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.     

Influence of post-flowering temperature on seed development, and subsequent performance of crisp lettuce

Crisp lettuce plants cv. Saladin were grown from the time they started flowering, at 20/10°C (16 h day, 8 h night), 25/15°C and 30/20°C in glasshouses on two occasions in 1985. Yields of seed increased from, on average, 15 g to 27 g and then fell to 20 g per plant with progressive increases in temperature. The number of mature florets per plant increased with temperature but the number of seeds per mature floret was lower at 20/10°C and 30/20°C than at 25/15°C. An increase in temperature reduced mean seed weight by up to 45%, seed volume by 15%, cell numerical volume density (N_v) by 27% and the number of cells per seed by 39%. Percentage seed germination reached a maximum early in seed development at the stage when the pappus appeared through the involucral bracts. Differences in percentage germination and vigour of seeds (slope test) from different temperatures were accounted for largely by the effects on mean seed weight. However, when germinated at 30°C seeds produced at 30/20°C germinated more readily than those produced at 25/15°C or 20/10°C. Seed vigour gradually increased with an increase in the length of storage after harvest, reaching a maximum after 260 days. In general, seeds produced at 25/15°C exhibited a greater variation in numbers of seeds per floret, N_v, seed weight, times of seedling emergence, seedling and mature head weight than seeds produced at lower or higher temperatures.     

N]NMR determination of asparagine and glutamine nitrogen utilization for synthesis of storage protein in developing cotyledons of soybean in culture

Solid-state [¹⁵N]NMR was used to measure the use of the amide and amino nitrogens of glutamine and asparagine for synthesis of storage protein in cotyledons of soybean (Glycine max L. cv. Elf) in culture. No major discrimination in the incorporation of the amide or amino nitrogens of glutamine into protein is apparent, but the same nitrogens of asparagine are used with a degree of specificity. During the first seven days in culture with asparagine as the sole nitrogen source, the amino nitrogen donates approximately twice as much nitrogen to protein as does the amide nitrogen. The use of the amide nitrogen increases with longer periods of culture. The reduced use of the amide nitrogen was confirmed by its early appearance as ammonium in the culture medium. The amide nitrogen of asparagine was found at all times to be an essential precursor for protein because of its appearance in protein in residues whose nitrogens were not supplied by the amino nitrogen. In addition, methionine sulfoximine inhibited growth completely on asparagine, indicating that some ammonium assimilation is essential for storage protein synthesis. These results indicate that in a developing cotyledon, a transaminase reaction is of major importance in the utilization of asparagine for synthesis of storage protein and that, at least in the early stages of cotyledon development, reduced activities of ammonium-assimilating enzymes in the cotyledon tissue or in other tissues of the seed or pod may be a limiting factor in the use of asparagine-amide nitrogen.     

Asparagine metabolism and asparaginase activity in a euryhaline Chlamydomonas species.

A Chlamydomonas species isolated from a marine environment possesses an L-asparaginase, an enzyme not yet reported in the microalgae. This enzyme enabled the organism to grow as well with asparagine as sole nitrogen source as with inorganic nitrogen sources (NO3-, NH4+). Only the amide nitrogen was used for growth since growth did not occur on aspartate and aspartate accumulated in the media when cells were either grown on asparagine or during short-term incubations with L-[U-14C]asparagine. Cells grown on NO3-, NH4+, or L-asparagine in batch culture possessed equivalent asparaginase activities. However, nitrogen-limited cells possessed four times the activity of cells grown with sufficient nitrogen for normal growth, regardless of the possessed the lowest activity per cell, while lag phase and stationary phase cells possessed greater activity. The enzyme behaved like a periplasmic space enzyme since (1) breaking the cells did not release into solution more activity than was shown by whole cells and (2) whole cells converted L-[U-14C]asparagine to [14C]aspartate with little intracellular accumulation of radioactivity. Cell-free preparations of the enzyme possessed a Km value for asparagine of 1.1 x 10-4 M, with no glutaminase activity.     

