Embryoless Half-seeds Research Articles

Effect of gibberellic acid on α-amylase and ribonuclease activities in rye endosperm after various periods of grain storage

α-Amylase and ribonuclease activities were determined in embryoless rye half-seeds exhibiting different viaibility after various times of storage (2, 5 and 8 years). The half-seeds were incubated for 24, 48, 72 and 96 h with and without GA<sub>3</sub>. α-Amylase activity in the endosperm of rye grain stored for 2 and 5 years and incubated without the hormone increased considerably, whereas in 8-year grain, practically incapable of germination, it remained almost unchanged. GA3caused a considerable increase of α-amylase activity in all the tested samples. In grain stored for 2 and 5 years the enzyme activity reached its peak under the influence of the hormone earlier than in the control samples. The differences in ribonuclease activity between the particular samples were less pronounced, and the reaction to the hormone much weaker. The results indicate that the reaction of rye endosperm to GA3depends on the physiological properties of the grain.

Phosphorylation of d-allose by hexokinase involved in regulation of OsABF1 expression for growth inhibition in Oryza sativa L.

We previously reported that a rare sugar D-allose, which is the D-glucose epimer at C3, inhibits the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half seeds in rice (Fukumoto et al. 2011). D-Allose suppresses expressions of gibberellin-responsive genes downstream of SLR1 protein in the gibberellin-signaling through hexokinase (HXK)-dependent pathway. In this study, we discovered that D-allose induced expression of ABA-related genes including OsNCED1-3 and OsABA8ox1-3 in rice. Interestingly, D-allose also up-regulated expression of OsABF1, encoding a conserved bZIP transcription factor in ABA signaling, in rice. The D-allose-induced expression of OsABF1 was diminished by a hexokinase inhibitor, D-mannoheptulose (MNH). Consistently, D-allose also inhibited Arabidopsis growth, but failed to trigger growth retardation in the glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1. D-Allose activated AtABI5 expression in transgenic gin2 over-expressing wild-type AtHXK1 but not in gin2 over-expressing the catalytic mutant AtHXK1(S177A), indicating that the D-allose phosphorylation by HXK to D-allose 6-phosphate (A6P) is the first step for the up-regulation of AtABI5 gene expression as well as D-allose-induced growth inhibition. Moreover, overexpression of OsABF1 showed increased sensitivity to D-allose in rice. These findings indicated that the phosphorylation of D-allose at C6 by hexokinase is essential and OsABF1 is involved in the signal transduction for D-allose-induced growth inhibition.

Gibberellin homeostasis and plant height control by EUI and a role for gibberellin in root gravity responses in rice

The rice Eui (ELONGATED UPPERMOST INTERNODE) gene encodes a cytochrome P450 monooxygenase that deactivates bioactive gibberellins (GAs). In this study, we investigated controlled expression of the Eui gene and its role in plant development. We found that Eui was differentially induced by exogenous GAs and that the Eui promoter had the highest activity in the vascular bundles. The eui mutant was defective in starch granule development in root caps and Eui overexpression enhanced starch granule generation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Experiments using embryoless half-seeds revealed that RAmy1A and GAmyb were highly upregulated in eui aleurone cells in the absence of exogenous GA. In addition, the GA biosynthesis genes GA3ox1 and GA20ox2 were downregulated and GA2ox1 was upregulated in eui seedlings. These results indicate that EUI is involved in GA homeostasis, not only in the internodes at the heading stage, but also in the seedling stage, roots and seeds. Disturbing GA homeostasis affected the expression of the GA signaling genes GID1 (GIBBERELLIN INSENSITIVE DWARF 1), GID2 and SLR1. Transgenic RNA interference of the Eui gene effectively increased plant height and improved heading performance. By contrast, the ectopic expression of Eui under the promoters of the rice GA biosynthesis genes GA3ox2 and GA20ox2 significantly reduced plant height. These results demonstrate that a slight increase in Eui expression could dramatically change rice morphology, indicating the practical application of the Eui gene in rice molecular breeding for a high yield potential.

