Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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 120 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 122 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 124 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 126 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 128 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 130 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) 132 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 REFERENCES T. & MIYATA, S. (1982). Biosynthesis and secretion of a-amylase and other hydrolates in germinating cereal seeds. In Essays in Biochemistry, vol 18 (eds P. N. Campbell & R. D. Marshall), pp. 40-78. London: Academic Press. ATZORN, R. & WEILER, E. W. (1983). The role of endogenous gibberellins in the formation of a-amylase by the aleurone layers of germinating barley caryopses. Planta Berl. 159, 289-299. BAULCOMBE, D. C. (1983). Wheat a-amylase genes: Cloning of a developmentally regulated gene family. In Genetic Engineering V (eds J. Setlow & A. Hollaender), pp. 93-108. BAULCOMBE, D. C.& BUFFARD, D. (1983). Gibberellic acid-regulated expression of a-amylase and six other genes in wheat aleurone layers. Planta Berl. 157, 493-501. BERNAL-LUGO, I. (1983). Relationship of polyamines, endogenous content and GA3 effect in barley aleurone layer. Plant Physiol. 73, (Supp). 83. BROWN, A. H. D. & JACOBSEN, J. V. (1982). Genetic basis and natural variation of a-amylase isozymes in barley. Genetical Res. Camb. 40, 315-324. CHANDLER, P. M., ZWAR, J. A., JACOBSEN, J. B., HIGGINS, T. J. V. & INGLIS, A. S. (1984). The effects of gibberellic acid and abscisic acid on a:-amylase mRNA levels in barley aleurone layers: Studies using an a-amylase cDNA clone. Plant Molecular Biology (in press). CHANDRA, G. R. & DUYNSTEE, E. E. (1968). Hormonal regulation of nucleic acid metabolism in aleurone cells. In: Biochemistry and Physiology of Plant Growth Substances (eds F. Wightman & G. Setterfield), pp. 793-814. Ottawa: Runge Press. DAVIDSON, E. H., THOMAS, T. L., SCHELLER, R. H. & BRITTEN, R. J. (1982). The sea urchin actin genes, and a speculation on the evolutionary significance of small gene families. In Genome Evolution (eds G. Dover & R. B. Flavell), pp. 177-192. Academic Press. FLINTHAM, J. E. (1981). The physiological role and plant breeding potential of the Tom Thumb dwarfing gene in wheat. Ph.D Thesis University of Cambridge. GALE, M. D. & AINSWORTH, C. C. (1984). The relationship between a-amylase species found in developing and germinating wheat grains. Biochem. Genet, (in press). GALE, M. D. & MARSHALL, G. A. (1973). Insensitivity to gibberellin in dwarf wheat. Annals of Botany 37, 729-735. GALE, M. D., LAW, C. N., CHOJECKI, A. J. & KEMPTON, R. A. (1983). Genetic control of a-amylase production in wheat. Theor. Appl. Genet. 64, 309-316. GIBBONS, G. C. (1979). On the localisation and transport of a-amylase during germination and early seedling growth of Hordeum vulgare. Carlsberg Res. Commun. 44, 353-366. GIBBONS, G. C. (1981). On the relative role of the scutellum and aleurone in the production of hydrolases during germination of barley. Carlsberg Res. Commun. 46, 215-225. HARDIE, D. G. (1975). Control of carbohydrate formation by gibberellic acid in barley endosperm. Phytochem. 14, 1719-1722. HIGGINS, T. J. V., ZWAR, J. A. & JACOBSEN (1976). Gibberellic acid enhances the level of translatable mRNA for a-amylase in barley aleurone cells. Nature. 260, 166-169. Ho, T.