We previously described the purification and characterization of a 37,000 Mr cysteine proteinase, designated EP-A, from gibberellic acid (GA3)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M, of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pi of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and Nterminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M, as determined by gradient SDS-PAGE.Barley aleurone layers have been used as a model system to analyze the hormonal regulation of endosperm mobilization to support early seedling growth. Gibberellic acid (GA3) induces the synthesis and/or secretion of several hydrolytic enzymes from isolated aleurone layers including a-amylase isoenzymes (14), nuclease (7), f3-1,3;1,4-glucanase (29), and proteases (10). GA3 increases the steady-state mRNA levels of f3-1,3;1,4-glucanase (29), a putative cysteine proteinase designated aleurain (26), and a-amylase isoenzymes (8,21,25), and at least for a-amylase this is mainly due to an increase in the rate of a-amylase gene transcription (13). In most cases, ABA antagonizes most of the effects of GA3 on the level of steady-state mRNA, protein synthesis, and secretion (12). ' Supported by National Science Foundation grant DCB-8702299. In contrast to a-amylase, 3-amylase, which is also necessary for starch degradation, is present in the endosperm of dry seeds primarily in a protein-bound form (28). The hydrolysis of bound ,B-amylase into a more active, soluble form by endosperm extracts from germinating grains is inhibited by cysteine proteinase inhibitors. In this case, GA3 may regulate 13-amylase activity by inducing the synthesis and secretion of cysteine proteinases, which can account for up to 95% of proteinase activity in the starchy endosperm (28). The GA3-induced cysteine proteinases are probably also responsible for the extensive proteolysis of seed storage proteins, hordeins, into small peptides (24). These can then be utilized by an active peptide transport system in the scutellar epithelium (1 1), or alternatively hydrolyzed by carboxypeptidase whose se...

Barley aleurone layers synthesize and secrete severa1 proteases in response to gibberellic acid (GA,). Two major cysteine proteinases designated EP-A (37,000 M,) and EP-B (30,000 M,) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-1031. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA,-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEPl and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.

Using in series ammonium sulfate precipitation, gel ifitration, and DEAE anion exchange high performance liquid chromatography, we have purified to homogeneity a protease of M, 37,000 secreted from barley (Hor-deum 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 (GA3)-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 halfseeds between 72 to 96 hours of incubation in the presence of GA3 as detected on activity gels containing hemoglobin as the substrate. Analysis of digestion products by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel riltration 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°C. Comparison of cyanogen bromide generated peptide fragments and NH2-terminal sequence analyses of the three individual EP-A isozymes demonstrates that they are very similar to each other. The NH2-terminal sequence shows extensive sequence homology to the NH2-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 GA3-induced barley cDNA clone (JC Rogers, D Dean, GR Heck 1985 Proc Natl Acad Sci USA 82:6512-6516).In germinating barley, gibberellins produced by the embryo diffuse to the aleurone layer where they induce the synthesis and secretion of several hydrolytic enzymes including a-amylase (7), nuclease (6), (31,3-1,4 glucanase (28), and protease (13). Proteases, particularly those secreted into the endosperm, are necessary for the mobilization of seed storage proteins during seedling growth (24), and they may also be involved in the activation of latent, protein-bound enzymes such as, 8-amylase (14). Many types of proteases have been characterized in germinating barley, some of which have been purified. These include five carboxypeptidases, four neutral aminopeptidases (naphthylamidases), and three alkaline amino-or di-peptidases (for a review, see Mikola and Mikola [18]). Of these exo-or di-peptidase activities, only carboxypeptidase activity is secreted from aleurone layers and/or scutella into the starchy endosperm (17). Sundblom and Mikola (29) demonstrated that at least four different endoproteases are present in and secreted by GA3-treated aleurone layers. Based on assays with gelatin as a substrate, a major enzyme with a pH optimum of 3.9 and two others with higher pH optima ISupported by National Science Foundation Grant DCB-8316319.are active in the presence of 8IME.2 The fourth enzyme was metal activated with a pH optimum of 7.0. Others have also described acid cysteine proteinases and metal-activated proteina...

Abscission, organ separation, is accompanied by cell wall breakdown in separation layer cells. In tomato (Lycopersicon esculentum), ethylene-induced abscission is correlated with an increase in polygalacturonase (PG) and endo-beta-1,4-D-glucanase (cellulase) activity. We have identified a putative, abscission-specific cDNA clone for PG, pTAPG1. The TAPG1 cDNA has 43% identity at the amino acid level with the tomato fruit PG. Genomic blot analysis suggests that the gene for TAPG1 is a member of a small subfamily of PG genes that is distinct from the tomato fruit PG. The TAPG1 cDNA hybridizes to mRNA expressed during the course of ethylene-induced leaf and flower abscission. A high level of PG transcript accumulation coincides with the occurrence of abscission. Auxin, an abscission inhibitor, and silver thiosulfate, an ethylene action inhibitor, suppressed accumulation of mRNA in leaf abscission zones complementary to the TAPG1 cDNA. Expression of TAPG1 transcripts is several-fold higher in flower abscission zones than in leaf abscission zones. The identification of cDNAs that encode abscission-specific PG provide and additional tool to study the regulation of abscission and cell wall dissolution in separation layer cells.

Barley aleurone layers synthesize and secrete severa1 proteases in response to gibberellic acid (GA,). Two major cysteine proteinases designated EP-A (37,000 M,) and EP-B (30,000 M,) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-1031. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA,-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEPl and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.