Saroj Parmar

The effects of sulphur starvation on the amino acid and protein compositions of barley grain

Sulphur starvation resulted in decreases in the total sulphur content, and in the proportions of the sulphur-containing amino acids, cysteine and methionine, in the grain of pot-grown plants of the barley cultivars Sundance and Athos. The S-deficient grain contained a decreased proportion of storage protein (hordein) and increased non-protein nitrogen (NPN). Comparison of the amino acid compositions of the whole grain, and of the individual protein fractions, indicated that aspartate or asparagine was a major component of the increased NPN. Electrophoretic analyses showed that the hordein fraction was depleted in S-rich ‘B’ and ‘D’ hordein polypeptides, which was consistent with its low content of sulphur-containing amino acids. The salt-soluble protein fraction also contained low levels of cysteine and methionine, and SDS-PAGE showed that some bands, notably those of low molecular weight, were either greatly reduced in amount or were absent. It is suggested that these are non-essential components, possibly storage proteins. In contrast there was little effect on the amino acid or polypeptide compositions of the glutelin fraction.

The purification and N-terminal amino acid sequence analysis of the high molecular weight gluten polypeptides of wheat

Nine high molecular weight gluten subunits have been purified from cultivars of bread wheat (Triticum aestivum). They have broadly similar amino acid compositions with 34–39 mol% Glx, 12–17 mol% Pro and 14–19 mol% Gly. Some differences in the contents of other amino acids were apparent; notably cysteine varied from 0.4 to 1.5 mol%. On the basis of these compositions, the peptide maps with V8 proteinase and N-terminal amino acid sequences the subunits could be divided into four groups. Two of these contained subunits encoded by structural loci on chromosomes 1A and 1B, respectively, while the other two probably represented the products of two subfamilies of genes at a single locus on chromosome 1D. The N-terminal amino acid sequences were homologous, but differed in substitutions of single amino acids and in deletions of three and seven amino acids. Two subunits purified from Triticum monococcum and Aegilops squarrosa had N-terminal sequences closely related or identical to those of the 1A subunits and one group of 1D subunits respectively. Two cysteine residues were present in the N-terminal sequences of all the subunits, but the distances between these were affected by the deletions and varied from 7 to 14 residues. The results are discussed in relation to the ability of the subunits to form elastic disulphide-linked aggregates, and the importance of these in the structure and functionality of gluten.

Identification of γ-type hordeins in barley

The barley mutant Risϕ 56 has a deletion of at least 85 kb of DNA at the Hor 2 locus, resulting in the absence from the seeds of almost all of the major B hordein group of storage proteins. We have purified two fractions containing the three remaining ‘B hordein‐like’ bands and determined their N‐terminal amino acid sequences. One fraction, containing the two bands with higher M r, gave a single major N‐terminal sequence which was closely related to those reported for γ‐type prolamins of wheat and rye. The sequence of the third band was also homologous with those of the γ‐type prolamins, but less closely so. We consider that the γ‐type prolamins are most closely related to the ancestral S‐rich prolamins of the Triticeae, and this is the first report of their presence in barley.

Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity.

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulansSynechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the β/α barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in Vmax but did not alter the enzymes relative specificity towards either of its gaseous substrates, CO2 and O2. However replacement of alanine 340 with glutamate decreased the enzymes specificity for CO2 but had no significant effect on either the Km for ribulose-1,5-bisphosphate or CO2 or on Vmax. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.

The presence of high molecular weight aggregates in the protein bodies of developing endosperms of wheat and other cereals

Protein bodies have been isolated from developing grain of wheat, barley and rye. The protein has been solubilised in a solvent consisting of 0·01- M acetic acid, 6- M urea and 55-m M cetyltrimethylammonium bromide and chromatographed on columns of controlled pore glass. In each case a proportion of the protein was eluted in the excluded volume of the column, indicating the presence of multimeric aggregates in the protein bodies. In all three cereals these aggregates contained an enhanced proportion of high molecular weight polypeptides and certain sulphur-rich prolamins. The proportion of aggregated material in wheat protein bodies was similar to that in gluten preparations and two-dimensional electrophoretic analyses showed that protein bodies and gluten consisted of almost identical polypeptides. We therefore suggest that the aggregates of gluten proteins, which have been implicated as important in determining bread-making quality, are laid down in protein bodies shortly after synthesis in the developing seed. Similar studies have also been carried out with maize protein bodies. Although these contained some aggregated proteins they did not appear to contain the very large complexes observed in the other three cereals.

Analysis of a rare recombination event within the multigenic Hor 2 locus of barley (Hordeum vulgare L.).