-H. D. & VARNER, J. E. (1974). Hormonal control of messenger RNA metabolism in barley aleurone layers. Proc. natn. Acad. Sci., U.S.A. 71, 4783-4786. Ho, T.-H. D., NOLAN, R. C. & SHUTE, D. E. (1981). Characterisation of a gibberellin insensitive dwarf wheat, D 6899: Evidence for a regulatory step common to many diverse responses to gibberellins. Plant Physiol. 67, 1026-1031. JACOBSEN, J. V. & HIGGINS, T. J. V. (1982). Characterisation of the a-amylases synthesised by aleurone layers of Himalaya barley in response to gibberellic acid. Plant Physiol. 70, 1647-1653. JACOBSEN, J. V. & VARNER, J. E. (1967). Gibberellic acid-induced synthesis of protease by isolated aleurone layers of barley. Plant Physiol. 42, 1596-1610. JOHNSON, K. D. & KENDE, H. (1971). Hormonal control of lecithin biosynthesis in barley aleurone cells: Regulation of the CDP-choline pathway by gibberellin. Proc. natn. Acad. Sci., U.S.A. 68, 2674-2677. AKAZAWA, Gibberellins and gene control 135 C. M. & BAULCOMBE, D. C. (1984). Nucleotide sequence analysis of a-amylase cDNA clones from wheat (in preparation). LAZARUS, C. M. BAULCOMBE, D. C. & MARTIENSSEN, R. A. (1984). The structure and evolution of a-amylase gene loci in wheat (submitted for publication). MOTOJIMA, K. & SAKAGUCHI, K. (1982). Part of the lysyl residues in wheat a-amylase is methylated as N-E-Trimethyl Lysine. Plant & Cell Physiol. 23, 709-712. MARTIN, C. & NORTH COTE, D. H. (1982). The action of exogenous gibberellic acid on protein and mRNA in germinating castor bean seeds. Planta Berl. 154, 168-173. MUTUKRISHNAN, S., CHANDRA, G. R. & MAXWELL, E. S. (1983). Hormonal control of a-amylase gene expression in barley. /. biol. Chem. 258, 2370-2375. NICHOLLS, P. B. (1982). Influence of temperature during grain growth and ripening of barley on the subsequent response to exogenous gibberellic acid. Aust. J. Plant Physiol. 9, 373-383. NISHIKAWA, K. & NOBUHARA, M. (1971). Genetic studies of a-amylase isozymes in wheat. I. Location of genes and variation in tetra and hexaploid wheat. Japan J. Genetics 46, 345-353. NORMAN, H. A., BLACK, M. & CHAPMAN, J. M. (1982). The induction to sensitivity of gibberellin in aleurone tissue of developing wheat grains. Planta Berl. 154, 578-586. RADLEY, M. (1967). Site of production of gibberellin-like substances in germinating barley embryos. Planta Berl. 75, 164-171. RODAWAY, S. J. (1978). Composition of a-amylase secreted by aleurone layers of grains of Himalaya barley. Phytochemistry 17, 385-389. ROGERS, J. C. & MILLIMAN, C. (1983). Isolation and sequence analysis of a barley a-amylase cDNA clone. /. biol. Chem. 258, 8169-8174. SINGH, S. P. & PALEG, L. G. (1984). Low temperature induction of hormone insensitivity in genotypically gibberellic acid-insensitive aleurone tissue. Plant Physiol. 74, 437-438. LAZARUS, TOMKINS, G. M., MARTIN, D. W., STEWWAGEN, R. H., BAXTER, J. D., MAMONT, P. & LEVIN- SON, B. B. (1970). Regulation of specific protein synthesis in eukaryotic cells. Cold Spring Harbor Symp. quant. Biol. XXXV, 635-640. TREWAVAS, A. J. (1982). Growth substances. The limiting factor in plant development. Physiologia Plantarum 55, 60-72. VIGIL, E. L. & RUDDAT, M. (1973). Effect of gibberellic acid and actinomycin D on the formation and distribution of rough endoplasmic reticulum in barley aleurone cells. Plant Physiol. 51, 549-558. YOMO, H. & VARNER, J. E. (1971). Hormonal control in secretory tissues. In Current Topics in Developmental Biology, (eds A. Moscona & A. Monroy), 6, 111-114. New York: Academic Press. YAMAMOTO, K. R. & ALBERTS, B. M. (1976). Steroid receptors: elements for modulation of eukaryotic transcription. Ann. Rev. Biochem. 45, 721-746.