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J. Embryol. exp. Morph. 83, Supplement, 119-135 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
H9
Gibberellins and gene control in cereal aleurone
cells
By DAVID BAULCOMBE, COLIN LAZARUS AND
ROBERT MARTIENSSEN
Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ,
U.K.
TABLE OF CONTENTS
Summary
Introduction
Coordination of aleurone cell activity with the onset of germination: the role of
gibberellin
Gibberellin action at the cellular level
a-amylase gene families in wheat
The origin of variation in a-amylase gene families
The expression of Gibb-ons
Conclusions
References
SUMMARY
The production of hydrolytic enzymes in the germinating cereal grain is considered as a
model for plant cell differentiation. Recent literature is reviewed which suggests that gibberellins are involved in this process, but in a less straightforward manner than considered
previously. It seems likely that only a subfraction of gibberellin is active and that production
of this gibberellin is actually in the hydrolase-producing cells. These include aleurone cells and
also the scutellar epithelial cells.
At the intracellular level the action of gibberellin results in the accumulation of a-amylase
mRNA and also mRNA for other unidentified proteins, referred to as gibb-ons. The aamylase mRNAs are transcribed from two distinct gene families. The pattern of expression
of a-amylase and gibb-on mRNAs is consistent with a common gibberellin-stimulated
mechanism of control of for all of these genes. However it seems likely from experiments with
ABA and from observations on gibb-on gene expression in non-aleurone cells that gibberellin
does not have a role determining the specificity of gene expression, but rather acts as a general
stimulator of mRNA accumulation.
INTRODUCTION
The aleurone cells of cereal grains surround the starchy endosperm and secrete
a-amylase and a range of hydrolytic enzymes into this tissue following the onset
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D. BAULCOMBE, C. LAZARUS AND R. MARTIENSSEN
of germination. The physiology and biochemistry of aleurone cells have been the
subject of much experimental investigation for three primary reasons. First, the
aleurone cells may be dissected free of any other living tissue simply by excision
of the embryo. Thus, incubation of de-embryonated grains is an unusually simple
organ culture, comprising a single cell type which, at the beginning of germination is almost completely differentiated. Second, it is widely considered
(although see below) that many activities of aleurone cells, including the production of a-amylase, are under the control of a plant growth regulator, gibberellin.
A third reason for much of the interest in this tissue results from the demonstration that a-amylase production in these cells results from a new accumulation of
mRNA (Higgins, Zwar & Jacobsen, 1976). These three features, morphological
uniformity, a well-characterized developmental stimulus and a defined response
at the level of gene expression all commend aleurone cells of germinating cereals
as a tissue in which to study the details of a small developmental progression in
a higher plant system.
The picture which has built up from this research is of a highly specialized cell
type undergoing the final stage of its differentiation as germination begins. Most
of the changes observed relate to the secretory activity of the tissue or to the
general increased rate of metabolism. Thus, the earliest events include an increase of the enzymes involved in lecithin biosynthesis (Johnson & Kende, 1971)
and a subsequent proliferation and vesiculation of rough endoplasmic reticulum
(Vigil & Ruddat, 1973). Later, there is an elevated level of ribosomal RNA
(Chandra & Duynstee, 1968) and polyadenylated (poly(A)+) mRNA (Ho &
Varner, 1974) synthesis, including the mRNA for a-amylase (Higgins et al.
1976), which results in an increased rate of protein synthesis. Much of this
synthesis may involve proteins which are secreted and which are synthesized on
membrane-bound polyribosomes. The fully mature aleurone cells are producing
and secreting a range of hydrolytic enzymes include a- and /?-amylases,
glucanases for degradation of cell walls and the enzymes of ^-oxidation of fatty
acids (Akazawa & Miyata, 1982). As the germinating plantlet becomes
autonomous, and photosynthetic leaf tissue develops, the aleurone cells senesce.
The processes considered in this article are thus the terminal stages of aleurone
differentiation. Main emphasis is given to how the activities of the aleurone layer
might be integrated, both with each other at the cell level and also with the
development of the rest of the plant.
