Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.     

Vicilin genes ofPisum

Summary We describe four genomic clones of pea 7S storage protein gene, one of which corresponds to convicilin, and the others to vicilin. Hybridization studies exploiting these clones, and previously identified cDNA clones, have enabled us to define six different loci. Three of these loci have been mapped to positions on chromosome 7.     

Convicilin mRNA from pea (Pisum sativum L.) has sequence homology with other legume 7S storage protein mRNA species.

Nucleotide-sequence analysis of a complementary-DNA clone for convicilin, one of the storage proteins from pea (Pisum sativum L.) seeds, shows it to be homologous with the 7S legume seed storage proteins vicilin, conglycinin and phaseolin. Convicilin is more similar to vicilin than to phaseolin or to conglycinin. Significant areas of sequence difference are discussed.     

Molecular analysis of a null mutant for pea (Pisum sativum L.) seed lipoxygenase-2

A mutant line of Pisum fulvum was identified that lacked seed lipoxygenase-2 (LOX-2). The mutant phenotype was introgressed into a standard Pisum sativum cv. Birte to provide near-isogenic lines with or without seed LOX-2. Genetic analyses showed the mutation to behave as a single, recessive Mendelian gene. Northern and dot-blot analyses showed a large reduction in LOX-2 mRNA from developing seeds of the LOX-2-null mutant. A restriction fragment length polymorphism associated with the 5 end of the LOX-2 gene(s) co-segregated with the null phenotype, indicating that the reduction of LOX-2 mRNA was neither a consequence of deletion of the LOX genes nor a consequence of the action of a genetically distant regulatory gene. Analysis of the 5-flanking sequences of LOX-2 genes from Birte and the near-isogenic LOX-2-null mutant revealed a number of insertions, deletions and substitutions within the promoter from the LOX-2-null mutant that could be responsible for the null phenotype. Incubation of crude seed LOX preparations from Birte and the LOX-2-null mutant showed that the latter generated relatively less 13-hydroperoxides and also produced relatively more hydroxy- and ketoacid compounds that have implications for the fresh-frozen pea industry.     

Three classes of proteinase inhibitor gene have distinct but overlapping patterns of expression in Pisum sativum plants

Genes representative of three gene classes encoding proteinase inhibitor proteins, with distinct spatial expression patterns, were isolated and characterized from Pisum.Under standard plant growth conditions, one class is expressed exclusively in seeds, whereas the other two make minor contributions to seed inhibitor proteins but are also expressed in other organs, predominantly in root endodermal and floral reproductive tissues. Two of the gene classes contain few genes and are genetically linked at the Tri locus, whereas the third class displays complex hybridization patterns to genomic DNA and maps to diverse genetic loci. Expression analysis of this last class suggests that only a small number of these genes are expressed. The quantitative effect of the Tri locus on root and floral inhibitor gene expression was examined in near-isogenic lines of pea. The proteins encoded by the three classes are all members of the same family (Bowman-Birk) of enzyme inhibitors but are distinct in terms of overall sequence, active site sequences and inhibitor function.     

Inheritance of qualitative and quantitative trypsin inhibitor variants in Pisum

A trypsin inhibitor locus (Tri) has been mapped close to Vc-2 on Pisum (pea) linkage group 5 using recombinant inbred lines derived from crosses of genotypes showing qualitative variation in seed trypsin inhibitors. F₂ seed populations derived from crosses between lines showing qualitative variation in trypsin inhibitors as well as quantitative variation in inhibitor activity showed an association between the segregation of the structural variation and relative activity levels. Clones complementary to Pisum trypsin inhibitor mRNA were used in hybridization analyses which showed that the segregation of protein polymorphisms reflected directly the segregation of polymorphisms associated with the structural genes.     

Responses of the pea (Pisum sativum L.) leaf metabolome to drought stress assessed by nuclear magnetic resonance spectroscopy

While many compounds have been reported to change in laboratory based drought-stress experiments, little is known about how such compounds change, and are significant, under field conditions. The Pisum sativum L. (pea) leaf metabolome has been profiled, using 1D and 2D NMR spectroscopy, to monitor the changes induced by drought-stress, under both glasshouse and simulated field conditions. Significant changes in resonances were attributed to a range of compounds, identified as both primary and secondary metabolites, highlighting metabolic pathways that are stress-responsive. Importantly, these effects were largely consistent among different experiments with highly diverse conditions. The metabolites that were present at significantly higher concentrations in drought-stressed plants under all growth conditions included proline, valine, threonine, homoserine, myoinositol, γ-aminobutyrate (GABA) and trigonelline (nicotinic acid betaine). Metabolites that were altered in relative amounts in different experiments, but not specifically associated with drought-stress, were also identified. These included glutamate, asparagine and malate, with the last being present at up to 5-fold higher concentrations in plants grown in field experiments. Such changes may be expected to impact both on plant performance and crop end-use. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pisum lipoxygenase genes

Summary The copy number, genomic arrangement and linkage relationships of two classes of lipoxygenase gene have been investigated in Pisum(pea) lines. Each of the two classes contained two to three members in P. sativum lines. RFLPs associated with genomic fragments containing the 5 sequences of one gene class permitted its correlation in genetical analyses with a lipoxygenase locus on linkage group 4, which was previously identified through polypeptide variation. Genetical analyses of RFLPs associated with other fragments identified by low- and medium-stringency hybridization to lipoxygenase cDNAs indicate the existence of other unlinked lipoxygenase gene loci.     

The initiation of legumin synthesis in immature embryos of Pisum sativum L. grown in vivo and in vitro

A highly sensitive immunoassay has been used for the detection of a major storage protein, legumin, in embryos of Pisum sativum L.; with this technique nanogram quantities could be measured. In the two varieties tested, legumin could be detected in embryos in vivo, when they had attained a fresh weight of 2·10^-3 g and 3·10^-3 g, respectively. Contrary to earlier claims, embryos cultured in vitro were shown to be capable of initiating legumin synthesis. This capacity to initiate legumin synthesis was confirmed by two-dimensional isoelectric focusing-electrophoresis and fluorography; embryos harvested before initiation of legumin synthesis and cultured in radioactive medium were shown to have synthesized legumin subunits. The amounts of legumin and total protein synthesized per unit fresh weight were consistently greater in vitro than in equivalent embryos grown in vivo.Abbreviations ELISA Enzyme-linked immunosorbent assay - BSA bovine serum albumin - IgG immunoglobulin - SDS sodium dodecyl sulphate - DSP Pisum cv. Dark Skinned Perfection