Protein content and amino acid spectrum of finger millet [Eleusine coracana (L.) Gaertn.] as influenced by nitrogen and sulphur fertilizers

Summary The effect of N and S fertilizers on yield, protein content and amino acid composition of the tropical cereal finger millet was studied in field and sand culture experiments. In the field the grain yield measured from 2400 to 4100 kg/ha, and the seed crude protein from 7 to 13% with rates of N up to 150 kg/ha. Seed crude protein was high in methionine and cystine, but contained less sulphur amino acids when the seed was high in protein.Supplies in excess failed to increase the lower methionine and cystine contents of the crude protein.Sulphur deficiency drastically reduced the synthesis of sulphur amino acids, concomitantly raising the aspartic acid/asparagine content of the leaves up to 45 per cent of their total N content. An S:N weight ratio of 1:10 in the seeds and 1:15 in the leaves indicates adequate supply of sulphur. The importance of finger millet for human nutrition is considered in relation to increasing substitution with other crops, use of nitrogen fertilizers, and the actual sulphur status of soils in East Africa.Sponsored by the Norwegian Agency for International Development.Sponsored by the Norwegian Agency for International Development.     

Changes in accumulation of seed nitrogen compounds in maize under conditions of sulphur deficiency

Maize ( Zea mays L., hybrid INRA 260) was grown in the greenhouse with mineral nutrition of different sulphate concentrations. Mature seeds from these plants were compared for their free amino acid and protein N forms. For the most S-deficient sample, the Asx (asparagine + aspartic acid) content increased by 30% as compared with control, while methionine and cysteine decreased (by 25 and 30%, respectively), as well as glycine, lysine, histidine, arginine and tryptophan. In seeds lowest in S the non-protein N to total N ratio was 77% higher than in the control. Free asparagine dominated in starved seeds (50 mol % of total free amino acids) and was ten-fold more concentrated than in the control, where proline was the predominant free amino acid. Thus the Asx of non-protein N reached 28% of the total mol Asx of the whole starved seed. Altered S nutrition had virtually no effect on the amino acid composition of the main protein fractions, but it significantly changed their ratios. Zeins, which are poor in S-containing amino acids, showed 25% higher level than in seeds supplied with normal S. As a counterbalance, two glutelin subfractions rich in S-containing amino acids, decreased by 36–71% under limiting S nutrition.
It is concluded that the plant reacts against S deficiency by modifying its N metabolism. Significant accumulation occurred of free asparagine, which is the main form of N transportation. The biosynthesis of seed storage protein occurred through the accumulation of the highest possible protein quantity allowed by the available S-containing amino acids, i.e. proteins low in S-containing amino acids were preferentially synthesized.     

