Four major basic proteins of barley grain. Purification and partial characterization

Four major basic proteins termed C, K, N and Q, which are synthesized very late in grain development, have been isolated from barley ( Hordum vulgare L.) mutant Bomi 1508. Immunoelectrophoretic monitoring assured a high degree of purity after a few ion exchange and gel filtration steps. Charge microheterogeneity of two of the four antigenically distinct proteins was observed. Some physico-chemical properties were determined, including molecular mass (C ∼ 28 000; K ∼ 30 000; N ∼ 11 000; Q ∼ 60000), isoelectric point(s) (C ∼ 9.7; K ∼ 10.1–10.3; N ∼ 9.3; Q ∼ 8.9–9.1 at 25°C), and amino acid composition. In total, the four proteins represent ∼ 5% of the salt-soluble protein in grains of some cultivated barleys. The most basic protein K is rich in lysine (∼ 7.9 mol %) and may account for ∼1% of the grain lysine content in these barleys.     

Inhibitors of chymotrypsin and microbial serine proteases in barley grains

Barley grains contain two imrnunochemically distinct inhibitors of chymotrypsin and microbial serine proteases. Both inhibitors are rich in lysine (9.5 and 11.5 g Lys/g protein). Hiproly high-lysine barley contains twenty-fold higher, high-lysine mutant 1508 five-fold higher amounts of these inhibitors than normally cultivated varieties. Inhibitors were extracted from Hiproly barley, and ammonium sulfate fractionation followed by gel filtration resulted in a neariy complete separation of the two inhibitors. No inactive protein impurities could be detected in a number of isoinhibitor preparations obtained in subsequent cation exchange chrotnatography steps. One inhibitor (CI-1) was composed of at leas# 4 molecular forms with isoelecfric points in the range 4.75–5.55 and a monomer molecular size of about 9 000. Most of this inhibitor was apparently present as dimer forms in grain extracts. The other inhibitor (CI-2) included at least 7 different molecular forms with isoelectric points in the range 6.05–7.90 and different molecular sizes in the range 6 500–9 000. Both dimer and monomer forms were present in grain extracts. In contrast to previously purified protease inhibitors of plant origin, the two barley inhibitors contain no cysteine. No interactions between the two inhibitors and trypsin were observed, but the inhibitors were immediately inactivated by pepsin at pH 2.0. Monospecific antibodies towards the two inhibitors were obtained after immunization with glutaraldehyde-polymerized inhibitor.
Inhibitor CI-1 is identical with an inhibitor of microbial alkaline proteases previously purified (Mikola and Suolinna 1971. Arch. Biochem. Biophys. 144: 566–575).     

Divergence in properties of two closely related α-amylase inhibitors of barley

The only inhibitor of human salivary α-amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N-terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu-C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α-amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α-amylase inhibitor family.     

Divergence in properties of two closely related α-amylase inhibitors of barley

The only inhibitor of human salivary α-amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N-terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu-C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α-amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α-amylase inhibitor family.     

Changes in the levels of seven proteins involved in polypeptide folding and transport during endosperm development of two barley genotypes differing in storage protein localisation

The Russian barley cultivar Nevsky lacks ₃ hordein and accumulates most of its hordein in the lumen of the endoplasmic reticulum and only a minor portion in the vacuole. In wild type barley and all other temperate cereals, storage proteins are deposited in the vacuole. F1 crosses revealed that the Nevsky phenotype is recessive; but the extent of hordein accumulation in the endoplasmic reticulum in F2 endosperm lacking ₃ hordein was very much less than in the Nevsky parent. In order to study the Nevsky endosperm phenotype we have measured the levels of seven proteins and two mRNAs involved in protein folding in the ER lumen or ER to Golgi transport during endosperm development. The protein levels were unaltered in Nevsky as compared to the wild-type variety Bomi. When the levels of these seven proteins were correlated with the rate of hordein accumulation, four of these (HSP70, PDI, Sar1p and Sec18p) were consistently up-regulated with hordein synthesis. Accumulation of hordein in the endoplasmic reticulum appears to be determined by the absence of ₃ hordein, or the product of a gene closely linked to it, plus one or more other recessive genes.     