Logo Genetic purity testing of two-line hybrid rice seeds by ultrathin-layer isoelectric focusing of proteins

To test for inbreds among F1 seeds of two-line hybrid rice (Oryza sativa L.) in the laboratory, ultrathin-layer isoelectric focusing (UTLIEF) of seed proteins was used. One female marker band in the photo-thermo-sensitive genic male sterile rice (cv. Peiai 64S) and one common male marker band in the four restorer lines (cv. G67, Peifu, I9-1 and Minkezhan) were found in the pH range of 5-8. Embryoless half-seeds contained the same male marker proteins as whole seeds and could be used for UTLIEF to test inbreds in the laboratory. The corresponding embryo-containing half-seeds could germinate to form seedlings on vermiculite and seedlings could be transplanted into the field for grow-out tests. The genetic purity determined by UTLIEF of proteins from embryoless half-seeds was confirmed by grow-out tests of the corresponding embryo-containing half-seeds. This work shows that UTLIEF of seed proteins could replace grow-out tests to determine F1 genetic purity of two-line hybrid rice. Using half-seeds, the comparison between laboratory and field tests was more objective and accurate than using whole seeds.

Biochemical Responses of Rice Cells to Mastoparan 7, an Activator of Heterotrimeric G Proteins.

In order to estimate whether mastoparan 7 specifically activates plant heterotrimeric G proteins, comparisons were made between the responses of a rice normal cultivar and a rice dwarf mutant, Daikoku d1, which has a mutation in the gene of the α subunit of a heterotrimeric G protein, to mastoparan 7. First, we found that mastoparan 7 induced the activity of the α-amylase via the heterotrimeric G protein in rice embryo-less half seeds. This observation indicated that mastoparan 7 activated the G proteins in plant cells, as does it in mammalian cells. Second, mastoparan 7 seemed to cause damages in rice cells in a G protein-independent manner under some experimental conditions. We propose that mastoparan 7 should carefully be used for study on the plant G protein-mediated signaling pathways.

Characterization of β-amylase and its deficiency in various rice cultivars

β-Amylase deficiency in various cultivars of rice was examined at the molecular level. Using an antibody against β-amylase purified from germinating seeds of rice, we were able to demonstrate the expression and organization of the β-amylase gene in normal and deficient cultivars. Although β-amylase is a starch-hydrolyzing enzyme, as is α-amylase, the β-amylase protein/gene is expressed differently from the α-amylase protein/gene; i.e. (1) β-amylase is synthesized only in aleurone cells, (2) the enzyme production in the embryo-less half-seeds is not under hormonal control. We identified some cultivars of rice that are deficient for β-amylase activity. We present new evidence that synthesis is blocked at the level of mRNA synthesis in the deficient cultivars. The usefulness of β-amylase as a crop trait is also discussed.

BEN1 and ZEN1 cDNAs encoding S1-type DNases that are associated with programmed cell death in plants

We describe the isolation and characterization of cDNAs encoding two DNases that may be involved in the programmed death of plant cells: a 35-kDa nuclease of barley, which had been reported to be secreted from the aleurone layer into the endosperm during germination and may be responsible for the digestion of nuclear DNA in the course of degradation (cell death) of endosperm, and a 43-kDa nuclease of zinnia, which appears transiently in association with differentiation to tracheary elements and is likely to participate in the autolysis at the final step of the differentiation. Genes for these nucleases of barley and zinnia were designated BEN1 and ZEN1, respectively. The amino acid sequence of BEN1 protein deduced from the nucleotide sequence of BEN1 cDNA consisted of 288 residues with a putative signal sequence of 23 residues. RNA gel blot analysis revealed that BEN1 mRNA increased in the embryo-less half seeds of barley in response to the application of gibberellic acid. The deduced amino acid sequence of ZEN1 protein consisted of 303 residues with a putative signal sequence of 25 residues. Temporal accumulation of ZEN1 mRNA was detected during transdifferentiation of zinnia mesophyll cells into tracheary elements. Significant similarities were found among the amino acid sequences of BEN1, ZEN1, nuclease S1 from Aspergillus oryzae, and two other S1-type nucleases.

Effects of (+)-8',8',8'-trifluoroabscisic acid on alpha-amylase expression and sugar accumulation in rice cells.

