panel shows a typical
mutant selection plate containing luM paclobutrazol. No resistant plants are apparent.

83
Surface sterilized M2 seed suspended in 0.1% agar was spread on cheesecloth

lined plates at a density of 5-10,000 seed per 13.5 cm diameter plate (Figure. 5-1). The
plates were placed in a growth room at 21 °C under cool white fluorescent lights (50-100
umol 1n‘2 s'l PFD) for 5-10 days. Unusually large, light green seedlings were transplanted .
to individual (4 cm wide) square plastic pots containing Arabidopsis mixture (equal parts
by volume of perlite, vermiculite, and potting soil). The pots had been wetted with
mineral nutrient solution (Somerville and Ogren, 1982) and topped with 4 mm fine
vermiculite. Transplants were covered with plastic wrap and placed in a growth
chamber,where they experienced a temperature of 22 i 1°C and a continuous light
intensity of 110 i 10 umol m'2 s‘1 PFD emitted by cool white fluorescent bulbs. After
self-fertilization,-these putative paclobutrazol resistant plants were retested in the next
generation. The resulting seed was plated on minimal, 1 ,uM paclobutrazol, and 0. 114M
paclobutrazol containing media and compared for germination and growth.

Phenotypic Characterization. The gas] mutant was backcrossed to Landsberg cream
5 times before characterization. For height measurements, plants were grown in
individual test tubes, each containing 5 ml Arabidopsis nutrient agar supplemented with
10 g per liter sucrose with or without paclobutrazol. Flowering time was measured on
plants grown in individual pots in short days (8 hr. fluorescent light, 16 hr darkness) as
has been previously described (Wilson et a]. , 1992). Flowering was recorded when floral
buds became visible to the naked eye. ‘

RESULTS
Mutant Frequency. Paclobutrazol resistant mutants appear to be rare in Arabidopsis.

Only one such mutant, gas], was recovered out of a total of more than 100,000 EMS

84
mutagenized M2 seed (5-15,000 from each of 19 subpopulations) of the Landsberg erecta

strain. Three additional mutants which show paclobutrazol resistance were recovered from
approximately 106 EMS mutagenized M2 seed of ecotype Columbia. (The Columbia
mutants await further characterization). By contrast, simple loss of function mutations
generally occur quite frequently in EMS mutagenized Arabidopsis. One survey of 15 loci
revealed that on average, approximately 1/2,000 EMS mutagenized M, plants carried a
mutation at an individual locus (Koornneef et al., 1982).

Characterization of the gasl Mutant. A mutant of the Landsberg erecta (WT) strain of
Arabidopsis which shows resistance to paclobutrazol (gas]) was isolated as a potential GA
super-responder. When grown on paclobutrazol, this mutant appears quite different from
WT, showing greater stem elongation, somewhat earlier flowering, and a lighter green
color (Figure 5-2). The mutant can grow several fold taller than WT on paclobutrazol
(Figure 5-3), but surprisingly, when grown on minimal medium, it actually shows slightly
poorer growth than WT (Figure 5-3). Because alterations in GA levels or response can
affect flowering time in short days (Wilson et al., 1992), the gas] mutant was examined
for this response. The gas] mutant clearly flowers earlier than WT when grown under

short day conditions (Figure 5-4).

 

85

A

0L6

8
lllllllllllllllllll‘lllllllllllllllllll’lllllllllllllll

 

Figure 5-2. The gas] Mutant Is Resistant to Paclobutrazol. WT is on the left and gas]
on the right. Plants were grown on 0.1uM paclobutrazol in individual test tubes. The
roots are not completely intact. Scale is in cm. .

86

, Resistance to Paclobutrazol

‘2‘. l

10']

 

Plant Height (cm)
0)

 

 

 

 

 

2%

PAC .

 

i:

M

Figure 5-3. Plant Height in the Presence of Paclobutrazol. Bars represent mean 1 SE
(n=9-10). PAC=0.1 11M paclobutrazol. Plants were measured after 27 days growth in
test tubes.

 

 

 

 

 

 

 

 

 

 

eeeeeeeeeee
ooooooo

88
DISCUSSION

It is not clear why slender-like mutants were recovered so infrequently in these
experiments. One possible explanation is that a single simple loss of function mutation
is not sufficient to induce a constitutive GA response phenotype in Arabidopsis. In pea,
two mutations in different genes are necessary to produce the slender phenotype (Potts
et al.,1985), although only one mutation is nwded in barley. (Lanahan and Ho, 1988).
Perhaps both pea and Arabidopsis contain a duplication of a gene that normally functions
to inhibit GA responses. In that case, inactivation of both c0pies by random mutagenesis
would be predicted to be extremely unlikely. Depending on the size of the gene, a
frequency of at most l/106 EMS M2 seed might be expected. It may also be that a rare
type of single mutation, such as a particular base pair substitution within a gene, could
somehow elevate GA responses. Preliminarily, the gas] mutant phenotype appears to be
determined by a single recessive nuclear gene (data not shown).