CO-ORDINATION OF ALEURONE CELL ACTIVITY WITH THE ONSET OF
GERMINATION: THE ROLE OF GIBBERELLIN
It is obviously important that mobilization of seed reserves will not be initiated
until the embryo is ready to start growth and it is predicted therefore that a signal
be passed, at the appropriate time, from the embryo to the site of hydrolytic
enzyme production. The early experiments of Paleg and Yomo (reviewed in
Gibberellins and gene control
121
Yomo & Varner, 1971) confirmed this by showing that hydration of isolated
aleurones was not sufficient stimulus, but that a diffusible factor was released
from embryos which served to actuate the production of a-amylase and other
enzymes. It was also shown that gibberellin was released during germination of
isolated embryos (Radley, 1967) and that isolated aleurones would undergo the
normal differentiation into a secretory tissue in the presence of low concentrations (10~ 6 M) gibberellic acid (GA3). This suggested a hormonal model in which
gibberellin produced by the embryo diffused to the aleurone cells and induced
the terminal development. However and perhaps inevitably, further investigation has shown that the communication between embryo and site of hydrolytic
enzyme production is more complicated than suggested by this attractively simple model. In particular, the precise role of gibberellins in this process has been
questioned, and the aleurone layer is no longer thought to be the major site of
hydrolytic enzyme production.
Evidence for the latter comes from the work of Gibbons (1979) who used an
in situ immunological staining procedure to show that the major site of a-amylase
production in barley grains was not the aleurone layer, but was in fact the
scutellar epithelium. Okamoto (described in Akazawa & Miyata, 1982) has
shown essentially the same in rice grains using a starch film method to detect
amylolytic activity in tissue sections. Similarly, a stain for undegraded cell wall
material was used to locate the site of /3-glucanase production to the scutellar
epithelium (Gibbons, 1981). On the basis of these data, a modified scheme of
seed reserve breakdown was proposed by Akazawa & Miyata (1982) in which
a-amylase and other enzyme activities is produced from the scutellar epithelium
in a process which is insensitive to gibberellin. However, in lines of wheat which
show a constitutively reduced sensitivity to gibberellin as a result of the presence
of the Rht3 dwarfing gene (Ho, Nolan & Shute, 1981) the degree of inhibition
of a-amylase is such that all sources must be affected, including the scutellar
epithelium (Ho, et al. 1981; Gale & Marshall, 1973). It seems likely therefore
that, in order to accommodate the data of Gibbons and Okamoto, the hormonal
model need only be changed to include the scutellar epithelium.
However, Trewavas (1982) has described a number of inconsistencies between
observation and the predictions of the hormonal model which require a more
radical revision. He shows that production of gibberellins by embryo tissue is
inadequate to account for levels of gibberellin in the intact grain and suggests the
source of active gibberellin in the grain is a reserve of conjugated gibberellins in
the endosperm. In addition, he reviews a large body of work which shows that
the levels of free gibberellin in germinating grains do not parallel, and may even
lag behind the rates of synthesis of a-amylase. The gibberellin dose/a-amylase
response curve presents similar difficulties. Trewavas points out that in experiments with de-embryonated half grains, the range of gibberellin concentrations
(GA3) which affect a-amylase production vary over at least four orders of magnitude. In intact grains the changes in free gibberellin levels span less than a
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D. BAULCOMBE, C. LAZARUS AND R. MARTIENSSEN
factor of ten and are clearly inadequate to account for the changes in a-amylase
production. A recent paper by Atzorn & Weiler (1983) goes someway towards
a resolution of these problems. They used a highly sensitive and specific immunological assay of chromatographic fractionations to identify and quantify different
gibberellins present in various regions of germinating barley grains and produced
evidence that GA4 may be the active fraction. GA4 was the only gibberellin
which showed concentration differences bearing any relationship to the pattern
of a-amylase synthesis, and reached a maximum just before the maximum rate
of a-amylase synthesis. In de-embryonated grains, applied GA4 was active in
stimulating a-amylase synthesis, an effect which was not inhibited by the
presence of inhibitors of gibberellin synthesis. Other gibberellins only produced
stimulation of a-amylase production in the absence of the inhibitors. It was also
shown that GAi applied to de-embryonated grains stimulated a transient increase in GA4 levels. In addition a strong correlation was established between
the level of a-amylase production and the amount of extractable GA4. In the
light of these data it does not seem necessary to suggest, as does Trewavas, that
the sensitivity to gibberellin changes during final stages of aleurone cell differentiation, but rather that levels of GA4 are the limiting factor. These experiments do suggest however that the active gibberellin is produced not in the
embryos, but actually in the aleurone cells. If this is the case, then a different
diffusible factor between the embryo and site of hydrolase production must exist.