GLUTAMYL, ASPARTYL AND AMIDE MOIETIES OF CEREBRAL PROTEINS: METABOLIC ASPECTS IN VITRO

Abstract— The total mixed proteins (excluding proteolipids) were isolated from cat cerebral cortex and subjected to acid and enzymic hydrolyses. Analyses on the hydrolysates were carried out by specific enzymic procedures to determine the glutamyl, glutaminyl, aspartyl and asparaginyl composition. The content of total glutamyl and total aspartyl residues was the same in all types of protein samples, with average values of 78 and 58 /miol/100 mg of protein, respectively. In biopsy samples approximately 45 per cent of each total was in the amide form. Preparation of slices of cerebral cortex for incubation was associated with deamidation in situ of 16 per cent of the protein-bound glutaminyl residues. The extent of deamidation was not increased by incubation or by prolonged hypoxia and was unaffected by prior anaesthesia or by incubation of slices with 10 mM-NH₄Cl or 40 mM-malonate. Slices prepared from animals intoxicated with methionine sulphoximine exhibited no deamidation. No deamidation was observed for slices of subcortical white matter, liver, kidney, testis or diaphragm of the cat. Cortical proteins from other species appeared to behave similarly to those of the cat. The 5-4 μmol of NH₃ released/g of fresh cortex could account for about 85 per cent of the endogenous free ammonia regularly encountered in such slices. Hence the labile fraction of protein-bound glutaminyl amide groups represents, as previously suspected, a major source of endogenous cerebral NH₃. Proteins isolated from cerebral cortical slices incubated with L-[U-¹⁴C]glutamic acid or L-[U-¹⁴C]glutamine contained 105 (±0.095) per cent of the total ¹⁴C metabolized. The ratios (x 100) of protein to free pool specific radioactivities (c.p.m.μmol) of glutamic acid and of glutamine were in the range 0-22 to 0-42, or of the same order as previously reported for other amino acids. Comparable results were obtained with proteins isolated from cerebral cortical slices incubated with 10 mM-¹⁵NH₄Cl or L-[amide-¹⁵N]glutamine or both. In the amide N of protein-bound glutaminyl residues the atoms per cent excess ¹⁵N ranged from 007 to 0-42. This degree of labelling could be accounted for completely by the turnover of the entire glutaminyl moiety, as indicated by the ¹⁴C studies. Simultaneous analyses of free pool NH₃ and glutamine suggested that transfer of glutamine from medium to slice involves deamidation as it is taken up and reamidation after entry.     

The Significance of Transpirationally Derived Nitrogen in Protein Synthesis in Fruiting Plants of Pea (Pisum sativum L.)

Feeding of ¹⁵N-nitrate, ¹⁵N(amide)-L-glutamine, or ¹⁵N-L-glutamicacid to detached shoots of pea through the transpiration streamresults in the soluble and insoluble nitrogen of stem, leaves,and fruits becoming extensively enriched with isotopic nitrogen.The time course of labelling suggests that non-reproductiveparts are the principal centres of uptake and assimilation andthat from them translocation takes place to the developing seeds. Distribution patterns for ¹⁵N in free and protein-bound aminoacids of leaf and seed indicate that each labelled source donatesnitrogen to a wide range of amino compounds, with no evidenceof consistent differences in the manner in which each is assimilated.Alanine, glutamic acid, homoserine, and -aminobutyric acid,are the main recipients of ¹⁵N in the soluble fraction of theleaves, whilst in the insoluble fraction nitrogen of the aminoacids serine, glycine, alanine, threonine, glutamic acid + glutamine,and aspartic acid + asparagine achieves high specific labelling.Amino acids of the seeds are labelled more uniformly with ¹⁵N. A complementary ¹⁴C-labelling experiment on the translocationof photosynthetically fixed carbon from leaf to seed is describedand the labelling patterns obtained for amino acids in leaf,seed, and phloem exudate are discussed in relation to thosefor ¹⁵N.     

Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of ¹⁵N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [¹⁴N]asparagine and [¹⁴N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-¹⁵N- and double-¹⁵N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[¹⁵N] abundance of these amino acids was determined. We demonstrate that this approach to ¹⁵N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-¹⁵N:double-¹⁵N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with ¹⁵N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [¹⁵N]H₄⁺ are ¹⁴N-enriched and must be derived primarily from protein turnover.     

Utilization of the amide groups of asparagine and 2-hydroxysuccinamic Acid by young pea leaves

The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [¹⁵N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no ¹⁵N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [¹⁵N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH₃ accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of ¹⁵N into glutamine-amide, NH₃, and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system.     