The effects of (+)-8',8',8'-trifluoroabscisic acid (trifluoro-ABA) on alpha-amylase expression were studied in rice embryoless half-seeds, scutella, and suspension-cultured cells derived from the embryo, and the effects of the analog on sugar accumulation were also studied in scutella and suspension-cultured cells. Treatment with (+)-trifluoro-ABA strongly inhibited the gibberellic acid-inducible expression of alpha-amylase I-1 encoded by RAmy1A in the aleurone layers of embryoless half-seeds at the levels of transcription, protein synthesis, and enzyme activity. It was also found that (+)-trifluoro-ABA stimulated (i) the uptake of glucose from the incubation medium and (ii) the synthesis of sucrose in scutellar tissues and suspension-cultured cells of rice. The biological activity of (+)-trifluoro-ABA was found to be more potent and persistent than that of natural ABA. We further examined the effects of trifluoro-ABA on the expression of alpha-amylase I-1 in scutellar tissues and suspension-cultured cells. It was found that (+)-trifluoro-ABA did not inhibit the formation of alpha-amylase I-1 in the absence of external glucose. However, glucose and (+)-trifluoro-ABA cooperatively suppressed the formation of alpha-amylase I-1. Judging from these results, we conclude that the regulatory mechanism for the expression of alpha-amylase I-1 in the scutellar epithelium is distinguishable from that operating in the aleurone layer.

Developmental and Hormonal Regulation of Rice [alpha]-Amylase(RAmy1A)-gusA Fusion Genes in Transgenic Rice Seeds.

Transgenic seeds of rice (Oryza sativa L.) were used to investigate temporal, spatial, and hormonal regulation of a rice [alpha]-amylase gene, RAmy1A. Two overlapping segments of the RAmy1A promoter were fused to the coding region of the bacterial reporter gene, gusA. The resulting promoter-gusA fusions, pE4/GUS (-232 to +31) and pH4/GUS (-748 to +31), were used separately to transform rice protoplasts. [beta]-Glucuronidase (GUS) activity was detected in germinated transgenic seeds, although the two constructs showed no significant difference in timing or location of GUS expression. Both constructs first expressed GUS in the scutellar epithelium and then in the aleurone layer. Aleurone expression of GUS activity was strongly induced when embryoless half-seeds were treated with gibberellic acid. GUS expression in the aleurone layer was also suppressed by abscisic acid. These results indicate that the 5[prime] regulatory region from -232 to +31 is sufficient for temporal, spatial, and hormonal regulation of RAmy1A gene expression.

Slender barley, an extension growth mutant

Abstract Slender barley arises from a mutation in a single nuclear-gene and has pleiotropic effects on growth and development. Plants homozygous for the mutant allele have a greatly increased leaf extension rate, resulting in long and attenuated leaves. The difference in growth rate is accentuated at low temperature. Individual epidermal cells are both longer and narrower in slender, so the whole-plant phenotype is mirrored at the cell level. Whilst the slender phenotype can be mimicked in normal plants by the application of gibberellin, endogenous levels are low. In addition, unlike the normal plants, embryo-less half-seeds of slender show GA-independent secretion of α-amylase. Unlike mature leaf tissue of normal and slender plants which have similar cell wall rheology, young slender leaves are much more plastic than the corresponding normal. Differential screening of cDNA libraries has led to the identification of at least five genes with altered patterns of expression in slender. Described in detail are expression patterns for two of these; pcD1311 encoding a shoot peroxidase, and WPRP1 encoding a prolinerich protein thought to have a structural role in the cell wall. Gene expression at low temperatures suggests that slender is abnormally insensitive to temperature reduction at the molecular level. This is consistent with leaf extension continuing at lower than normal temperatures in slender compared with normal.

Aleurones from a Barley with Low [alpha]-Amylase Activity Become Highly Responsive to Gibberellin When Detached from the Starchy Endosperm.