Possible candidates for this role include other gibberellins, which as described
above were shown by Atzorn and Weiler to stimulate production of GA4. Alternatively the diffusible factor may be an inactivator of abscisic acid. Abscisic acid
(ABA) is a naturally occurring plant growth regulator and antagonist of the
action of gibberellin. The involvement of ABA in aleurone cell development is
suggested by the demonstration that ABA prevents the accumulation of GA4,
and therefore of a-amylase, in germinating barley (Atzorn & Weiler, 1983). In
addition it was shown that ABA levels decline following the onset of germination.
It is proposed therefore that the original model (Yomo & Varner, 1971) is still
appropriate, but with the modification that an additional step, triggered by an
unidentified factor, occurs in the hydrolase-producing cells. This step is the
production of active gibberellin. A major challenge now is to identify the nature
of the unidentified factor and to establish how it interacts with the target cell.
GIBBERELLIN ACTION AT THE CELLULAR LEVEL
It is proposed that gibberellins might interact with target aleurone cells in
either of two ways. They may serve to stimulate, in a non-specific manner,
cellular processes which are programmed during a preceding stage of development. Alternatively, the action of gibberellin might be a programming event in
itself, acting at the level of gene expression. An example of the former process
Gibberellins and gene control
123
may be the action of gibberellins on the cotyledonary cells of castor bean seeds
(Martin & Northcote, 1982). In this tissue the response to gibberellin is the
production of enzymes involved in lipid metabolism and glyoxalate cycle.
Isocitrate lyase is the most prominent, and its increase is dependent on an increased level of translatable mRNA. However this is not a specific increase.
Within the limits of resolution offered by single-dimension electrophoresis of
translation products, it seems that all mRNA species increase in the same way.
Thus, gibberellin serves only to stimulate mRNA in a non-specific manner and
is effective only after an initial, gibberellin-independent mechanism has allowed
the amount of new mRNA species to increase to detectable levels. If such a
mechanism acts in aleurone cells, the gibberellin stimulus would act after the
inbuilt development switch had activated (for example) a-amylase gene expression, but before accumulation of a-amylase mRNA.
Unfortunately our understanding of gene control during plant development is
still rudimentary and there are no good examples of genetic reprogramming as
a result of action by gibberellin, or any other plant hormone. However a
frequently cited example which may serve as a useful analogy, is the action of
steroid hormones on gene expression in animal cells. In this example it is thought
that hormone receptor complexes bind in the DNA sequences flank which hormone responsive genes and result in transcription of those genes (Yamomoto &
Alberts, 1976), although other mechanisms of reprogramming are possible involving post-transcriptional or mRNA stability changes.
In order to resolve these alternatives of either stimulatory or deterministic
action of gibberellin, several laboratories are investigating the effect of gibberellin on gene expression in isolated aleurones. Most of this work is with
barley, although the experiments described below from this laboratory used deembryonated grains of wheat. The incubations started with dry tissue, a-amylase
production commenced at approximately 12 h and reached a maximal rate after
approximately 24 h of incubation, dependent on the presence of gibberellic acid
(GA3 10~ 5 M). This rate was maintained for up to 96h of incubation. In this
system, the effects of gibberellin on mRNA production have been classified into
three groups (Baulcombe & Buffard, 1983). One group comprises mRNAs
which are present at high level in aleurones of dry grain and which show reduced
levels of expression during the incubation in GA3. A second group are mRNA
species which, expressed as a proportion of poly(A) + mRNA, are present at
similar levels in both the presence or absence of GA3. However, because there
is an increase in the level of poly(A) + mRNA in aleurones incubated in GA3,
these mRNAs are actually present in a greater number of copies per cell. Finally,
there are several abundant mRNA species which are present at both increased
absolute and relative levels in mRNA of GA3-treated aleurones. These are
referred to as gibb-on mRNAs. For only one gibb-on mRNA, the a-amylase
mRNA, is the identity known. It is probable that the others include the mRNAs
for protease and for a-glucosidase, both of which increase as a result of de novo
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D. BAULCOMBE, C. LAZARUS AND R. MARTIENSSEN
synthesis in the aleurones of barley (Jacobson & Varner, 1967; Hardie, 1975).