Phosphoenolpyruvate carboxykinase in developing pea seeds is associated with tissues involved in solute transport and is nitrogen-responsive

The aim of this work was to investigate the occurrence of phosphoenolpyruvate carboxykinase (PEPCK) in developing pea (Pisum sativum) seeds in relation to their nitrogen supply. PEPCK was present throughout development, with the peak of PEPCK protein and activity in the seed coat and cotyledons preceding protein accumulation in the cotyledons. It showed a different developmental pattern from enzymes involved in amino acid metabolism (phosphoenolpyruvate carboxylase, glutamine synthetase and glutamate dehydrogenase). Immunolocalization showed that PEPCK was present in parts of the developing seed that are involved in the transport and metabolism of assimilates. Early in development, it was associated with the inner integument of the ovule, the endospermic cytoplasm and the outer cells of the embryo. In the middle of development, around the peak of activity, PEPCK was abundant at the outer surface of the developing cotyledons, in the embryonic axis and in the vasculature of the seed coat. Later in development, PEPCK was associated with the embryonic leaf primordia and meristem and cortex of the radicle. PEPCK protein was strongly induced in vitro in the seed coat by nitrate, ammonium and asparagine, in the cotyledons by asparagine and in planta by the supply of nitrogen, which led to an increase in asparagine secretion by empty seed coats. It is suggested that PEPCK is involved in the metabolism of nitrogenous solutes in developing pea seeds.     

Morphophysiological dormancy in seeds of the ANA grade angiosperm Schisandra arisanensis (Schisandraceae)

We determined the kind of seed dormancy in Schisandra arisanensis, an ANA grade ([A]mborellales [N]ymphaeales [A]ustrobaileyales) angiosperm with medicinal value. Seeds have small underdeveloped embryos, and following seed maturity their length increased approximately 360% before radicle emergence. Germination was delayed 6–8 weeks, and the percentage and rate were much higher at 15/6, 20/10 and 25/15°C than at 30/20°C. For seeds incubated at 5/5°C (8 weeks) → 15/6°C (4 weeks) → 20/10°C (8 weeks) → 25/15°C (12 weeks) → 20/10°C (5 weeks), embryos grew at 15/6°C → 20/10°C, and almost all seeds that germinated (89%) did so at 20/10°C → 25/15°C. When seeds were incubated in a complementary temperature sequence, 25/15°C (12 weeks) → 20/10°C (8 weeks) → 15/6°C (4 weeks) → 5/5°C (9 weeks) → 15/6°C (4 weeks), embryos grew at 25/15°C → 20/10°C. Nearly all seeds that germinated (93%) did so at 25/15°C → 20/10°C and at 15/6°C following 9 weeks at 5/5°C. Based on the temperature requirements for embryo growth and seed germination, seeds of this species have non‐deep simple morphophysiological dormancy (C_1bB).     

Can sucrose content in the phloem sap reaching field pea seeds (Pisum sativum L.) be an accurate indicator of seed growth potential?

The composition of the translocates reaching the seeds of pea plants having various nitrogen (N) nutrition regimes was investigated under field situations. Sucrose flow in the phloem sap increased with the node number, but was not significantly different between N nutrition levels. Because N deficiency reduced the number of flowering nodes and the number of seeds per pod, the sucrose flow bleeding from cut peduncles was divided by the number of seeds to give the amount of assimilates available per seed. The sucrose concentration in phloem sap supplied to seeds at the upper nodes was higher than that at the lower nodes. The flow of sucrose delivered to the seeds during the cell division period was correlated with seed growth potential. Seeds from the more N-stressed plants had both the highest seed growth rate and received a higher sucrose flux per seed during the cell division period. As seed growth rate is highly correlated with the number of cotyledonary cells produced during the cell division period, sucrose flow in phloem sap is proposed to be an important determinant of mitotic activity in seed embryos. The carbon (C)/N ratio of the flow of translocates towards seeds was higher under conditions of N-deficiency than with optimal N nutrition, indicating that N flux towards seeds, in itself, is not the main determinant of seed growth potential.     