Hormonal regulation of barley nuclease: investigation using a monoclonal antibody

Abstract. A monoclonal antibody prepared against barley ( Hordeum vulgare L., cv. Himalaya) nuclease (EC 3.1.30.2) was characterized with solid-state enzyme-linked immunosorbent assays and immuno-blotting. The antibody was specific for intracellular and secreted nuclease. Hormonal regulation of the synthesis and secretion of nuclease in isolated aleurone layers was investigated by immunoprecipitation of biosynthetically-labelled nuclease using polyclonal antibodies and by immunoblot analyses using the monoclonal antibody, respectively. Gibberellic acid (GA₃) induced the de novo synthesis and secretion of nuclease in a time-and concentration-dependent manner. Nuclease was detected in aleurone layers incubated in 1 mmol m⁻³ GA₃, after 24 h. The maximum rates of nuclease synthesis and secretion occurred 36–48 h after hormone treatment. A minimum concentration of 10⁻⁶ mol m⁻³ GA₃ was required for nuclease synthesis and secretion, whereas the maximum rate of nuclease secretion occurred at concentrations of 10⁻⁵ mol m⁻³ and higher. In the presence of abscisic acid, the synthesis and secretion of nuclease from GA₃-treated aleurone layers was almost completely inhibited. Based on these findings, the authors conclude that all nuclease within and secreted from aleurone layers treated with GA₃ is the result of its de novo synthesis.     

Purification and properties of a β-glucosidase from Penicillium oxalicum autolysates

A beta-glucosidase from the medium of an autolyzed culture of Penicillium oxalicum has been purified by tannic acid precipitation, sephacryl S-200, DEAE-Biogel, CM-Biogel and Mono Q successively. The purification process produced a homogeneous band in the SDS-PAGE that correspond to a Mr of 133,500. The enzyme had a pl of 4, and the active optima were found at pH 5.5 and 55 degrees C. The enzyme hydrolyzed different substrates showing maximum affinity against p-nitrophenyl-beta-D-glucoside with a Km value of 0.37 mM. The beta-glucosidase was inhibited by Glucono-D-lactone but not by glucose in the concentration range of 1 to 10 mM. The enzyme was adsorbed by Concanavalin-A-Sepharose.     

An abscisic-acid-responsive, low temperature barley gene has homology with a maize phospholipid transfer protein

bltA is a barley gene which, as measured by steady state mRNA levels, is induced by a low positive temperature treatment of shoot meristems. The gene is also induced in shoot meristems by drought stress. We now report the response of this gene to foliar applications of abscisic acid. The striking similarity between the predicted amino acid sequence of bltA and two maize phospholipid transfer proteins indicates a biochemical function for the bltA gene product. This homology also demonstrates the hitherto unreported environmental regulation of expression of a phospholipid transfer protein which may involve abscisic acid in the signal transduction pathway.     

Expression of β-galactosidase multiple forms during barley (Hordeum vulgare) seed germination. Separation and characterization of enzyme isoforms

β-Galactosidase (EC 3.2.1.23) activity in barley ( Hordeum vulgare ) seedlings increases moderately during the first stages of germination. The level of activity in the whole seedling is the result of increasing activity of β-galactosidase in the roots and shoots and of declining enzyme activity in the grain. β-Galactosidase was purified during different developmental stages and from various parts of the barley seedling using affinity chromatography and was resolved into multiple forms by isoelectric focusing on polyacrylamide gels. The expression of the isoforms was shown to be under temporal and tissue-specific control. Four sets of isozymes were separated by DEAE-cellulose chromatography and were shown to be functionally similar. β-Galactosidase isoforms also exhibit size microheterogeneity, the more acidic entities having higher molecular masses. The differences in molecular weight are mainly restricted to the size of the small subunit. Multiplicity can not be attributed to glycosylation, since treatment of the enzyme preparation with N- or O-glycanase did not alter the isoelectric points or the molecular weights of the isoforms.     

