Proteinase Inhibitors in Germinating Barley Embryos

The activities of trypsin inhibitors and Aspergillus-proteinase inhibitors have been assayed in barley embryos germinating in different conditions. The Aspergillus-proteinase inhibitor activity increased about 20-fold during 6 days at 20°C both in separated embryos germinating on nutrient agar and in the embryos of undissected grains germinating on agar with or without mineral salts. Significant inhibitor activity was also present in root tips at later stages of vegetative growth. Whether any changes occurred in the molecular weight distribution of the inhibitors present in the seedlings was studied by gel filtration on Sephadex G-75; only minor changes were observed. The trypsin inhibitor activity per seedling remained roughly constant in all cultures. In gel filtration experiments all the activity appeared to be due to the inhibitor species present in resting embryos.     

Gibberellin-like substances and indole type auxins in developing grains of normal- and high-lysine genotypes of barley

Changes in gibberellin-like activity and content of indole type auxins were investigated during grain development of the two high-lysine barley (Hordeum vulgare L.) genotypes Sv 73608 and Risø 1508, and their corresponding normal cultivars Mona and Bomi. A peak in gibberellin-like activity was found in developing grains of the normal cultivars about 18 days after anthesis, whereas the grains of the high-lysine genotypes showed a two to five times higher maximum about 3–4 days later. The auxin content of the cultivar Bomi showed a maximum between the 22nd and the 29th day after anthesis, whereas, throughout their development the grains of the mutant Risø 1508 exhibited only about ¹/10 of the maximum level of auxin found in the grains of Bomi. The normal cultivar Mona also displayed higher contents of auxin than the high-lysine genotypes Sv 73608, particularly at the later stages of grain growth, but the differences in concentration were considerable smaller than for the pair ‘Bomi’—‘Risø 1508’. It is suggested that auxins play an important role in the development of barley grains.     

Occurrence of proteolytic inhibitors in various tissues of barley

Summary The three groups of proteolytic inhibitors present in resting barley grains, namely, trypsin inhibitors, Aspergillus-proteinase inhibitors, and inhibitors of endogenous proteinases, occur in both the embryo and the two endosperm tissues. There are pronounced quantitative differences, however. The three inhibitor activities in the embryo are, respectively, 6-, 0.1-, and 6-fold of those in the endosperm.During germination at 20° all inhibitor activities disappear from the endosperms in 4–5 days. Young rootlets and coleoptiles contain inhibitors of trypsin and Aspergillus proteinase, but these disappear after 4–5 days' germination. However, the trypsin inhibitor content per seedlings remains roughly constant through the whole period. The Aspergillus-proteinase inhibitors, in contrast, exhibit a pronounced increase of activity per seedling.No inhibitor activities were detected in leaves and roots at later stages of growth.The trypsin inhibitor which we have earlier purified from resting grains occurs exclusively in the two endospermal tissues and is immunologically entirely different from the trypsin inhibitors present in embryos and young seedlings.     

Initial proteome analysis of mature barley seeds and malt

Several barley (Hordeum vulgare) cultivars are used in the production of malt for brewing. The malt quality depends on the cultivar, its growth and storage conditions, and the industrial process. To enhance studies on malt quality, we embarked on a proteome analysis approach for barley seeds and malt. The proteome analysis includes two-dimensional (2-D) gel electrophoresis, mass spectrometry, and bioinformatics for identification of selected proteins. This project initially focused on proteins in major spots in the neutral isoelectric point range (pI 4-7) including selected spots that differ between four barley cultivars. The excellent malting barley cultivar Barke was used as reference. Cultivar differences in the 2-D gel spot patterns are observed both at the seed and the malt level. In seed extracts one of the proteins causing variations has been identified as an alpha-amylase/trypsin inhibitor. In malt extracts multiple forms of the alpha-amylase isozyme 2 have been identified in varying cultivar characteristic spot patterns. The present identification of proteins in major spots from 2-D gels includes 27 different proteins from 42 spots from mature seed extract, while only three specific proteins were identified by analysing 13 different spots from the corresponding malt extract. It is suggested that post-translational processing causes the same protein to occur in different spots.     

