Amylase aleurone synthesis

In the germination of cereal seeds, it was long known by brewers that if the embryo was excised (or dead) the endosperm would not be hydrolyzed and sugars would not be released. In 1960, Paleg showed that amylolytic activity in the embryo-less half could be fully restored in the presence of gibberellin. In other words, the substance that passed from the embryo to the endosperm (or rather, to the living cells of the aleurone layer that encloses the dead endosperm) induces there the synthesis of a-amylase which is responsible for hydrolysis of the stored starch reserves held in the endosperm. The extent to which the a-amylase was induced became another bioassay for gibberellin. 

The antagonism of GA action by ABA can be seen in studies of steady-state mRNA accumulation in aleurone cells, a major site of a-amylase synthesis in germinating cereal seeds (Fig. 3). GA treatment promotes the accumulation of high levels of a-amylase, shown by immunoprecipi-tation, while ABA, alone or with GA, completely represses this accumulation. In contrast, ABA clearly promotes the accumulation of a-amylase inhibitor mRNA, as determined by specific immunoprecipi-tation, while GA alone slightly reduces the level of a-amylase inhibitor mRNA. However, GA, together with ABA, does not decrease the level... 

Abscisic acid is a negative regulator in that it primarily antagonizes the action of cytokinins, auxins, and in particular, gibberellins. Abscisic acid decreased the activity of polymerase in radishes (52), peas (53), maize coleoptiles (54), and pear embryos (55). More detailed studies are needed before the question of ABA-induced "modification" of RNA polymerase (54) or "alterations" in the number of sites for template activity (56) can be answered. In barley aleurone cells, ABA-induced suppression of GA-induced <-amylase formation was presumed to involve the continuous synthesis of a short-lived RNA (57). 

For a hormone to have a specific effect on gene activity, any increase in enzyme activity must result from de novo synthesis by newly formed mRNA. This increase in enzyme activity may or may not precede any general increase in metabolic activity. From the foregoing discussion on chromatin activity, it is clear that plant hormones largely either increase the activity of polymerase I or increase the synthesis of total RNA s. Claims that the hormones "activate" chromatin-bound polymerases and "modulate" the number of active sites on the chromatin (21) have not been substantiated. There are only two known examples of hormone-induced synthesis of specific mRNA s. The classic example is the barley aleurone cells, in which GA treatment induces de novo synthesis and release of K-amylase (58, 59, 60), protease (61), and possibly as many as ten proteins (62). 

A large number of studies (33) support the thesis (59) that the function of GA may be that of a derepressor of gene activity. Giberellic acid has significant effect on the synthesis of all species of RNA s (63, 64), but the formation of < —amylase does not depend on new synthesis of ribosomal and transfer RNA s. Unequivocal proof for GA-induced formation of transcripts was provided by the in vitro synthesis of peptides that are immuno-logically similar to -amylase on poly A+RNA templates that were. isolated from hormone-treated aleurone cells (65, 66, 67). Of particular significance is the finding that detectable levels of -amylase mRNA s were formed within 2 hr of treatment with GA. 

We (68) measured Jv-amylase mRNA in aleurone tissue treated with GA for 2 to 24 hr at 25°C. The level of amylase mRNA increased to a maximum value at 12 hr of incubation and decreased thereafter. The mRNA level of a tissue treated with GA for 24 hr is comparable to the level of < -amylase mRNA in tissue treated with GA for 4 hr. From previous studies (69, 70), we know that d.-amylase synthesis is maximum between 8 to 24 hr of incubation with GA. Thus, the rate of synthesis of -amylase mRNA s reaches its maximum in the first 12 hr of incubation and reflects the later rate of tk-amylase synthesis during the 12-24 hr incubation. A direct correlation of the level of mRNA with the amount of amylase produced at any given time interval (65) would imply that the template molecules either remain intact after translation... 

Control of the synthesis of amylase ntRNA s in barley aleurone cells and the synthesis of cellulase mRNAs in pea epicotyl cells are similar in some respects. The control of cellulase activity in pea epicotyl is the only known example of auxin-induced formation of specific mRNA molecules. The formation of cellulase mRNA was demonstrated by the isolation of poly A + RNA s and in vitro synthesis of cellulase (71) using the protein-synthesizing system of wheat germ (72). The formation of cellulase mRNA precedes the increase in cellulase levels by more than 12 hr. Thus, it appears that the increase in rate of synthesis of translatable cellulase mRNA s in the pea epicotyl (71) and that of -amylase mRNA s in barley aleurone cells (65,... 

Higgins, T. J. V. Zwar, J. A. Jacobsen, J. V. Hormonal control of the level of translatable mRNA for barley aleurone layers. In Nucleic Acids and Protein Synthesis in Plants, Ed. J. H. Well L. Bogorad, Paris ... 

Chrispeels, M. J. Varner, J. E. Gibberellic acid-enhanced synthesis and release of -amylase and ribonuclease by isolated barley aleurone layers. Plant Physiol., 1967, 42(3), 398-406. 

Chandra, G. R. Duynstee, E. E. Methylation of ribonucleic acids and hormone-induced amylase synthesis in the aleurone cells. Biochem. Biophys. Acta., 1971, 232, 514-523. 