Purification and properties of diamine oxidase from pea epicotyls

Diamine oxidase (DAO) (EC 1.4.3.6) was purified from pea epicotyls to homogeneity by the criterion of polyacrylamide gel electrophoresis (PAGE). The pu     

Effects of fungal infection and wounding on the expression of chitinases and β-1,3 glucanases in near-isogenic lines of barley

Chitinases (EC 3.2.1.14) and β -1.3 glucanases (EC 3.2.1.39) have been known to play a vital role in the defense of plants against fungal pathogens. The pattern of induction of these two enzymes subsequent to infection by powdery mildew was studied in 10 pairs of near-isogenic lines of barley ( Hordeum vulgare L.) which possess powdery mildew resistance genes. These isogenic lines have been grotiped according to their reaction to the fungus. The induction patterns varied between the resistant and the susceptible cultivars within each group and between different groups. More tsozymcs were induced in susceptible varieties of highly resistant groups and the overall levels and the number of isozymes of chitinases and β -1.3 glucanases were lower in groups with low resistance. The effect of powdery mildew infection and mechanical wounding on the cellular localization of chitinases and β -1.3 glucanases in barley leaves has also been studied. The 31 kDa leaf chitinase, L-CH2, and trace amounts of a 25 kDa chitinase. L-CH3. were present in healthy leaves. Wounding increased the levels of L-CH3 within I ft h. Powdery mildew infection increased the levels of L-CH3 both in intercellular fluid and in intracellular extract of leaves. A /3-I.3 glucanase. GH, also increased after infection and wounding. In infected barley leaves, GL-1 was present both in intercellular space and intracellular extract. It is concluded that powdery mildew resistance genes exhibit qualitative and quantitative differences in the expression of chitinases and β -1.3 glucanases. Further, chitinases and β -1.3 glucanases appear to be a response to active infection rather than the factors responsible for disease resistance.     

Some physico-chemical properties of a major cationic peroxidase from cultured peanut cells

A major cationic peroxidase had been isolated by CMC chromatography from protein isolate of suspension medium that had supported growth of cultured peanut cells. This major cationic peroxidase proved to be antigenically different from both the anionic and the minor cationic peroxidase. Affinity for Concanavalin A found earler for the anionic peroxidase could not be detected for the major cationic peroxidase. The carbohydrate content of the major cationic peroxidase is nearly 15%. The molecular mass of the overall molecule is close to 40,000. Amino acid analysis of the hydrolysate of this major peroxidase showed similarities to amino acids of the hydrolysates of the cationic horseradish peroxidases, but no immunological relatedness could be detected between the major peanut peroxidase and the horseradish peroxidase.     

Verification of yield QTL through realized molecular marker- assisted selection responses in a barley cross