BIOPOTENCY OF SERINE PROTEASE INHIBITORS FROM COWPEA (Vigna unguiculata) SEEDS ON DIGESTIVE PROTEASES AND THE DEVELOPMENT OF Spodoptera littoralis (BOISDUVAL)

Serine protease inhibitors (PIs) have been described in many plant species and are universal throughout the plant kingdom, where trypsin inhibitors is the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of 13 selected cultivars/accessions of cowpea. Two cowpea cultivars, Cream7 and Buff, were found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested cultivars for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE‐Sephadex A‐25 column. Cream7‐purified proteins showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) corresponding to molecular mass of 17.10 and 14.90 kDa, while the purified protein from Buff cultivar showed a single band corresponding mass of 16.50 kDa. The purified inhibitors were stable at temperature below 60°C and were active at wide range of pH from 2 to 12. The kinetic analysis revealed noncompetitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (K_i) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis, where Buff PI was more effective than Cream7 PI. It may be concluded that cowpea PI gene(s) could be potential insect control protein for future studies in developing insect‐resistant transgenic plants.     

Protease inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

Plant protease inhibitors have been implicated in defense against insect pests. Podborer and pod fly are major pests of developing seeds of pigeonpea ( Cajanus cajan L. Millsp.). Therefore, we studied the presence of protease inhibitors in seeds of pigeonpea and its wild relatives. Seed extracts were analyzed for protease inhibitor activities by caseinolytic assay, and the number of protease inhibitors determined by polyacrylamide gel electrophoresis. Besides trypsin and chymotrypsin inhibitors, seed extracts contained weak papain inhibitor(s) but no bromelain inhibitor. Treatment of seed extract with bromelain generated new active forms of trypsin inhibitors. The relative amounts of different trypsin inhibitors and the total trypsin inhibitor activity varied with different extraction media. Trypsin inhibitors were not detectable in pigeonpea leaves. The profiles of trypsin and chymotrypsin inhibitors in almost all the cultivars of pigeonpea analyzed were similar; however, those in wild relatives were quite variable.     

Studies on Trypsin Inhibitor in Bean (Phaseolus vulgaris L) Cultivars of Himalayan Region

Eight Phaseolus vulgaris L cultivars of Himalayan region were analyzed for trypsin inhibitor activity and inhibition of gut trypsin enzyme extracted from Spodoptera littoralis larvae. Trypsin unit inhibited per gram seed weight was maximum in local yellow cultivar. The trypsin inhibitor was purified to 65.9-fold with 55.6% recovery from seeds of selected cultivar. The purified protein had a molecular weight of 14,130 Daltons and was found to be a monomer by SIDS-PAGE. It was heat stable at 100°C for 10 minutes and had a pH optimum of 7.5. Hence, the purified inhibitor appears to be of Bowman-Birk type. It lost its activity on exposure to 0.2M 2-mercaptoethanol. The inhibition pattern was of non-competitive type and the Ki value was 0.8μM. The KM value of trypsin enzyme for the substrate BApMA was 2.2mM.     

Grain sink strength may be related to the poor grain filling of indica-japonica rice (Oryza sativa) hybrids

The physiological and biochemical factors contributing to poor grain filling of indica-japonica rice ( Oryza sativa L.) hybrids were studied by analyzing the role of grain sink strength in dry matter accumulation of grains of two types of rice cultivars, Yayou 2 (an indica-japonica hybrid) and Yanjing 2 (a japonica cultivar). Carbon dioxide enrichment and plant hormone application were imposed at anthesis and the number of endosperm cells, dry matter accumulation and the activities of some sugar-metabolizing enzymes of grains were measured during grain filling. In Yayou 2, strong-potential grains (SPGs) accumulated dry weight much earlier than weak-potential grains (WPGs), but this difference was not obvious for Yanjing 2. Carbon dioxide enrichment imposed after heading significantly stimulated dry matter accumulation of WPGs of Yayou 2, but had little influence on WPGs of Yanjing 2 and SPGs of both cultivars. Leaf sheath dry matter decreased steadily in both cultivars during early stages of grain filling and accumulated during the later stages. Carbon dioxide enrichment increased leaf sheath dry matter. Dry matter accumulated by grains was linearly related to the increases in endosperm cell numbers and the activities of sucrose synthase (SS) and ADP-glucose pyrophosphorylase (AGPase) in SPGs and WPGs of both cultivars. Application of either 6-benzyladenine or abscisic acid had no significant influences on both endosperm cell number and grain dry matter accumulation. These results suggest that grain sink strength, determined by both cell numbers, SS and AGPase activities in the endosperm control the dry matter accumulation of grains.     