It is also possible that the concentration of paclobutrazol used for mutant screening
was too high. Paclobutrazol is not a completely specific inhibitor of GA synthesis, and
can interfere with sterol synthesis and other metabolic processes, particularly at high
concentrations (Davis and Curry, 1991). However at the concentration used for screening
( 1 11M), most visible affects of the chemical could be counteracted by the addition of GA3
(1 11M), during short term growth on petri plates (Figure 5-1). It may be that most GA
superresponse mutants are ”leaky", and would not have appeared resistant to 1 11M
paclobutrazol under the growth conditions used, but would have been detectable on a
lower concentration of the chemical.

The nature of the gas] mutation is not yet known. It may be that the mutant

89
simply accumulates higher GA levels than WT, or is specifically altered in paclobutraml

uptake or binding. Two pieces of preliminary data argue against these possibilities
however. When grown in the absence of paclobutrazol, the gas] mutant may have lower
GA levels than normal (Manuel Talon, personal communication). The mutant also appears
to show some resistance to AMO—l618 (data not shown), an inhibitor of GA biosynthesis
which acts on an earlier step in the pathway than paclobutrazol (Figure 5-5). Thus, it
seems likely that the gas] mutation does not raise GA levels or directly nullify the
effectiveness of paclobutrazol in the plant.

The gas] mutant does nOt show cemplete resistance to even a relatively low (0.1
aM) concentration of paclobutrazol (Figure. 5-3). It may be that GA response is

somewhat elevated but not constitutive in this mutant.

9O

mevalonate ,

geranylgeranyll pyrophosphate

 

 

 

 

} Amo-1618
copallyl pyrophosphate
ent -kaurene
ent -l<aurenol . >paclobutrazol

wt

 

 
   

ent -ka ren

ent -ka renoic acid

GA 12-aldehyde

Gibberellins

Figure 5-5. Early Steps in Gibberellin Biosynthesis That Can Be Inhibited by the Growth
Retardants Paclobutrazol and AMO-l618. Adapted from Davis and Curry, 1991.

9 1
REFERENCES

Bowman J .L., Yanofsky M.F., Meyerowitz E.M.(l988). Arabidopsis thaliana: a
review. In Oxford Surveys of Plant Molecular an Cell Biology. Vol 5, 57-87

Davis T.D. and Curry E.A. (1991). Chemical regulation of vegetative growth. Crit.
Rev. Plant Sci. 10(2), 151-188

Koornneef M., Dellaert L.W.M., van der Veen J.H. (1982). EMS and radiation
induced mutation frequencies at individual loci in Arabidopsis thaliana (L. ) Heynh.
Mutation Res. 93, 109-123.

Lanahan M.B., and Ho T.J.D. (1988) Slender barley: a constitutive gibberellin -
response mutant. Planta 175, 107-114. '

Moffat B. and Somerville C. (1988). Positive selection for male-sterile mutants of
Arabidopsis lacking adenine phosphoribosyl transferase activity. Plant. Physiol. 86, 1150-
1154 ‘

Potts W.C., Reid J.B.,Murfet LC. (1985). Intemode length in Pisum. Gibberellins and
.the slender phenotype. Physiol.Plant. 63, 357-364

Scott I.M. (1990). Plant hormone response mutants. Physiol.Plant. 78, 147-152
Somerville C.R. and Ogren W.L. (1982). Isolation of photorespiration mutants in
Arabidopsis thaliana. In Methods in Chloroplast Molecular Biology. M. Edelman et al.
eds (New York: Elsevier)

Wilson R.N., Heckman J.W., Somerville C.R. (1992). Gibberellin is required for
flowering in Arabidopsis thaliana under short days. Plant Physiol. 100, 403-408

92
CHAPTER 6

POLLEN MUTAGENESIS

INTRODUCTION
It was hoped that pollen mutagenesis of Arabidopsis thaliana might become a useful
technique for isolating new alleles of existing mutations. If the existing mutation is
recessive, wild-type (WT) pollen can be mutagenized, and then crossed, on to a mutant
female. Most resulting plants will appear WT. A new mutation in the gene of interest
can cause the mutant phenotype to appear in a rare plant.