The physiological and molecular bases for contrasting [alpha]-amylase phenotypes were examined in germinating seeds of two barley (Hordeum vulgare L.) cultivars, Morex and Steptoe. Morex is a high-quality malting barley that develops high [alpha]-amylase activity soon after germination. Steptoe is a feed barley that develops only low [alpha]-amylase activity levels during this period. The expression of all high- and low-isoelectric point (pl) [alpha]-amylase isozymes is reduced in Steptoe. The amount of [alpha]-amylase mRNA per gram of seedling tissue is correspondingly lower in Steptoe. Southern blot analysis revealed that the cultivars have the same copy number and organization for most high- and low-pl genes. Steptoe seedlings or embryoless half-seeds produce little [alpha]-amylase in response to exogenous applications of gibberellic acid (GA3) compared with Morex. However, when isolated aleurones of both cultivars are treated with GA3, they produce similar amounts of high- and low-pl [alpha]-amylase RNAs. This suggests that a factor in the starchy endosperm is responsible for lowered [alpha]-amylase response in Steptoe. The factor is probably not abscisic acid (ABA), since the two cultivars have similar concentrations of ABA during germination.

Some Physiological Effects and The Possible Mechanism of Action of Juglone in Plants.

The effects of juglone, an allelochemical, on cell elongation, respiration, the content of protein and α-amylase activity induced by gibberellin in aleurone cells of barley seeds, and its interactions with SH groups in amino acid, peptide, protein, and plant extracts were studied. Juglone inhibited the cell elongation in epicotyls of etiolated bean seedlings by 50 and 80%, and inhibited the elongation of coleoptiles of etiolated barley seedlings by 75 and 96% at 10-4M and 10-3M, respectively. The inhibition of the elongation of root cells of bean and barley were stronger. 10-3 M of juglone solution decreased the respiration (O2 uptake) of roots of bean and lettuce to a significant degree 10 and 30min after the beginning of treatment and reduced the O2 uptake to 0 after 2 and 6hrs treatment, respectively. It de creased the content of total soluble protein and α-amylase activity induced by gibberellin by 74% and 78%, respectively, in the aleurone cells of barley embryoless half seeds at 10-3M. Juglone at 10-3M could completely react with the SH groups in cysteine, glutathione, bovine serum albumin and decreased the contents of SH groups in crude extracts from germinated barley seeds and epicotyls of etiolated seedlings of bean by 60 and 55%, respectively. These results suggest that juglone may be a metabolic inhibitor which prevents many (if not all) physiological and biochemical processes involving in the function of SH groups by interfering with the SH-group bearing substances including amino acids, peptides and enzymes.

The α-amylase genes in Oryza sativa: Characterization of cDNA clones and mRNA expression during seed germination

Two cDNA clones, pOS103 and pOS137, were isolated which code for distinct alpha-amylase isozymes in germinating rice seeds. Sequence analysis indicated that the clones encode polypeptides of approximately 48 kDa, both of which possess a signal peptide involved in directing secretion of the protein. Comparison of the two rice alpha-amylase amino acid sequence showed that they are 76% similar to each other, while showing 85% to 90% similarity with other cereal alpha-amylases. A comparison of eleven cereal alpha-amylases also revealed three new conserved regions (I', II', and IV') not previously identified in the animal, bacterial, and fungal alpha-amylases. Regions I' and IV' are sites for intron splicing while region II' is probably involved in calcium binding. One of the rice alpha-amylase cDNAs, pOS103, encodes a protein that has two potential N-glycosylation sites, one in the signal peptide and the other in the mature portion of the protein. The cDNA clone, pOS137, encodes an alpha-amylase with a single glycosylation site in the signal peptide, suggesting that the mature OS137 isozyme is not glycosylated. Analysis of the expression of these genes in germinating rice seeds indicated that mRNA corresponding to pOS103 and pOS137 could be detected throughout a 48 h period of seed imbibition. RNA levels, however, were dramatically stimulated by treatment of embryoless half-seeds with exogenous GA3. Our results demonstrate that at least two forms of alpha-amylase are expressed in germinating rice seeds and that the expression of these genes is regulated by the phytohormone GA3.

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA3 and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied.α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA3 solution or 100 μM GA3+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA3+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA3+10 μM ABA.