Localization to chromosomes of structural genes for the major protease inhibitors of barley grains

Summary Wheat-barley chromosome addition lines were compared by isoelectric focusing of protein extracts to identify chromosomes carrying loci for the major immunochemically distinct protease inhibitors of barley grains. Structural genes for the following inhibitors were localized: an inhibitor of both endogenous -amylase 2 and subtilisin (ASI) on chromosome 2, two chymotrypsin/subtilisin inhibitors (CI-1 and CI-2) on chromosome 5 (long arm) and the major trypsin inhibitor (TI-1) on chromosome 3.     

A possible evolutionary relationship between plant trypsin inhibitor, alpha-amylase inhibitor, and mammalian pancreatic secretory trypsin inhibitor (Kazal)

The amino acid sequence of the carboxyl-terminal half of barley trypsin inhibitor was found to be significantly similar to the whole sequence of bovine pancreatic secretory trypsin inhibitor (Kazal). Kazal type inhibitors and related proteins are known for the extraordinary mode of divergence among animals, and the present observation extends this to a plant for the first time. The present observation together with our previous finding of sequence homology between barley trypsin inhibitor and wheat alpha-amylase inhibitor (Odani, S., Koide, T., & Ono, T. (1982) FEBS Lett. 141, 279-282) suggest an unusual evolutionary relationship between cereal enzyme inhibitors and animal proteinase inhibitors of the Kazal type.     

Antisense downregulation of the barley limit dextrinase inhibitor modulates starch granule size distribution, starch composition and amylopectin structure

The barley protein limit dextrinase inhibitor (LDI), structurally related to the alpha-amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, alpha- and beta-amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B-type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10-15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.     

Effect of soil salinity on grain filling and grain development in barley

Experiments were conducted with five barley cultivars with the aim of ascertaining the effect of salt stress on grain filling, grain development and bioohemical composition of developing grains. Reduced grain yield under salt stress was found to be due to reduced efficiency per day to fill the grains and consequent more effective days and also due to disturbed starch-sugar balance.     

Immunogold localization of chymotrypsin inhibitor-2, a lysine-rich protein,in developing barley endosperm

Chymotrypsin inhibitor-2, a lysine-rich protein in the barley endosperm, has been localized at the ultrastructural level by immunocytochemistry in developing barley endosperm cells 14 days post anthesis. The protein is deposited in the protein bodies. Two morphologically distinct types of protein bodies, small spherical and large irregularly shaped, are present. Golgi-apparatus-derived vesicles whose content is labelled by chymotrypsin inhibitor-2 antibody-gold particles are observed at the Golgi complex and around the vacuoles. These observations indicate that the transport of the protein to the site of deposition is mediated by the Golgi apparatus.Abbreviations CI chymotrypsin inhibitor - DPA days post anthesis - ER endoplasmic reticulum The authors wish to thank Dr. V.R. Franceschi (Department of Botany, Washington State University, Pullman, USA) for many helpful discussions and advice during the work, and the staff at the Electron Microscope Center at Washington State University for technical assistance.     

Barley proteinase inhibitors: A possible role in grasshopper control?

Young growing barley seedlings contain trypsin and chymotrypsin proteinase inhibitors in their leaf juices. The amount of chymotrypsin inhibitor varies greatly while trypsin inhibitor content is nearly the same in all varieties tested. Compana (CI5438) barley has much more total inhibitor than does Trebi (CI 936), Titan (CI 7055), Horn (CI 926), Hiproly (CI 3947) and Hiproly Normal (CI 4362). The distribution of inhibitors in different barley varieties correlates with the severity of grasshopper damage observed by other workers. Barley leaves could not be induced to accumulate proteinase inhibitors after excision and incubation, wounding, or absorption of crude “proteinase inhibitor inducing factor” preparations. Grasshopper damage experiments as related to proteinase inhibitor should be done in the field using yield as the correlative factor.     

