Characterisation of the storage protein subunits synthesised in vitro by polyribosomes and RNA from developing pea (Pisum sativum L.)

Evidence is presented to show that legumin, the major storage protein in Pisum, is synthesised in vitro by the wheat germ and reticulocyte lysate systems, from polyribosomes and mRNA isolated from developing pea seeds. While legumin isolated from mature pea seeds consists of 40,000 and 20,000 MW subunits, the in vitro legumin is synthesised as a 60,000 MW precursor consisting of covalently linked 40,000 and 20,000 MW subunits. The implications of these findings are discussed in relationship to studies with other systems.Abbreviations SDS-PAGE SDS-polyacrylamide gel electrophoresis - PBS phosphate buffered saline - MW molecular weight     

Temporary storage compounds and sucrose-starch metabolism in seed coats during pea seed development (Pisum sativum)

Quantitative data for growth, carbohydrate, protein and free amino acid nitrogen content of pea ( Pisum sativum L. cv. Finale) seed coat were obtained during the main stage of seed development. These data allowed us to define the role of the seed coat storage compounds. High amounts of arginine were measured in the seed coat and this amino acid is hypothesized to be synthesized de novo in the seed coat cells. Starch appeared to be stored in a specific parenchyma layer of the seed coat. Starch storage was shown to occur from phloem-unloaded sucrose and high activities of some enzymes of sucrose-starch metabolism (sucrose synthase, EC 2.4.1.13 and ADP glucose pyrophosphorylase, EC 2.7.7.27) were measured. The contribution of seed storage compounds is discussed in terms of buffering embryo nutrition. The sink strength of the young pea seed may be located within the seed coat.     

Cellular localization of a plant protein PSAM 1 in arbuscular mycorrhizas of Pisum sativum

Psam 1 is a single-copy gene which is activated during early plant-fungal interaction in wild-type pea inoculated with Glomus mosseae and which codes for PSAM 1, a putative protein of 108 amino acids. A synthetic peptide was designed in an antigenic region of this protein to produce a polyclonal antibody against PSAM 1 and to investigate its cellular localization. Western blot analysis revealed that a polypeptide of about 14.5 kDa accumulated more in mycorrhizal than non-mycorrhizal pea roots. The PSAM 1 antigen was immunolocated in planta in arbuscule-containing cells of mycorrhizal roots and especially in the cytoplasm surrounding young arbuscules in cortical cells, which suggests that its accumulation is somehow related to the symbiotic state of these cells. Received: 27 June 1998 / Accepted: 27 July 1998     

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     

Molecular heterogeneity and genetics of Vicia faba seed storage proteins

Summary Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different -major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.Contribution no. 55 from the Center of Vegetable Breeding, Portici, Italy     

The effect of the r a and r b loci on the lipid content of the seed of Pisum sativum

Summary The lipid content of seed from a set of isogenic lines for the R _a : r _a locus has been determined; the results show that this locus as well as affecting the starch, sugar and storage protein content and composition, also has a marked effect on the lipid content of the seed. Genotypes having different combinations of alleles at the r _a and r _b loci have also been examined; an r _a r _a r _b r _b , genotype had 5.57% purified total lipid in its seed — a more than 2-fold increase over that found in the round-seeded varieties (R _a R _a R _b R _b ) usually grown for the dry sed crop.     

Legumin-like and vicilin-like seed storage proteins: Evidence for a common single-domain ancestral gene