Okita, T. W. DeCaleya, R. Rappaport, L. Synthesis of a possible precursor of -amylase in wheat aleurone cells. Plant Physiol., 1979, 63, 195-200. 

When isolated aleurone layers of barley are incubated in a solution containing GA, they produce and secrete several hydrolytic enzymes. GA-dependent de novo synthesis has been demonstrated for a-amylase (13), protease (14), and 6-1,3-glucanase and ribonuclease (15). In addition, a GA-dependent release of ribonuclease and 6-1,3-glucanase has been demonstrated. The increase in activity of at least one hydrolase, 6-amylase, in the presence of GA is due to release of preformed enzyme and not to de novo synthesis. 

Induction of de novo synthesis of a-amylase by GA in isolated aleurone layers is evident after a lag period of approximately 8 hr following administration of the hormone. In keeping with hormone responses generally, GA must be present continuously if the de novo synthesis of hydrolases is to be sustained. Synthesis of new RNA is essential to the GA-induction of de novo synthesis of hydrolases. Actinomycin D, an inhibitor of RNA synthesis, inhibits the synthesis and release of a-amylase if the inhibitor is presented during the first 7 to 8 hr after treatment. Inhibitors of protein synthesis, such as cycloheximide, also inhibit GA-induction of hydrolases. And, interestingly, abscisic acid, a growth-inhibiting hormone, inhibits GA-induced a-amylase synthesis as well. 

Higgins at al. (17) provided a quite direct link between GA-stimulated dtt novo enzyme synthesis and appearance of the complementary mRNA. They demonstrated convincingly that the level of translatable a-amylase mRNA increased in GA-treated tissue in parallel with the increased rate of enzyme synthesis. These results provide still more evidence that GA acts to induce selective mRNA and de novo enzyme synthesis in aleurone cells. 

The mechanism of action of abscisic acid (ABA) has been studied to the greatest extent in the barley aleurone system (29), in which ABA counteracts the effect of GA in the induction of hydrolases. This action of ABA has largely been the basis for speculating that ABA may act specifically to inhibit, by some unknown mechanism, DNA-dependent RNA synthesis. Much evidence indicates that ABA acts at the transcriptional level, but it also has been proposed that the inhibition of induction of a-amylase synthesis is caused, at least in part, by an effect on translation because ABA still inhibited the formation of a-amylase at 12 hr when cordycepin (an inhibitor of RNA synthesis) no longer had an effect (30). 

Preliminary data further suggest the involvement of phosphoinositides in G A3-induced synthesis and secretion of a-amylase in aleurone cells [13]. GA3 activation of protein kinase C-like enzyme has been shown in potato tubers [10]. The in vitro effect of 2,4-D on protein phosphorylation in a membrane fraction isolated from etiolated soybean hypocotyls may also be associated with this system [24]. Further investigation in this field may be valuable in the elucidation of molecular mechanism of phytohormone signal transduction and amplification. 

Definitive proof for the de novo synthesis of a-amylase by GA-stimulated barley aleurone tissue was obtained by Filner and Varner [36] in 1967. They used a density-labelling technique which involved incubation of aleurone layers in (plus GA). The in the water becomes introduced as a density-label in the carboxyl oxygen of amino acids formed during breakdown of protein in the aleurone cells. 

They argued that if the extracted a-amylase contained (and was consequently heavier) it must have been synthesized from the amino acids. This would be proof of de novo synthesis of the enzyme which could be separated from light ( O) enzyme on the basis of its density. In order to obtain a convenient label for the light enzyme, Filner and Varner first incubated aleurone layers in GA3, plus H-lysine. The H-labelled a-amylase-produced was collected and placed on an equilibrium density gradient (cesium chloride). An initial experiment showed a coincidence between the radioactive ( H) peak and that of assayable a-amylase activity (Fig. 7.2A). The fact that a-amylase could incorporate H-lysine was, of course, good evidence for its de novo synthesis, but the final proof came when it was shown that assayed a-amylase synthesized in GA3-treated aleurone layers incubated in H2 0 was heavier than the radioactive marker ( H) peak (Fig. 7.2B). 

Isolated barley aleurone tissue requires about 8 h of exposure to GA before secretion of a-amylase commences (Fig. 7.1 A). The obvious question which arises is what events must occur in this lag period to culminate in enzyme secretion There is good evidence that there is no large accumulation of a-amylase within aleurone cells prior to its secretion (e.g. Fig. 7.1 A) so the lag period must consist of events which are preparative for synthesis of this enzyme, one of which might involve the transcription of the appropriate mRNA. This particular event could be the rate-limiting process. 

Some workers have claimed that the number of ribosomes present in isolated barley aleurone cells increases in response to GA [35, 65] although others have shown no GA-stimulated synthesis of ribosomal RNA (or transfer RNA) [59, 109]. Since rRNA synthesis can be stopped in GA-treated aleurone layers without significant effect on either a-amylase synthesis or cellular rRNA levels [59] this can be taken as evidence that synthesis of new ribosomes is not... 

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