Differences in Grain Ultrastructure,Phytochemical and Proteomic Profiles between the Two Contrasting Grain Cd-Accumulation Barley Genotypes

To reveal grain physio-chemical and proteomic differences between two barley genotypes, Zhenong8 and W6nk2 of high- and low- grain-Cd-accumulation, grain profiles of ultrastructure, amino acid and proteins were compared. Results showed that W6nk2 possesses significantly lower protein content, with hordein depicting the greatest genotypic difference, compared with Zhenong8, and lower amino acid contents with especially lower proportion of Glu, Tyr, Phe and Pro. Both scanning and transmission electron microscopy observation declared that the size of A-type starch molecule in W6nk2 was considerably larger than that of Zhenong8. Grains of Zhenong8 exhibited more protein-rich deposits around starch granules, with some A-type granules having surface pits. Seventeen proteins were identified in grains, using 2-DE coupled with mass spectrometry, with higher expression in Zhenong8 than that in W6nk2; including z-type serpin, serpin-Z7 and alpha-amylase/trypsin inhibitor CM, carbohydrate metabolism, protein synthesis and signal transduction related proteins. Twelve proteins were less expressed in Zhenong8 than that in W6nk2; including barley trypsin inhibitor chloroform/methanol-soluble protein (BTI-CMe2.1, BTI-CMe2.2), trypsin inhibitor, dehydroascorbate reductase (DHAR), pericentrin, dynein heavy chain and some antiviral related proteins. The data extend our understanding of mechanisms underlying Cd accumulation/tolerance and provides possible utilization of elite genetic resources in developing low-grain-Cd barley cultivars.     

Abscisic acid and jasmonic acid affect proteinase inhibitor activities in barley leaves

Proteinase inhibitor (PI) accumulation has been described as a plant defense response against insects and pathogens. The induction of PIs is known to be regulated by endogenous chemical factors including phytohormones. We studied the induction of barley chymotrypsin and trypsin inhibitory activities by aphid infestation, mechanical wounding, abscisic acid (ABA) and jasmonic acid (JA). Wounding experiments led to a minimal accumulation of PI activity (16% over controls) compared to that found in barley seedlings infested by aphids, where chymotrypsin inhibitor activity showed a two-fold increment. No systemic induction could be detected in healthy leaves of an infested or mechanically injured plant. Exogenous ABA applied on barley leaves increased the chymotrypsin inhibitory activity, while JA only increased trypsin inhibitory activity locally and systemically when applied exogenously. Our data suggest that two different mechanisms may be regulating the induction of these two types of inhibitors.     

Pyruvate orthophosphate dikinase from the immature grains of cereal grasses

Pyruvate orthophosphate dikinase has been identified in the green grains of eight cereal grasses, most of which are classified as C(3) plants. The wheat (Triticum aestivum L. cv. Lerma Rojo) grain enzyme was further investigated: activity was low in very young grains, increased to a maximum at about 25 days after anthesis, then returned to a low level as the grain matured. It appeared to be located in the aleurone layer. A procedure was developed for obtaining partially purified preparations of pyruvate orthophosphate dikinase from the ears of wheat, oat (Avena sativa L.), barley (Hordeum distichum L.), and rye (Secale cereale L.). These preparations were suitable for measuring activities in both the forward and reverse reaction directions. The affinities of these enzymes for the six substrates (pyruvate, orthophosphate, and ATP in the forward reaction; phosphoenolpyruvate, pyrophosphate, and AMP in the reverse reaction) were determined and found to be similar to the reported affinities of the enzyme from the leaves of the C(4) plant Zea mays. A possible role for pyruvate orthophosphate dikinase in cereal grains is considered briefly.     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Isolation and characterization of soybean Bowman-Birk inhibitor from different sources

A chromatographic procedure for isolation of different isoforms of Bowman--Birk soybean trypsin inhibitors was developed. The number of isoforms was shown to depend on soybean cultivar. The amount of the classical Bowman--Birk inhibitor (BBI) in different soybean cultivars, commercial flour, and processing products was analyzed. BBI reaches its highest concentration in freshly milled seeds. Storage conditions optimum for preservation of maximum inhibitory activity in soybean raw material were developed. The use of indirect enzyme immunoassay for BBI detection during its isolation from different sources was demonstrated.