Effects of Nutrient Supply on Seed Growth of Field Bean (Vicia faba L.). 1. Effects of Different Sulphur and Methionine Regimes on Cotyledon Growth, Protein and Uncombined Amino Acids In vitro

The effects of differemt S and methionine regimes on growthof developing Vicia faba cotyledons in vitro were studied. Basalmedium (containing adequate S) supplemented with 05 mM methioninemarginally increased d. wt and uncombined amino acid accumulationbut adding 1–5 mM methionine inhibited both growth andprotein accumulation. Sulphur deficiency reduced both d. wtand protein accumulation but incresed accumulation of uncombinedamino acids. Adding 1 mM methionine to the S-deficient mediumrestored growth, normal protein and uncombined amino acid acnunulation.High sulphate medium (7.5 mM ) decreased d. wt, protein anduncombined amino acid accumulation. High sulphate medium or basal medium+methionine (05 mM) changedthe proportions of the seed proteins; legumin increased butvicilin decreased. Sulphur deficiency caused a relative increasein vicilin but a decrease in legumin. The different S and methionineregimes markedly changed the composition of the uncombined aminoacids, especially those derived from aspartic acid but not thecomposition of the protein fraction, except during S deficiency. The data presented indicates a flexibility in the storage proteincomposition of developing cotyledons grown in vitro, with theS and methionine status having a regulatory effect. Vicia faba L., field bean, cotyledon, growth, in vitro culture, uncombined amino acids, protein composition, legumin, vicilin, methionine, sulphur     

Amino acid sequence of a crystalline seed albumin (winged bean albumin-1) from Psophocarpus tetragonolobus (L.) DC. Sequence similarity with Kunitz-type seed inhibitors and 7S storage globulins

The complete amino acid sequence of winged bean albumin-1 (WBA-1) of Psophocarpus tetragonolobus (L.) DC has been determined. The protein consists of a single polypeptide chain of 175 amino acid residues, with one disulfide bond, corresponding to a molecular mass of 19333 Da. WBA-1 was found to be homologous with the Kunitz-type seed trypsin inhibitors. The similarity between WBA-1 and the trypsin inhibitors from soybean and winged bean was 38% and 28%, respectively; similarity was most marked in the C-terminal third of the sequence with identities of 47% and 37%, respectively. Significant similarity was found also between the 2S Kunitz-type proteins and the carboxy-terminal region of the 7S storage globulins, suggesting that these two groups of proteins are related and may have evolved from a common ancestral precursor. Circular dichroism measurements suggest a high content of beta sheet (52%) while secondary structure predictions based on amino acid sequence indicate a similar content and distribution of beta sheet to that found for soybean trypsin inhibitor by X-ray diffraction studies.     

Advances in the molecular biology of plant seed storage proteins

Plant seed storage proteins were among the first proteins to be isolated (20); however, only recently, as a result of using molecular biology techniques, have the amino acid sequences of many of these proteins been determined. With the accumulation of amino acid sequence data for many vicilin-type storage proteins much has been learned concerning the location of conserved amino acid regions and other regions which can tolerate amino acid sequence variation. Combining this knowledge with recent advances in plant gene transfer technologies will allow molecular biologists to correct (by using amino acid replacement mutations) the sulfur amino acid deficiency inherent to bean seed storage proteins. The development of more nutritious soybean and common bean seeds will be of benefit to programs involving human and animal nutrition.     

Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency: Rates of Legumin Synthesis and Levels of Legumin mRNA

It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in legumin breakdown.

Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S-deficient plants indicated a reduction of 60 to 70% in synthesis of legumin-related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyledons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants.

When restored to an adequate S supply, S-deficient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA.

     

Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake

We previously reported on Vicia narbonensis seeds with largely decreased alpha- D-glucose-1-phosphate adenyltransferase (AGP; EC 2.7.7.27) due to antisense inhibition [H. Weber et al. (2000) Plant J 24:33-43]. In an extended biochemical analysis we show here that in transgenic seeds both AGP activity and ADP-glucose levels were strongly decreased but starch was only moderately reduced and contained less amylose. The flux control coefficient of AGP to starch accumulation was as low as 0.08, i.e. AGP exerts low control on starch biosynthesis in Vicia seeds. Mature cotyledons of antisense seeds had increased contents of lipids, nitrogen and sulfur. The protein content was higher due, in particular, to increased sulfur-rich albumins. Globulin fractions of storage proteins had a lower ratio of legumin to vicilin. Isolated cotyledons partitioned less [14C]sucrose into starch and more into soluble sugars with no change in the protein fraction. Respiration of isolated cotyledons and activities of the major glycolytic and carbohydrate-metabolizing enzymes were not affected. Sucrose and the hexose-phosphate pool were increased but UDP-glucose, 3-phosphoglyceric acid, phospho enolpyruvate, pyruvate, ATP and ADP were unchanged or even lower, indicating that carbon partitioning changed from starch to sucrose without affecting the glycolytic and respiratory pathways. Soluble compounds were increased but osmolality remained unchanged, indicating compensatory water influx resulting in higher water contents. Developmental patterns of water and nitrogen accumulation suggest a coupled uptake of amino acids and water into cotyledons. We conclude that, due to higher water uptake, transgenic cotyledons take up more amino acids, which become available for protein biosynthesis leading to a higher protein content. Obviously, a substantial part of amino acid uptake into Vicia seeds occurs passively and is osmotically controlled and driven by water influx.     