The mRNA for a-amylase and for six other gibb-ons have now been cloned as
complementary DNA (cDNA) from wheat and barley (Baulcombe & Buffard,
1983; Mutukrishnan, Chandra & Maxwell, 1983; Chandler etal. 1984; Rogers &
Milliman, 1983). These cDNA clones have been used to investigate the nuclear
DNA organization and the pattern of expression of gibb-on sequences. The data,
described below, suggest that in wheat the gibb-ons are expressed from small
gene families which are all co-regulated in a precise manner.
a-Amylase gene families in wheat
The degree of multiplicity of the gibb-on sequences is the result not only of the
involvement of different genes, but also of multiple variants within gene families.
This is especially complex for a-amylase which is encoded within the wheat and
barley genomes as two related gene families (Nishikawa & Nobuhara, 1971).
These were originally identified by isoelectric focussing of isozyme variants.
Subsequently, genetic mapping has shown that the two gene families in wheat,
called a-Amyl and a-Amy2 are encoded on the group 6 and group 7
chromosomes respectively. The a-Amyl and a-Amy2 polypeptides do not show
immunological cross reactivity. The multigene nature of the a-Amyl and
a-Amy2 gene families is indicated by several lines of evidence. Isoelectric focussing of isozymes suggests as many as 11 a-Amyl and 16 a-Amy2 alleles (Gale,
Law, Chojecki & Kempton, 1983), although it is not yet clear how many of these
variants are the product of post-translational glycosylation (Rodaway, 1978) or
methylation (Motojima & Sakaguchi, 1982) events. A similar analysis of in vitro
translation products showed at least six isoforms which were precipitated by
antiserum prepared with the product of a-Amyl genes (Baulcombe, 1983). Since
there is not likely to be extensive post-translational modification in vitro this is
probably a lower estimate of the number of genes. An upper estimate of gene
number is provided by hybridization of cloned cDNA to wheat DNA which was
digested with restriction endonucleases, fractionated by gel electrophoresis and
blotted onto a nitrocellulose membrane. Fig. 1 shows the autoradiograph of such
a membrane and onto which was blotted EcoRI digested wheat DNA samples
and which was hybridized with a cDNA probe for the a-Amyl genes. The wheat
DNA samples were euploid wheat, or aneuploid lines which are nulli-somic for
each of the group-6 chromosomes and in which the deficiency is compensated by
a tetra-somic dose of a different group-6 chromosome. In this analysis, to an
approximate degree, each fragment of hybridizing DNA may be considered to
Fig. 1. Hybridization of a-Amy cDNA to wheat DNA digested with restriction
endonuclease EcoRI. DNA from Chinese spring euploid wheat (a) or from lines
which are nullisomic tetrasomic for group-6 chromosomes (N6AT6B (b), N6BT6D
(c), N6DT6B (d)) was digested with EcoRI, fractionated on by electrophoresis in an
agarose gel and blotted onto a nitrocellulose filter. Thefilterwas hybridized with an
a-Amyl cDNA probe, washed and autoradiographed as shown.