If the existing mutation is dominant, pollen from the homozygous mutant plantcan
be mutagenized, andthen crossed on to a wild—type female. Most of the resulting plants
will appear mutant. If the dominance in this case is caused by overactivity, novel
activity, or ”subunit poisoning" of the mutant gene product, then a second mutation
which abolishes activity of the mutant gene should cause a phenotypically WT plant to
appear. In this case, both the dominant allele and the recessive one which replaces it are
mutant, while in the first case the dominant allele was WT.

Pollen mutagenesis could also be a useful method to isolate a variety of dominant
mutations readily, some of which might be difficult to recover from mutagenized seed.

Preliminary experiments had shown that Arabidopsis md set and in vitro pollen
tube germination remain fairly constant, when pollen is treated with doses of gamma
radiation ranging from 0-100 KRads. Percent seed germination, however, decreases

substantially at high doses (M. Estelle, personal communication). Mutagenesis

. . 93
experiments in which Colombia WT pollen was irradiated (with approximately 50 Krad.

gamma) and then applied to stigmas of the W1 strain (Koornneef and Stam, 1992), had
shown that mutants which showed a visible phenotype corresponding to one of the 5
recessive mutations present in the female parent occurred at an average frequency of
U292 per locus among viable seedlings (Mark Estelle, personal communication).

It was thought that a male-sterile (ms), thiamine auxotrophic (py), adh strain might
be very useful for testing pollen mutagenesis. New mutations which uncover the adh
mutation can be readily selected at the level of seed germination by allyl alcohol
treatment (Dolferus and Jacobs, 1984), and the presence of thems mutation makes cross-
fertilization easier. In practice, seed of the recessive ms mutant must always be obtained
from fertile MS/ms heterozygotes. Because the ms phenotype can not be readily
recognized until. after flowering, pollen cross-contamination sometimes occurs between
fertile plants and their male-sterile neighbors. If the ms tester strain also carries the py
mutation however, seed which results from cross-contamination of such plants will
develop into thiamine-requiring seedlings, which will soon bleach and die in the absence
of the vitamin. Thus, if mutagenized WT pollen is used to fertilize the stigmas of adh,
py, ms plants, only F, seeds which carry new adh mutations should survive allyl alcohol
selection and grow into green plants.

MATERIALS AND METHODS

The inflorescences of healthy, maximally fertile, Arabidopsis thaliana plants were
cut off and placed in a petri dish. The dish was rapidly (within one hour) inserted into
the sample cavity of a sealed-source irradiator, where the flowers were subjected to

various doses of gamma rays. Open flowers, which appeared to be shedding pollen, were

94

used to pollinate male-sterile tester plants over a period of 1-8 hours.The resulting seeds
were harvested when mature.

An adh mutant, thiamine—requiring strain which segregates male-sterile plants was
obtained from a cross of an adh mutant in ecotype Bensheim (Dolferus and Jacobs, 1984)
by the Landsberg strain W1. WT Landsberg (erecta) flowers were irradiated with a °°Co
source, and their pollen was applied to male-sterile plants of the adh strain. Homozygous
ADH null seeds were selected by treating dry F1 seeds with 35 mM allyl alcohol for 2
hours with agitation, and subsequently washing four times with sterile water. The seeds
were resuspended in 0.1% agar and spread on Arabidopsis nutrient (Somerville and
Ogren, 1982) agar plates. The plates were chilled at 4C for one week, and then
transferred to a growth room. Green seedlings were counted 12 days later. These
seedlings were transplanted to soil, and those which developed into fertile plants were
allowed to self-fertilize. The resulting. F2 seed was tested for germination with and
without allyl alcohol selection. A

RESULTS

Mutations which uncovered the recessive adh mutation were induced frequently
by gamma irradiation of pollen, and the fraction of adh mutants seemed to increase with
dose (Table 6-1). Six of the mutants obtained in this experiment gave rise to selfed seed.
All batches of F2 seed showed 97-100% germination when untreated (n=35-36 wd).
After allyl alcohol treatment, 5 of the seed batches showed 100% germination, while one
batch showed only 42% germination (n=36). WT control seed showed 100% germination
before allyl. alcohol, and 0% after. All batches of seed segregated white (py) mutant

seedlings.