Regulation of acid phosphatase by gibberellic acid in embryo-less half-seeds of wheat

Approximately 4–5-fold stimulation of acid phosphatase activity was witnessed in embryo-less half-seeds of wheat in response to gibberellic acid (GA 3) (48 h). This enzyme activity was secreted into the incubation medium. The increase in enzyme activity in GA 3-treated wheat half-seeds was inhibited significantly by cordycepin, cycloheximide (CHI) and amino acid analogues. This indicated the requirement of fresh transcription and translation for the GA 3-regulated acid phosphatase activity. Abscisic acid completely abolished the GA 3-induced acid phosphatase activity. Conclusive proof for de novo synthesis of acid phosphatase in GA 3-treated half-seeds was achieved by the in vivo labelling of enzyme with [ 35S]sulphate. The label was recovered in the cysteine residues of the purified enzyme. Acid phosphatase was purified (109-fold) to electrophoretic homogeneity by Con A-agarose affinity chromatography. The molecular weight of the native enzyme is 185 000 as determined by molecular sieve chromatography on Sepharose CL-6B. The K m of the enzyme for p-nitrophenyl phosphate is 5 × 10 −4 M. The pH optimum of the purified enzyme was 5.0. The enzyme activity was completely inhibited by NaF (2 mM). Both β-mercapto-ethanol (10 mM) and dithiothreitol (10 mM) brought about a 2.5–3-fold activation of acid phosphatase, thereby indicating the requirement of free sulfhydryl groups for the catalytic efficiency of the enzyme.

Purification and Characterization of Gibberellic Acid-Induced Cysteine Endoproteases in Barley Aleurone Layers

Using in series ammonium sulfate precipitation, gel filtration, and DEAE anion exchange high performance liquid chromatography, we have purified to homogeneity a protease of M(r) 37,000 secreted from barley (Hordeum vulgare L. cv Himalaya) embryoless half-seeds. This protease exists in three isozymic forms whose synthesis and secretion from barley aleurone layers was shown to be a gibberellic acid (GA(3))-dependent process (R Hammerton, T-HD Ho 1986 Plant Physiol 80: 692-697). This protease constitutes a major portion of the protease activity secreted from half-seeds between 72 to 96 hours of incubation in the presence of GA(3) as detected on activity gels containing hemoglobin as the substrate. Analysis of digestion products by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that this protease is an endoprotease, therefore it is designated as barley endoprotease-A (EP-A). Inhibitor studies demonstrated that EP-A belongs to the cysteine class of endoproteases. The optimum pH for EP-A activity was 5.0, and the temperature optimum was 45 degrees C. Comparison of cyanogen bromide generated peptide fragments and NH(2)-terminal sequence analyses of the three individual EP-A isozymes demonstrates that they are very similar to each other. The NH(2)-terminal sequence shows extensive sequence homology to the NH(2)-terminal sequence of papain and several other cysteine proteinases. We also provide evidence that EP-A is not ;aleurain,' a putative cysteine proteinase encoded by a GA(3)-induced barley cDNA clone (JC Rogers, D Dean, GR Heck 1985 Proc Natl Acad Sci USA 82:6512-6516).

Differential rates of α-amylase and acid phosphatase induction by Ga 3 in embryoless half-seeds and isolated aleurones of wheat

The induction of α-amylase and acid phosphatase by gibberellic acid (GA 3) was significantly higher (2–4-fold) in embryoless half-seeds of wheat than that observed in the excised aleurones. Addition of endosperm extract to excised aleurones enhanced the stimulatory effect of GA 3 on amylase activity by approximately 2-fold. Substitution of endosperm extract by 19 amino acids in GA 3-treated aleurones also brought about a 2–2.5-fold stimulation of α-amylase activity. Subsequent studies revealed that the addition of seven non-polar amino acids (0.5 mM each) was sufficient for the enhanced induction of α-amylase (1.8–2.5-fold) in GA 3-treated aleurones. A similatory effect of endosperm extract and amino acids on acid phosphatase activity was observed in GA 3-treated wheat aleurones. These observations are of physiological significance since an increased pool of free amino acids (5-fold) was also witnessed in the incubation medium of GA 3-treated half-seeds in comparison to the hormone-treated aleurones. The relative abundance of free amino acids in half-seed seems vital for the maximal induction of α-amylase and acid phosphatase. Thus, the presence of endosperm tissue associated with the aleurone layers is crucial for enhanced rate of production of GA 3-induced α-amylase and acid phosphatase in the wheat system.