Gibbertllins and gene control
a
b
e
125
d
EMB 83S
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D. BAULCOMBE, C. LAZARUS AND R. MARTIENSSEN
represent a different gene variant, since it is known that EcoRI does not cleave
the DNA of six different a-amylase genes which have been analysed at the level
of cloned DNA (D. Baulcombe etal., unpublished). According to this analysis,
therefore, the 12 fragments which hybridize correspond to 12 variants in the
a-Amyl gene family. The aneuploid analysis confirms the location on the group6 chromosomes and the intensity of hybridization suggests that each variant is
present once per haploid wheat genome. Hybridization does not, however, allow
inactive and active genes to be distinguished, and so it is possible that the number
of active genes is less than 12. A similar result is obtained for the a-Amy2 genes
using DNA from aneuploid lines of wheat which lack group-7 chromosomes.
The origin of variation in a-amylase gene families
It has been proposed that the origin of the variation within and between the
a-amylase gene families lies in a successive multiplication of ancestral gene types
(Lazarus & Baulcombe, 1984). The evidence for this is derived from nucleotide
sequence analysis of wheat and barley cDNA clones and also cross-hybridization
analysis. Comparing two cDNA clones from wheat it was established that within
the coding sequence, the a-Amyl and a-Amy2 mRNA sequences are closely
similar with only a single region producing more than five consecutive differences
in the deduced amino acid sequence. This region is at the carboxy-terminus,
where the a-Amy2 sequence encodes an additional nine amino acids. A similar
situation exists in barley where the two classes of gene are referred to as a-Amy A
and a-AmyB. The a-Amy A genes on barley chromosome 1 are homoeologous
with the wheat a-Amy2 and the barley a-AmyB genes on chromosome 6 similarly related to the wheat a-Amyl class (Brown & Jacobson, 1983). The nucleotide
sequence of a-AmyA (Rogers & Milliman, 1983) and a-AmyB (Chandler et al.
1984) mRNA species have been reported. It is interesting to note that, within the
coding regions the a-Amyl sequence (wheat) is more similar to the a-AmyB
sequence (barley) than it is to the a-Amy2 (wheat) sequence. A parallel relationship exists for the a-Amy2 (wheat) and a-AmyA (barley) sequences. For example, the carboxy-terminus feature of the wheat a-Amy2 sequence is shared with
the barley a-AmyA sequence (Rogers & Milliman, 1983). This suggests that the
two classes of a-amylase gene evolved by duplication of an ancestral gene at
sometime before the evolutionary divergence of wheat and barley. A comparison of the 3' non-coding regions presents a similar but exaggerated picture.
Within a gene family the 3' non-coding regions are similar in all members. This
has been shown by hybridization of probes which are specific for that region to
nuclear DNA clones of a-Amyl and a-Amy2 sequences from wheat and by
experiments in which the 3' non-coding region DNA was used to select by
hybridization the complementary mRNA species. These mRNAs were translated in vitro and analysed by isoelectric focussing to demonstrate that the
isoforms produced were the same as that produced by total a-Amyl mRNA
(Lazarus, Baulcombe & Martienssen, 1984). This extreme conservation of the
Gibberellins and gene control
127
3' non-coding region within a-amylase gene families is preserved even between
species. The barley a-Amy A cDNA sequence (Rogers & Milliman, 1983) is
more than 90 % homologous with the wheat a-Amy2 sequence in this region
(Lazarus & Baulcombe, 1984). However, there is no similarity detected either
by hybridization or nucleotide sequence analysis between the 3' non-coding
regions of a-Amyl and a-Amy2 gene families (Lazarus et al. 1984; Lazarus &
Baulcombe, 1984). These results confirm the proposed origin of a-Amyl and
a-Amy2 gene families before the evolutionary divergence of wheat and barley.
In addition, the extreme conservation within the 3' non-coding region suggests
constraint on the rate of evolution of this sequence, possibly imposed by a
sequence-dependent function. A similar situation has been reported for the
family of actin genes in sea urchin where different subfamilies show different
patterns of expression and are identified by the 3' non-coding region (Davidson,
Thomas, Scheller & Britten, 1982). It has been suggested that in this case there
may be an effect of the 3' non-coding region on mRNA stability (Davidson et al.
1982).