Sequence Peculiarity of Gnetalean Legumin-Like Seed Storage Proteins

The development of seeds as a specialized organ for the nutrition, protection, and dispersal of the next generation was an important step in the evolution of land plants. Seed maturation is accompanied by massive synthesis of storage compounds such as proteins, starch, and lipids. To study the processes of seed storage protein evolution we have partially sequenced storage proteins from maturing seeds of representatives from the gymnosperm genera Gnetum, Ephedra, and Welwitschia—morphologically diverse and unusual taxa that are grouped in most formal systems into the common order Gnetales. Based on partial N-terminal amino acid sequences, oligonucleotide primers were derived and used for PCR amplification and cloning of the corresponding cDNAs. We also describe the structure of the nuclear gene for legumin of Welwitschia mirabilis. This first gnetalean nuclear gene structure contains introns in only two of the four conserved positions previously characterized in other spermatophyte legumin genes. The distinct phylogenetic status of the gnetalean taxa is also reflected in a sequence peculiarity of their legumin genes. A comparative analysis of exon/intron sequences leads to the hypothesis that legumin genes from Gnetales belong to a monophyletic evolutionary branch clearly distinct from that of legumin genes of extant Ginkgoales and Coniferales as well as from all angiosperms. Received: 5 June 1997 / Accepted: 31 March 1998     

Inheritance of legumin and albumin contents in a cross between round and wrinkled peas

Summary Legumin and albumin are the fractions of pea seed proteins preferred to vicilin because of their high sulfur amino acid contents. The joint inheritance of legumin and albumin contents was studied in a cross between to contrasting lines of peas — one with high legumin and low albumin, and the other with low legumin and medium to high albumin. Single seed determinations were made in the parental, F₁; F₂ and backcross generations using rocket immunoelectrophoresis. In the non-segregating generations (P₁, P₂ and F₁), legumin and albumin contents were negatively correlated (r=–0.50). The estimates of correlation coefficients in the segregating generations (F₂, BC₁ and BC₂) were also about –0.5. However, the two estimates based on the round and on the wrinkled seeds separately in the F₂ generation were not significantly different from zero. At least four individual round F₂ seeds showed the desired recombination of high legumin with high albumin indicating that the unfavorable correlation can be broken. In this cross legumin content showed predominantly additive genetic variation whereas the dominance variance was the largest component for albumin content. A combined relative sulfur index, proposed as a convenient measure for selection, showed a narrow sense heritability of 47%. In general these results support the view that sulfur amino acid content of peas can be improved by breeding, but that the required selection regime must take both legumin and albumin content into account.     

OCCURRENCE OF LOW MOLECULAR WEIGHT AND HIGH CYSTEINE CONTAINING ALBUMIN STORAGE PROTEINS IN OILSEEDS OF DIVERSE SPECIES

The proteins in the oilseeds of species from 11 families, including sunflower, mustard, linseed, almond, lupin, peanut, cucumber, Brazil nut, hazelnut, yucca, castor bean, and cottonseed were studied. Sucrose gradient centrifugation showed that a substantial proportion of the total seed protein from each species migrated with a 2S sedimentation coefficient. The 2S proteins, being water-soluble and thus termed albumins, comprised 20–60% of the total seed proteins, while faster migrating globulins comprised the rest. The amino acid compositions of the 2S proteins were characterisitic of storage proteins by having a high amide content. However, the 2S proteins are different from the classical globulin storage proteins in having a high content of cysteine. It is proposed that 2S albumins are seed storage proteins with a wide distribution and with chemical properties distinct from those of the globulin storage proteins. They play an additional and unique role of providing sulfur reserve for germination.     