Legumin-like 11S and vicilin-like 7S globulins are the main storage proteins of most angiosperms and gymnosperms. The subunits of the hexameric legumin are synthesized as a precursor comprising a N-terminal acidic - and a C-terminal basic -chain. The trimeric vicilin molecule consists of subunits composed of two symmetrical N- and C-terminal structural domains.In a multiple alignment we have compared the N-terminal and C-terminal domains of 11 legumns and seven vicilins of several dicot, monocot, and gymnosperm species. The comparisons using all six possible pairwise combinations reveal that the N-terminal and C-terminal domains of both protein families are similar to each other. These results together with data on the distribution of variable and conserved regions, on the positions of susceptible sites for proteolytic attack, as well as on the published 7S protein tertiary structure suggest that both protein families share a common single-domain ancestor molecule and lead to the hypothesis that a triplication event has occurred during the evolution of a putative legumin/vicilin ancestor gene.Moreover, the comparison of the intron/exon pattern reveals that at least three out of five intron positions are precisely conserved between the genes of both protein families, further supporting the idea of a common evolutionary origin of recent legumin and vicilin encoding genes. Correspondence to: H. Bäumlein     

Changes in legumin messenger RNAs throughout seed development in Pisum sativum L.

The patterns of accumulation of three classes of legumin mRNA from Pisum sativum have been followed through seed development by cell-free translation and hybridization to complementary DNAs. Maximum amounts of mRNA were found at 19 days after flowering (DAF) for two classes and at 24 DAF for the third class. The proportions of the three classes varied through development: the RNA species which was 40% of the total legumin mRNA at 14 DAF represented 15–20% of the total at 25–27 DAF, whereas a second mRNA species represented approx. 30% and 70% at the same stages, respectively. Based on these results, some deductions about the possible contribution of individual genes within classes are made.Abbreviations cDNA complementary DNA - DAF days after flowering - IgG immunoglobulin G - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate)     

Immunoelectrophoretic analysis of seed proteins from Pisum sativum L.

Summary Soluble proteins of pea seed were investigated by quantitative immunological methods. Vicilin, legumin, pea seed lectin (PEA), 26 albumins and a globulin (B₁) were detected and observed during seed development, germination and under different extraction and fractionation procedures. Vicilin and legumin were found to be immunologically distinctly different. Legumin was found to be comprised of two similar proteins, Legumin species I and II. Vicilin, but no legumin, was detected in the embryonic axis.Three albumins, B₁ and PEA were found to be synthesized after the onset of legumin synthesis.Among the pea lines investigated, one line exhibited distinct differences with respect to the albumins and PEA.Some observations indicate that PEA might interact with other seed proteins of pea.     

In vitro clonal propagation of Pisum sativum L.

A system of in vitro clonal propagation has been developed in Pisum sativum L. (cv. Bohatýr). A modified MS-medium supplemented with 20 M 6-benzylaminopurine (BAP) and 0.1 M -naphthaleneacetic acid (NAA) was used to induce multiple shoot formation from shoot apices, axillary buds of the first normal leaf, axillary buds of the first and second primary scales and axillary buds of cotyledons of 4 to 6 day old pea seedlings. Meristem explants maintained a high proliferation ability in each subculture in the course of 20 months of the culture. Regenerated shoots were rooted in the same basal medium containing 5 M NAA. Rooted plants were cultured in hydroponic pots filled with half-strength MS-medium to attain anthesis and seed maturity. The phenotypic uniformity of the regenerants was evaluated. Cytological investigation confirmed the diploid stage (2n=14) of regenerants and their progeny. Histological studies revealed that proliferating shoots originated from axillary and adventitious buds. In vitro propagation is discussed as related to pea breeding.     

Heterogeneity of sulphur content in the storage proteins of pea cotyledons

By means of crossed immunoelectrophoresis of the cotyledonary storage proteins of Pisum sativum L. it was shown that reduced accumulation of the legumin fraction, resulting from severe sulphur deficiency during growth, is accompanied by relative suppression of a quantitatively minor storage protein (Peak 3) shown previously by subunit analysis to be related to the vicilin series of holoproteins. The pattern of isotopic labelling of the storage proteins after injection of [³⁵S]methionine into the pedicel during seed development under normal nutritional conditions indicated that Peak-3 protein, like legumin, has a relatively high content of sulphur amino-acids. Like certain of the vicilin molecules carrying the determinants responsible for Peak-4, Peak-3 protein binds selectively to concanavalin A.     

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.     

Matromorphy in Pisum sativum L.