The organization in the wheat nuclear genome of the other gibb-on sequences
may be much more straightforward. For two of these which encode polypeptide
products of relative molecular mass (gibb-on 35) 35 000 and 50 000 (gibb-on 50),
the hybridizations to wheat DNA digested with restriction enzymes BamHI or
EcoRI show only three fragments. Fig. 2 shows the data for gibb-on 50
hybridized to euploid wheat DNA, or to aneuploid wheat DNA which is
nullisomic for each of the group-6 chromosomes and illustrates that each of the
three fragments is encoded by a different group-6 chromosome. Also shown is
the hybridization to a ditelocentric line which lacked the short arms of the 6B
chromosomes. This DNA clearly does not contain the gibb-on 50 fragments
which are located on chromosome 6B and it is thus established that these
sequences are on the short arm of chromosome 6B. The gibb-on 35 sequences
have been shown in a similar analysis to be encoded on the group-4
chromosomes, and the a-amylase genes are known from isozyme analysis to be
located on the long arms of the group-6 and group-7 chromosomes (Gale et al.
1983). The common response of these genes to the gibberellin stimulus is clearly
not the result of gene clustering.
The expression of gibb-ons
Our initial approach to understanding the manner and mechanism of action of
gibberellin on gene expression in aleurones has been to measure the level of gibbon gene expression in tissues with different genetic backgrounds, or subject to
modified incubation conditions.
Within the a-amylase gene families, the degree of co-ordination is extremely
tight. Thus, the amount of isozyme variants relative to each other does not
change during development, nor in response to the presence of Rht3 genes
(Flintham, 1981). Analysis of in vitro translation products confirmed that this
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D. BAULCOMBE, C. LAZARUS AND R. MARTIENSSEN
a
b
c
d
e
Fig. 2. Hybridization of gibb-on 50 cDNA to wheat DNA digested with restriction
endonuclease BamHI. The DNA samples as described in Fig. 1 (a-d) and a
ditelocentric line which lacked the short arm of the 6B chromosomes (e) were digested with BamHI, fractionated by electrophoresis in an agarose gel and blotted onto
a nitrocellulose filter. The filter was hybridized with a gibb-on A50 cDNA probe and
autoradiographed as shown.
reflects relative constancy of mRNA variants. The translation products of
aleurone mRNA isolated at 2 or 4 days of incubation were precipitated by antia-Amyl serum and analysed by isoelectric focussing. Six a-Amyl isoforms were
detected which were present in similar amounts relative to each other in both
samples (Fig. 3). Since the aleurone tissue is producing a net accumulation of
a-Amyl mRNA at 2 days of incubation and a net decay at 4 days (Baulcombe &
Buffard, 1983), the implication of this result is that the regulation of different
a-Amyl mRNAs is co-ordinated both at the level of synthesis and at the level of
turnover.
This parallel pattern is not observed however in a comparison of a-Amyl and
a-Amy2 mRNAs. Each was measured by hybridization of the 3' non-coding
region probes, as described above, to RNA samples which were fractionated on
Gibberellins and gene control
2d
4d
129
Fig. 3. Analysis of a-Amyl mRNA variants. Poly (A) + RNA from aleurones which
were incubated for 2 d or 4 d in 10~5 M GA3 was translated in vitro in a cell-free
wheatgerm extract in the presence of [35S]methionine and the labelled products were
analysed by iso-electric focussing. T ' indicates total translation products and 'Im' the
products which were immunoprecipitated with anti-a-Amyi-serum.
agarose gels and blotted onto a nitrocellulose membrane. An autoradiograph of
the membrane (Fig. 4) following hybridization shows that the level of a-Amyl
mRNA reached a maximum level in aleurones which were incubated for 48 h in
GA3 and then declined slowly. The a-Amy2 mRNA showed a continued
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D. BAULCOMBE, C. LAZARUS AND R. MARTIENSSEN
increase in level up to 96 h of incubation. In both cases however, the onset of
GA3-induced accumulation was first detected between 12 h and 24 h of incubation. These data suggest therefore that the synthesis of these two mRNA classes
is co-ordinated, but that the a-Amyl mRNA species are less stable than the
a-Amy2 mRNAs. So, as germination proceeds and transcription slows down
before the onset of senescence in the aleurone tissue, the balance between
synthesis and degradation switches earlier for a-Amyl mRNAs. This pattern of
mRNA accumulation is reflected in the accumulation of a-Amyl and a-Amy2
enzyme levels (Flintham, 1981). Both a-Amyl and a-Amy2 mRNAs depend on
GA3 at similar concentration for expression (D. Baulcombe, unpublished). It is
likely that a similar situation exists in barley, with the difference that the switch
is reversed. The most prevalent early class are the low pi isozymes of the
a-AmyA class (homoeologous with wheat a-Amyl) (Jacobsen & Higgins, 1982).