Verification of putative quantitative trait loci (QTL) is an essential step towards implementing the use of marker-assisted selection (MAS) in cultivar improvement. In a previous study with 150 doubled haploid lines derived from the 6-row cross Steptoe/Morex (S/M), four regions (QTL1–4) of the barley genome were associated with differential genotypic expression for grain yield across environments. The objectives of this study were to verify the value of these four QTL for selection and to compare the efficiency of alternative MAS strategies using these QTL vs. conventional phenotypic selection for grain yield. A total of 92 DHLs derived from the S/M cross that were not used in the original mapping efforts were used for QTL verification. Confirmation of QTL effects was first accomplished by assessing yield differences between individuals carrying alternative alleles at each putative locus in three environments. QTL1 on chromosome 3 was confirmed as the most important and consistent locus to determine yield across sites, with the S allele being favorable. The M allele at QTL3 on chromosome 6 was beneficial for grain yield across sites, but to a lesser degree than QTL1. Magnitudes of allele effects at QTL2 (chromosome 2) and QTL4 (chromosome 7) were highly influenced by the environment where the genotypes were grown. Verification of QTL effects was best achieved by comparing realized selection response. Genotypic (MAS) and tandem genotypic and phenotypic selection were at least as good as phenotypic selection. Consistent selection responses were detected for QTL1 alone and together with QTL3. Genotypic selection for lines carrying the S allele at QTL1 resulted in the identification of high-yielding genotypes. Selection responses increased when the M allele at QTL3 was combined with the S allele at QTL1. Significant qualitative QTL × environment interactions for QTL2 and QTL4 were detected through differential realized selection responses at different sites. Without a thorough understanding of the physiological and agronomic particulars of any QTL and the target environment, MAS for QTL showing qualitative interactions should be minimized This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of light on abscisic acid‐induced proline accumulation in leaves: comparison between barley and wheat

Light enhanced the abscisic acid‐induced accumulation of proline in barley ( Hordeum vulgare L. cv. Georgie) and wheat ( Triticum durum L. cv. Valnova). In wheat ABA is ineffective in the dark. In both barley and wheat, the accumulation of proline in the light showed the same characteristics as those of the process that occurs in barley in the dark, namely a synergistic interaction between the hormone and K(Na)Cl, an enhancing effect of Cl⁻ anion in excess over K⁺ cation in the incubation medium, and an inhibiting effect of D ‐mannose and monensine. In wheat, furthermore, light is needed during treatment with ABA if proline is to accumulate. Light was effective in both wheat and barley during the second or accumulation phase of the hormonal process, whereas the events occurring in the first (or lag) phase did not require light. The results suggest that in wheat light induces a putative factor(s) involved in the proline accumulation pathway that is lost in the dark, whereas in barley it is present in the dark.     

Conversion of D-tryptophan to indole-3-acetic acid in coleoptiles of a normal and a semi-dwarf barley (Hordeum vulgare) strain

Exogenously applied D-tryptophan (D-Trp) was more effective than L-Trp in inducing elongation of coleoptile segments of a normal barley ( Hordeum vulgare L. cv. Akashinriki) strain and a semi-dwarf strain with lower endogenous indole-3-acetic acid (IAA) level. D-cycloserine, an inhibitor of D-aminotransferase, completely inhibited both the D- and L-Trp-induced elongation of both strains. Addition of D-Trp increased IAA levels in both strains 4-fold over endogenous levels. The increase in IAA level was completely inhibited by D-cycloserine. The endogenous L-Trp level of semi-dwarf coleoptiles was similar to that of normal ones. These results suggested that IAA is synthesized by the conversion of L-Trp to indole-3-pyruvic acid via D-Trp in both strains, and that the lower IAA level of the semi-dwarf strain probably is a result of the impeded IAA biosynthesis involved in D-Trp.     

Control by phytochrome of the synthesis of protein related to senescence in chloroplasts of barley (Hordeum vulgare)

Protein synthesis has been measured in chloroplast isolated from detached leaves of barley ( Hordeum vulgare L. cv. Hassan). The effects of hormone and light treatments of the leaves on chloroplast protein synthesis have been compared with effects on other senescence symptoms. Interruption of the dark with red light retards senescence and increases chloroplast protein synthesis. The effect of red light was reversed by far-red light. Red light did not act additively with kinetin, and it competed with ethylene and abscisic acid, accelerators of senescence, which decreased protein synthesis. In contrast to the interruption of the dark with red light, continuous light decreased chloroplast protein synthesis. It may be concluded effects on chloroplast protein synthesis. The retardation of senescence by continuous light is not necessarily related to P_u Instead, energy provided by photosynthesis may be an important factor.