Structure, production characteristics and fungal antagonism of tensin - a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578

AIM: To study the antagonistic activity by Pseudomonas fluorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani. METHODS AND RESULTS: Strain 96.578 produced a new cyclic lipopeptide, tensin. High tensin production per cell was detected in liquid media with glucose, mannitol or glutamate as growth substrate while fructose, sucrose and asparagine supported low production. Tensin production was nearly constant in media with different initial C levels, while low initial N contents reduced production. When applied to sugar beet seeds, strain 96.578 produced tensin during seed germination. When challenged with strain 96.578 or purified tensin, Rhizoctonia solani reduced radial mycelium extension but increased branching and rosette formation. CONCLUSION: The antagonistic activity of strain 96.578 towards Rhizoctonia solani was caused by tensin. SIGNIFICANCE AND IMPACT OF THE STUDY: When coated onto sugar beet seeds, tensin production by strain 96.578 could be of significant importance for inhibition of mycelial growth and seed infection by Rhizoctonia solani.     

Nitrogen metabolism of asparagine and glutamate in Vero cells studied by <Superscript>1</Superscript>H/<Superscript>15</Superscript>N NMR spectroscopy

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with l-[¹⁵N]glutamate, the ¹⁵N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with l-[2-¹⁵N]asparagine, the ¹⁵N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with l-[4-¹⁵N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of , showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.     

Asparagine formation in soybean nodules

¹⁵NH₄⁺ and [¹⁵N](amide)-glutamine externally supplied to detached nodules from soybean plants (cv. Tamanishiki) were incorporated within nodule tissues by vacuum infiltration and metabolized to various nitrogen compounds during 60 minutes of incubation time. In the case of ¹⁵NH₄⁺ - feeding, the ¹⁵N abundance ratio was highest in the amide nitrogen of glutamine, followed by glutamate and the amide nitrogen of asparagine. In ¹⁵N content (micrograms excess ¹⁵N), the amide nitrogen of asparagine was most highly enriched after 60 minutes. ¹⁵NH₄⁺ was also appreciably assimilated into alanine.     

4-methyleneglutamine synthetase: a new amide synthetase present in germinating peanuts

Enzymatic activity which catalyzes the synthesis of 4-methyleneglutamine from 4-methyleneglutamic acid + ammonia was detected in and partially purified from cotyledons of peanut seeds germinated 5 to 7 days. This activity was separated from glutamine and asparagine synthetases by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme is distinct from these other amide synthetases in its substrate specificity, lack of amide/hydroxylamine exchange, and use of ammonium ion as amide donor together with formation of AMP from ATP. The activity is quite labile in solution, but is retained as a precipitate in ammonium sulfate or when frozen in 12.5% glycerol at -77 degrees C. This activity might be responsible for catalyzing the rapid synthesis of 4-methyleneglutamine which occurs in germinating peanuts.     

Culture of soybean fruit explants: growth conditions and efficiency of nitrogen sources for reserve protein synthesis

A system was devised for the in vitro culture of soybean fruits. The culture system consisted of a single fruit attached to a short piece of stem through which the nutrients were supplied. The fruit explants were taken when pods were fully expanded and the seeds at initial stages of growth. During a 7-day culture period, the seeds accumulated dry matter and protein in quantities comparable to those in situ. Omission of the C source (sucrose) from the medium resulted in no dry matter accumulation in the seeds, but omission of the N source (glutamine) still led to some protein accumulation, indicating mobilization of N from other parts of the fruit explant. Optimum protein accumulation occurred when glutamine was supplied at 1.2 mg N ml^-1. Protein accumulation in the seeds was highly dependent on the nature of the N source. Glutamine, asparagine and the ureide, allantoin, were equally the most efficient sources, whereas several other amino acids tested showed lower degrees of efficiency. The data indicate a high metabolic capacity of the fruit tissues for principal N transport compounds of soybean, namely allantoin, asparagine and glutamine. The culture system described should prove useful for developmental and metabolic studies where the complex influence of the rest of the plant is to be avoided.Abbreviations ALN allantoin - ALC allantoic acid Preliminary report presented at the IV World Soybean Research Conference, Buenos Aires, Arggentina, March 1989.