The expression of the two classes of a-amylase mRNA has not been described
in detail in barley. However, it is clear that the a-AmyA mRNA shows quite a
a
b
e
d
e
aAMY1
aAMY2
Fig. 4. The expression of a-Amy and a-Amy2 mRNA. Total cellular RNA was
isolated from aleurones incubated for 12 h(a), 24 h(b), 48 h(c), 72 h(d), 96 h(e) in the
presence of 10~5 GA3, fractionated on by electrophoresis in agarose under denaturing conditions blotted onto a nitrocellulose filter. The filter was hybridized with
cDNA probes which are specific for either a-Amyl or a-Amy2 mRNA species,
washed and autoradiographed, as shown.
Gibberellins and gene control
131
different pattern of accumulation from the a-AmyB mRNA (Rogers &
Milliman, 1983; Chandler et al. 1984).
A similar analysis has, been carried out on gibb-on mRNAs by hybridization
of cDNA clones to RN A which had been fractionated on agarose gels and blotted
onto nitrocellulose. In wheat and barley the levels of gibb-on mRNAs were
shown to increase to a maximal level in advance of total a-amylase mRNA
(Baulcombe & Buffard, 1983; Chandler et al. 1984). However in these experiments the a-amylase hybridization probe would have produced a composite
result of the a-Amy I and a-Amy2 mRNA accumulations. In fact, the levels of
gibb-on mRNAs in wheat parallel the accumulation and decay of a-Amyll
mRNA (Lazarus et al. 1984). The same applies if the level of GA3 in the incubation medium is reduced, or if the gibberellin sensitivity of the plants is modified
by the presence or absence of the Rht3. These data all suggest therefore that the
gibb-ons considered in this analysis are all subject to the same regulatory
mechanism in aleurone cells. This may be either a common 'deterministic'
mechanism acting in trans onto unlinked genes or alternatively a non-specific,
stimulatory mechanism acting on every active gene. Several lines of evidence do
in fact support the 'non-specific stimulatory' alternative.
1) The magnitude of the response is independent of the level of gibberellin.
Gibberellin acts to accelerate rather than initiate new processes. This is
illustrated by the expression of two gibb-on mRNA species, represented by
cDNA clones which accumulate to high levels in isolated aleurones in the absence of GA3. The level of these mRNA species may even reach the same level
in the absence of GA3 as in its presence, with the difference that in the absence
of the GA3 the maximal level of accumulation is reached 48 h later (Baulcombe
& Buffard, 1983). Even a-amylase mRNA accumulation is detected at a very low
level in the absence of GA3, and is still increasing at 96 h of incubation in GA3.
Experiments using ABA at levels which only partly block the response of
aleurone cells to gibberellin serve to illustrate the same point. An analysis of the
time course of a -amylase enzyme levels shows that the isolated aleurones incubated in the presence of GA3 and ABA produce as much a-amylase as do
aleurones incubate with GA3 alone. Similarly the level of a-amylase and of
different gibb-on mRNAs all increase to the same levels or even higher levels but
48 h later in aleurones treated with GA3+ABA. This is shown in Fig. 5 which is
an autoradiograph of cDNA hybridization to poly(A)+ RNA which was fractionated by electrophoresis and blotted onto a nitrocellulose membrane. These
results which show an effect of gibberellin on the kinetics of response, contrast
with dose/response curve measurements for the action of steroid hormones on
animal cells, where the amount, rather than the timing, of the response is affected (Tomkins et al 1970).