A rare recombinant within the multigenic Hor 2 locus of barley was detected by SDS-PAGE of hordein fractions from F 2 grain from the cross Bormi × P12/3. Analysis of a homozygous F 4 line by 2-D IEF/SDS-PAGE showed that recombination between the class I/II and class III subfamilies of genes had occurred

The synthesis and deposition of the prolamin storage proteins (secalins) of rye

The synthesis and deposition of the endosperm storage proteins of rye, usually termed secalins, has been studied. The rate of accumulation of secalin in developing rye grain was at a maximum between 3 and 5 weeks after anthesis. Some changes in the proportions of the four major groups of secalin polypeptides were observed during maturation, notably an increase in γ-secalins of Mr 75k and a decrease in ω-secalins. In-vitro translation of mRNA fractions prepared from 4-week-old endosperms showed that secalin polypeptides were synthesised on membrane-bound polysomes. The secalin products were identified by their mobilities on SDS-PAGE and their relative incorporation of radioactive lysine, glycine, proline, leucine and methionine. Protein bodies prepared by sucrose density ultracentrifugation contained reduced amounts of γ-secalins of Mr 40k and ω-secalins compared with the total secalin fraction, but these components were present in the expected amounts when 1.0 M NaCl was added to the buffers. Treatment of the protein bodies with proteinase-k resulted in the digestion of their contents regardless of the presence of NaCl, indicating that the surrounding membrane was incomplete. It was concluded that the NaCl reduced the loss of secalins from the protein bodies by decreasing secalin solubility rather than by affecting the integrity of the protein body membrane. The results reported for the synthesis and deposition of secalins are consistent with the results of previous studies on the prolamins of wheat and barley.

The regulation of component processes of photosynthesis in transgenic tobacco with decreased phosphoribulokinase activity

Tobacco plants (Nicotiana tabacum L.) transformed with an inverted cDNA encoding ribulose 5-phosphate kinase (phosphoribulokinase,PRK; EC 2.7.1.19) were employed to study the in vivo relationship between photosynthetic electron transport and the partitioning of electron transport products to major carbon metabolism sinks under conditions of elevated ATP concentrations and limited ribulose 1,5-bisphosphate (RuBP) regeneration. Simultaneous measurements of room temperature chlorophyll fluorescence and CO2 gas exchange were conducted on intact leaves. Under ambient CO2 concentrations and light intensities above those at which the plants were grown, transformants with only 5% of PRK activity showed ‘down-regulation” of PS II activity and electron transport in response to a decrease in net carbon assimilation when compared to wild-type. This was manifested as a decline in the efficiency of PS II electron transport (ΦPS II), an increase in dissipation of excess absorbed light in the antennae of PS II and a decline in: total linear electron transport (J1), electron transport dedicated to carbon assimilation (JA) and electron transport allocated to photorespiration (JL). The transformants showed no alteration in the Rubisco specificity factor measured in vitro and calculated in vivo but had a relatively smaller ratio of RuBP oxygenation to carboxylation rates (vo/vc), due to a higher CO2 concentration at the carboxylation site (Cc). The relationship between ΦPS II and ΦCO2was similar in transformants and wild-type under photorespiratory conditions demonstrating no change in the intrinsic relationship between PS II function and carbon assimilation, however, a novel result of this study is that this similar relationship occurred at different values of quantum flux, J1, JA, JL and vo/vc in the transformant. For both wild-type and transformants, an assessment was made of the possible presence of a third major sink for electron transport products, beside RuBP oxygenation and carboxylation, the data provided no evidence for such a sink.

α-Type prolamins are encoded by genes on chromosomes 4Ha and 6Ha ofHaynaldia villosa schur (syn.Dasypyrum villosum L.)

Because of their high degree of polymorphism and structural diversity, the prolamin storage proteins of grasses have proved to be valuable markers in taxonomic studies. We have used this approach to suggest that the wild grass Haynaldia villosa is more closely related to cultivated wheat than to rye (Shewry et al., 1987), a result which conflicts with detailed numerical studies (Baum, 1977, 1978). In particular, seeds ofH. vitlosa contain o~-type gliadins, a group of prolamins previously reported only in cultivated wheat and closely related species of Triticum, Aegilops, andAgropyron (Elytrigia ) ( Au t ran et aL, 1979; Dvorak et al., 1986). Two-dimensional electrophoretic analyses of addition lines of H. villosa chromosomes in bread wheat (cv. Chinese Spring) showed the presence of prolamins encoded by chromosomes 1Ha and 6Ha, the latter being presumed to encode the oL-type gliadins (by homology with the Gli-2 loci on the group 6 chromosomes of bread wheat). However, analyses of the same series of addition lines by Montebove et al. (1987) failed to confirm the presence of gliadin genes on chromosome 6Ha but did show putative oL-type gliadins encoded by 4Ha. Furthermore, Blanco et al. (1991) showed a-type gliadins encoded by chromosomes 4Ha and 6Ha using a separate series of lines added into pasta wheat. The different results reported by Shewry et al. (1987) and Montebove et al. (1987) may relate to the electrophoretic systems which were used: two-dimensional (2-D) isoelectric focusing/sodium dodecyl sulfate-poly-