Use of synthetic oligodeoxyribonucleotides and primed cDNA as probes for pea (Pisum sativum L.) RNA and genomic DNA sequences

Two oligonucleotide sequences were synthesised by a solid-phase phosphotriester method. One of these sequences, A was a copy of part of a characterised cDNA clone encoding the basic subunit of legumin, a seed storage protein of Pisum sativum L. (garden pea); the other sequence B was predicted to be complementary to the 5 region of legumin mRNA on the basis of the amino acid sequence of legumin acidic subunits and most likely codon usage. Sequence A was shown to hybridise specifically to a legumin cDNA clone and to legumin mRNA. Sequence B did not hybridise specifically to legumin mRNA and was concluded not to be correctly complementary to legumin mRNA. Sequence A was used as a primer for cDNA synthesis using pea seed mRNA as a template. The cDNA so produced hybridised specifically to a legumin cDNA clone, to legumin mRNA, and to sequences encoding legumin in a restriction digest of pea genomic DNA. It is suggested that such oligonucleotide primed cDNAs may be of general value in probing eukaryotic genomic DNA.     

Characterization and evolutionary relationship of methionine-rich legumin-like protein from buckwheat.

We have isolated and characterized a full-length cDNA for legumin-like storage polypeptide from buckwheat seed (Fagopyrum esculentum Moench) and compared its deduced amino acid sequence with those from different representatives of dicots, monocots and gymnosperms. The cDNA sequence was reconstructed from two overlapping clones isolated from a cDNA library made on mRNA of buckwheat seed at the mid-maturation stage of development. Analysis of the deduced amino acid sequence revealed that this specific buckwheat storage polypeptide should be classified in the methionine-rich legumin subfamily present in the lower angiosperm clades, a representative of which was first characterized in Magnolia salicifolia (clone B 14). The fact that a methionine-rich legumin coexists together with methionine-poor legumins in buckwheat should be an important element regarding the evolutionary position of buckwheat. This may also be supporting evidence that the B14 ortholog was not lost in evolution but was protected under pressure of an increased need for sulfur. Using primers designed from characterized cDNA, we also isolated its corresponding gene from buckwheat genomic DNA and analyzed the characteristic exon/intron structure. The firstly identified two-intron structure of buckwheat legumin gene is an important contribution to study of methionine-rich legumins in lower angiosperms.     

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

Variation of the legumin seed storage protein amongst Vicia species

Variation in legumin, the major seed storage protein of Vicia faba was investigated in different (primitive and cultivated) varieties of V. faba and in other primitive Vicia species. Qualitative variation in legumin subunit patterns on gel electrophoresis was less within the species V. faba than the variation between Vicia species. However, the large seeded modern V. faba cultivars showed much increased levels of the ‘main’ legumin subunit pairs. Analyses of amino acid composition, and nitrogen and sulphur content did not show systematic variation between the samples tested, and suggested that breeding and selection had not decreased protein content or nutritional quality. It was concluded that the heterogeneities of legumin genes in the Vicia species examined are comparable, and that selection for a large-seeded phenotype in V. faba has had the effect of increasing the expression of a subset of legumin genes, those encoding the ‘main’ subunit pairs.     

An investigation of the dissociation and denaturation of legumin by salts using laser light scattering and circular dichroism spectroscopy

The dissociation of legumin, a 12 S seed storage globulin from Pisum sativum, has been studied by laser light scattering and circular dichroism spectroscopy. Salts from the Hofmeister series, in particular sodium perchlorate, were used as dissociating agents. The Mr 360,000 hexameric protein was found to dissociate first to trimers and further to monomers and the number of amino acids involved in the trimer-trimer interaction estimated to be 23(+/-4). Native legumin appears to be more strongly bound together than some analogous seed storage globulins from other plant species such as Arachis hypogaea or Sesamum indicum and the dissociation process was accompanied by some changes in conformation.     

Turnover of Storage Protein in Seeds of Soya Bean and Pea

We were interested in determining whether the low protein contentof pea seeds (Pisum sativum L.) as compared to soya bean seeds(Glycine max L. Merrill) might be due to faster degradationof the pea storage proteins during development of the seed.Pea and soya bean cotyledons were subjected to a ‘pulse-chase’experiment using [³H]glycine in in-vitro cultures. In peas,legumin had a half-life of 146 days, while vicilin had a half-lifeof 39 days. There was no measureable degradation of soya beanstorage proteins. Even with the pea storage proteins, the half-liveswere so much longer than the maturation time of seeds that degradationof storage proteins could not account for the lower proteincontent of peas as compared to soya beans. The validity of theseresults was indicated by the finding that non-storage proteinshad much shorter half-lives and that omission of a carbon ora nitrogen source greatly accelerated degradation. Labelledglycine was found to be a good probe for protein turnover studiesbecause it was very rapidly metabolized. Glycine max L. Merrill, soya bean, Pisum sativum, L. pea, protein turnover, storage proteins, legumin, vicilin     

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.