2) Genes respond differently to gibberellin in different cell types. Thus, although
a-amylase genes are expressed at a high level in aleurone cells in response to
gibberellin, a-amylase expression is not detected in leaf cells (Baulcombe, 1983)
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D. BAULCOMBE, C. LAZARUS AND R. MARTIENSSEN
a
b
c d e
GA-
GA 3
ABA
Fig. 5 The effect of ABA on gibb-on gene expression. Polyadenylated RNA was
isolated from aleurones incubated for 6 h(a), 12 h(b), 2 h(c), 48 h(d) or 96 h(e) in the
presence of 10~5GA3 or 1 0 ~ 5 G A 3 + 1 0 ~ ^ M ABA. The RNA was fractionated by
electrophoresis in agarose under denaturing conditions and blotted onto a
nitrocellulose filter. The filter was hybridized with a cDNA probe for gibbon-50,
washed and autoradiographed as shown.
which are capable of a gibberellin response (Flintham, 1981). A subset of
a-Amy2 isozymes are detected in developing grain, probably expressed in the
pericarp (Gale & Ainsworth, 1984). However this expression is not affected by
presence of Rht3 genes and so is not controlled by gibberellin in that tissue.
Similarly, the expression of other gibb-on sequences is detected at high levels in
leaf tissue of wheat (Baulcombe, 1983) but is unaffected by Rht3 genes. It is to
be predicted that if the action of gibberellins was to directly and specifically
activate gene expression analogously to steroid hormones, that the expression of
gibb-ons would be regulated by gibberellin in every cell which is sensitive to
gibberellins. Since this does not appear to be the case, it is concluded that
gibberellins do not control the specificity of gene expression, but rather serve as
quantitative regulators of expression.
CONCLUSIONS
There are still many questions to be answered, concerning the control of
Gibberellins and gene control
133
production of a-amylases and other hydrolases in germinating cereal grains. At
the intercellular level, the diffusible signal from the embryo to the site of enzyme
production in the aleurone and scutellar epithelium is still undefined. It is probable that gibberellins are involved in this but as links in the chain of communication. Different gibberellins may act differently and form different links. For
example, in barley it seems likely that GA4 may be a late link in the chain (Atzorn
& Weiler, 1983) and that other gibberellins may actually stimulate the production of GA4.
At the intracellular level many of the responses to gibberellin, most
prominantly the production of a-amylase, may result from an effect on mRNA
accumulation. It is not yet resolved how this is achieved, but it seems likely that
gibberellins do not determine the specificity of gene expression, but rather that
they accelerate preprogrammed events. In the near future it should be possible
to test this proposal by two approaches. A detailed analysis of nuclear DNA
clones for a-amylases and other gibb-ons which have been isolated in this
laboratory should allow the identification of DNA sequence elements which are
involved in the tissue specific regulation of gene expression. It should be
possible to use these sequences as probes for other cellular components involved in the regulatory process and to determine whether the action of these
components correlates with the action of gibberellins or with other developmental stimuli.
A second line of approach may lead to a more detailed understanding of how
gibberellins interact with aleurone cells, which cellular processes are the immediate consequence of this action and how these processes relate to effects on
mRNA accumulation. Examples of this approach include attempts to characterize gibberellin receptors and to identify agents or processes which modify the
gibberellin response. In this category are experiments which have shown that
high temperature pretreatment of immature grains leads to the development of
aleurones which produce a-amylase in the absence of added gibberellin
(Nicholls, 1982; Norman, Black & Chapman, 1982). In wheat which is insensitive to gibberellin as a result of the action of Rht3, low-temperature pretreatment induces gibberellin sensitivity (Singh & Paleg, 1984). These experiments
have been interpreted in terms of homeoviscous transitions in membrane structure, pointing to a role of membranes in the early gibberellin response (Norman
et al. 1982). An interesting report of preliminary results (Bernal-Lugo, 1984)
suggests a causal relationship between polyamine production in aleurones and
production of a-amylase. It is hoped that a development of these studies on the
cellular events leading to a-amylase and gibb-on production, combined with
molecular studies on the genes will eventually lead to a complete understanding
of the action of gibberellin in aleurone cells.
134
D. BAULCOMBE, C. LAZARUS AND R. MARTIENSSEN
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