95

 

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96
DISCUSSION
Because the ADH gene has been physically isolated (Chang and Meyerowitz,
1986), it is possible to directly determine whether this gene has been altered or deleted
by pollen mutagenesis. Apparently, gamma radiation can produce mutations in
Arabidopsis pollen very effectively. Because high energy radiation causes deletions and
other chromosome rearrangements, as well as point mutations, this method may prove
useful in generating new alleles. There are currently some problems with the technique
however. Some of the F1 plants produced by this method show infertility (perhaps due
to uncovering of the ms trait). Also, when the 6 adh mutants obtained by pollen
mutagenesis were sent to the laboratory of Dr. F. Ausubel, no homozygous deletions
were found to be present in the ADH gene (F. Ausubel, personal communication).
REFERENCES
Chang, C. and Meyerowitz, E.M. (1986). Molecular cloning and DNA sequence of the
.11 Zilzlzridopsis thaliana alcohol dehydrogenase gene. Proc. Natl. Acad. Sci. USA 83: 1408—
Dolferus, R. and Jacobs, M. (1984). Polymorphism of alcohol dehydrogenase in
Arabidopsis thaliana (L. ) Heynh: genetical and biochemical characterization. Biochemical
Genet. 22: 817-838 . .

Koornneef, M. and Stam, P. (1992). Genetic Analyses.1n Methods in Arabidopsis
Research. C. Koncz et al. eds. (Singapore: World Scientific) p.83-99

Somerville, C.R. and Ogren, W.L. (1982). Isolation of photorespiration mutants of
Arabidopsis thaliana.ln Methods in Chloroplast Molecular Biology. M.Bdelman et a1.
Eds.(New York: Elsevier) p.129-l38

97

CONCLUSION

In summary:
The gai mutation has been found to affect flower development, and initiation of flowering
in short days (SD). This mutant now appears to show alteration in every gibberellin (GA)
response known in Arabidopsis. ’
GA has been found to be necessary for Arabidopsis flowering in SD. Apparently, a cold
treatment cannot work to accelerate flowering under such conditions unless some minimal
level of the hormone is present. ,
Suppressor mutations of gai appear to be readily inducible. Most of these mutations are
semidominant for suppression. The garZ-I mutation is completely dominant and defines
a new locus involved in GA response.
Paclobutrazol resistant mutants appear to be very rare. The gas] mutant grows taller than
WT on paclobutrazol and flowers earlier in SD. .
The gai mutation maps 5.1 CM from the polymorphic DNA marker 219, and is located
further away from some other markers tested.
At least three different types of mutants which show altered GA responses are now known
in Arabidopsis; gai, garZ-I, and possibly gas].
Future research should have the following aims:

The gar2-I mutant should be further characterized after it has been crossedaway '
from the gai mutation. It should be determined whether this mutant really shows some
level of constitutive GA response. The gas] mutant should also be characterized in more

detail. If the gar2—I mutant proves to be recessive for a paclobutrazol resistance

98

phenotype, a complementation test could be performed between the two mutants. Both
mutations should be placed on the genetic map.

Double and triple mutant combinations between gai, gar2-1, gas], and perhaps
, garI-I should be constructed to try to determine if the different genes can be arranged
in a linear pathway, or perhaps parallel pathways in GA signal transduction.

Attempts should be made to isolate gene(s) from radiation induced mutants by
genomic subtractive hybridization, because it appears possible that these mutants may
contain sizable DNA deletions. The gai mutation may actually be a deletion, since it was
induced by X-rays. Gamma and Fast Neutron induced semidominant gar mutants may
also contain deletions in the gai gene, or some other gene nearby. Once isolated,.the gai
gene could be sequenced in hopes of learning about its function by comparison to known
genes. The gene product from mutant and~ normal GA] genes could be tested for the
ability to bind GA, and if that fails, for the ability to bind other cell components, such
as DNA.

Once mapped relative to DNA markers, attempts might be made to isolate the
gar2-I and gas] genes by chromosome walking. Ultimately one could test for interactions
between various GA signalling proteins in vitro.

All of the existing gar mutants and the gas mutant must eventually be located on
the Arabidopsis genetic map. >

It is possible that more genes involved in GA response could be identified if
mutant hunting was continued, although the existence of gene duplications or families in
Arabidopsis may make it impossible to find certain types of mutants.

A straightforward, but laborious strategy would be to look for new gibberellin-

99

insensitive mutants simply by screening for dwarfs which show little response to GA
treatment. Since properly dormant gai seeds germinate in the light but not in the dark,
another possible procedure for isolating gai-like mutants might be to germinate dormant
M2 seeds in the dark with GA, kill germinated mdlings by desiccation, and then rewet
non-germinated seeds in the light.

Other new mutants might be found by searching for mutants that flower early or
late in SD, and subsequently screening them for alterations in stem growth and other GA
responses. It also might be interesting to isolate more suppressors of gai from a large

EMS mutagenized population, to see if any other kinds of gar mutants can be found.

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