Summary The possibility of obtaining instant pure breeding lines by matromorph seed development in Pisum sativum L. has been investigated. Two types of maternal parents, namely, homozygous for the recessive marker genes and heterozygous for the dominant marker genes were pollinated with Lathyrus odoratus and the P174 variety of Pisum sativum L. carrying dominant markers. For both pollinators, induction of matromorphy by prickle pollination, irradiated pollen and IAA treatment was examined. Promising matromorphs were identified in the M₁ generation which were studied in the M₂ generation for assessing their genetic status with respect to homozygosis. The success of pod set varied from zero to 28% with a varying number of matromorphic seeds following different treatments. The possible mechanisms for matromorphic origin have been discussed. The evidence presented herein favours induction of matromorphy in peas for the production of homozygous stocks. In addition, the recovery of double recessive seed markers of the maternal parents along with plant markers from the paternals has prospective implications in plant breeding as an alternative tool to recurrent back crossing.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.     

Inheritance of seed yield and quality traits in peas (Pisum sativum L.)

Summary A half diallel analysis involving nine cultivars showed that additive as well as non-additive gene effects were important for the inheritance of seed yield per plant, 100-seed weights, protein content and potassium per cent. For remaining traits non-additive genetic components were important. Overdominance was observed for all traits except for 100-seed weight, which expressed partial dominance. Parents PMR-T10, EC21857, EC109182, T163 and EC109189 were good general combiners for seed yield, seed weight and quality traits. In general there was a good relationship between per se performance and the gca effects of the parents for all traits. Cross combinations such as LMR8 x EC109182,LMR8 x PMR-T10,LMR8 x EC21857,PMRT10 x EC21857 and P23 x EC21857 were found promising. The seed yield was positively correlated with other quality traits. Protein had a positive correlation with methionine and phosphorus. All the values of correlation co-efficients were non-significant except for yield with potassium, 100-seed weight and protein with methionine, indicating that yield and quality attributes can be improved simultaneously by simple selection procedures.     

Activation of carboxylesterases in root cortical parenchymal cells of Pisum sativum during xylem induction in vitro

A quantitative cytochemical study of naphthol AS-D esterase activity in explants from roots of Pisum sativum grown on basal medium for 3 or 6 days showed similar levels of activity to those seen in sections of cortex and stele from intact roots of similar ages. Explants grown in the presence of auxins or cytokinin alone showed a threefold or twofold increase in cortical parenchymal activity, respectively. On adding both hormones to initiate xylem element formation, there was also a threefold increase in activity in the cortex. In all three cases, the stimulated activity was totally inhibited by either 10(-4) M diisopropylfluorophosphate (DFP) or 10(-4) M diethyl p-nitrophenylphosphate (E600), indicating carboxylesterase activity. The low level of activity normally present in cortical cells was inhibitor resistant, so indicating acetylesterase activity. Thus, auxins and cytokinins appear to activate mainly similar carboxylesterases during the initiation of xylem elements from cortical parenchyma cells.     

Paraquat toxicity in Pisum sativum: Effects on soluble and membrane-bound proteins

The effects of paraquat (PQ) on Pisum sativum L. proteins were investigated in vivo in a new experimental system utilizing 10-day-old plant cuts.A marked decrease in the specific activity of membrane-bound Ca²⁺-dependent ATPase was recorded, while that of Mg²⁺-dependent ATPase remained unchanged. Concurrently with a drop in the total plant protein, the specific activities of the three cytoplasmic enzymes, malate dehydrogenase, hydroxypyruvate reductase and triose-phosphate isomerase, were also found to decrease. The effect on various enzymes involved in cellular defense mechanisms was also studied: glutathione reductase and superoxide dismutase activities increased, while ascorbate peroxidase was not affected.
These findings shed light on the selectivity of PQ-induced injurious processes, focusing on protein homeostasis mechanisms in the membrane and cytoplasmic compartments at the cellular level, as well as on the prominent role played by enzymatic defense systems against PQ poisoning.