Inorganic phosphate mimics the specific action of gibberellic acid in regulating the activity of monophenolase in embryo-less half-seeds of wheat

The GA 3-mediated activation of monophenolase (about 12-fold stimulation) in embryo-less half-seeds of wheat was mimicked by the addition of inorganic phosphate (Pi, 50 mM). Similarly, the hormone-induced altered molecular properties of monophenolase, such as the shift in the pH optimum towards alkalinity (pH 9.0), the relatively increased thermostability of the enzyme at 55° and the changed pattern of multiple forms of the enzyme, were also seen in Pi-treated half-seeds. Furthermore, the simultaneous addition of GA 3 and Pi to wheat half-seeds showed no cumulative effect on the enhancement of monophenolase activity. This indicated that both GA 3 and Pi regulated monophenolase activity through a common mechanism. Abscisic acid effectively blocked the GA 3- and Pi-directed stimulation of monophenolase. Since GA 3 treatment of half-seeds increased the free pool of Pi (2.5-fold), we envisage that the Pi-mediated activation of monophenolase is of physiological relevance in our system. However, the in vitro addition of Pi (50 mM) to water-imbibed control half-seeds (48 hr) during enzyme extraction failed to activate monophenolase. We thus consider that the high pool of Pi, generated by GA 3 in half-seeds, or even the direct addition of Pi to half-seeds in vivo, seems necessary for some metabolic events which eventually trigger the activation of monophenolase.

Hormonal regulation of α-amylase inhibitor synthesis in germinating barley

The in vivo synthesis of α-amylase and of a bifunctional inhibitor of endogenous α-amylase and subtilisin was studied in embryoless barley half grains treated with abscisic (ABA) and gibberellic (GA3) acids. Fluorography of newly synthesized proteins and of immunoprecipitated α-amylase and its inhibitor separated by SDS-PAGE showed that GA3 induces the production of large amounts of α-amylase while the synthesis of other proteins, including the α-amylase inhibitor, is reduced. In contrast, synthesis and/or accumulation of the inhibitor is induced in embryoless half seeds incubated with ABA. These results identify the α-amylase inhibitor as an ABA-induced protein in barley aleurone layers and suggest that it functions as an active mediator of amylase activity during the development and germination of barley seeds.

Multiple molecular forms of the gibberellin-induced α-amylase from the aleurone layers of barley seeds

A class of plant growth regulators, gibberellins, induce the synthesis of α-amylase (1,4-α- d-glucan glucanohydrolase, EC 3.2.1.1) in the aleurone layers of barley ( Hordeum vulgare L. var. Himalaya) seeds. The purified α-amylase is composed of multiple isozymic forms with indistinguishable molecular weights, but different net charges. These α-amylase isozymes separate on isoelectric focusing gels into two groups, each containing multiple species. One group has an apparent isoelectric point (p I) of approximately 5.8 (the high p I group). The other group's p I values are around 4.5 (the low p I group). On some gels a small amount of protein focuses between the high and low p I isozymes. These proteins comigrate with the low p I isozymes upon reelectrophoresis. The synthesis of these two groups is temporally regulated. The high p I group is the dominant set of isozymes secreted from embryoless half seeds during the first two days of gibberellin administration. After four days, however, the major isozymes are those of the low p I group. This shift in isozyme pattern is due to a shift in their relative rates of synthesis. Peptide analysis of these two groups of isozymes with Staphylococcus aureus V8 protease and cyanogen bromide shows amino acid sequence differences. However, members within the same group have similar peptide patterns. Both groups of isozymes are synthesized in vitro in a wheat germ extract primed with poly(A) + RNA isolated from gibberellin-treated aleurone layers. This indicates that the synthesis of the two groups of α-amylase isozymes is probably directed by two or more different populations of mature mRNA. A model that explains these observations and the available genetic information is that barley aleurone α-amylase isozymes are encoded by at least two sets of structural genes.