Effect of Nitrogen Nutrition on Remobilization of Protein Sulfur in the Leaves of Vegetative Soybean and Associated Changes in Soluble Sulfur Metabolites

The hypothesis that protein S is remobilized from mature leaves in response to N stress but not S stress was examined by transferring vegetative soybean (Glycine max L. Merr) plants grown with adequate sulfate and nitrate to nutrient medium with low sulfate (5 [mu]M) and nitrate at either 15, 7.5, 2, or 0.25 mM. Soluble S decreased to very low levels in mature and maturing leaves, especially in low-N plants. At high [N], insoluble S (protein) in mature leaves remained constant, but at low [N], after the soluble S declined, up to 40% of the insoluble S was exported. The losses were complemented by gains, initially in soluble S, but subsequently in insoluble S, in the expanding leaves and the root. In low-N plants, but not in high-N plants, the decrease in insoluble S in mature leaves was complemented by increases in homoglutathione (hGSH), Cys, and Met. At low [N], but not at high [N], the developing leaf, leaf 5, contained high amounts of soluble S, mostly hGSH. The results suggest that, at low [N], protein S is metabolized to hGSH, which serves as the principal transport compound for the export of organic S.     

Sulphur nutrition changes the sources of S in vegetative tissues of wheat during generative growth

During generative growth, developing wheat grains require nitrogen and sulphur to synthesize storage proteins. The hypothesis that the S required for grain growth can be derived from vegetative tissues was examined by growing plants in nutrient culture containing either 50 M S (low-S) or 200 M S (high-S) and terminating the nutrient supply at various times during generative growth. After terminating the nutrient supply, high-S plants redistributed soluble S to developing grains from pools in roots and leaves, whereas low-S plants remobilized insoluble S (protein-S) from the leaves to the grains. A model for the cycling of S within mature leaves during generative growth is presented.     

Effects of defoliation on growth and yield in groundnut (Arachis hypogea), cowpeas (Vigna unguiculata), soyabean (Glycine max) and green gram (Vigna aurens)

Defoliation reduced the dry weight of stems, pods, grains and size of individual grains in all four of the legume crops studied and the dry weight of flowering inflorescence stalk in the case of cowpeas and green gram only. The adverse effect of defoliation was more pronounced when defoliation was complete than when half of the number of leaves were removed. The greatest reduction in grain yield occurred when the plants were defoliated during the early podding stage, the percentage reduction being 59.7, 79.0, 86.4 and 95.3 in groundnut, cowpeas, soyabeans and green gram respectively when completely defoliated at this stage and 43.3, 14.0, 42.4 and 46.1 respectively when only half defoliated. The results show that assimilates produced by the leaves during the early stages of growth are used in the growth of stems and leaves, but the assimilates produced during the reproductive stage are used mainly for the growth of the pods. In groundnut, pod number and grain weight were positively correlated with stem weight. It appears that defoliation reduced pod number by depressing the growth of stems and this in turn reduced the number of flowering nodes. The reasons for the differences between the crops in their response to the defoliation treatments and the practical implications of the findings in relation to pest and disease control and plucking of leaves for human consumption are discussed.     

Properties and localization of the homoglutathione synthetase from Phaseolus coccineus leaves

The synthesis of homoglutathione (hGSH) by several plants of the tribe Phaseoleae is shown to be catalysed by a β-alanine-specific hGSH synthetase, Properties of the enzyme from Phaseolus coccineus L. cv. Preisgewinner were studied, using ammonium sulfate precipitates of primary leaf extracts. The hGSH synthetase showed a broad pH optimum at pH 8–9, an absolute requirement for Mg²⁺, a stimulation by K⁺, and a high affinity for γ-glutamylcysteine [K_m(app.) 73 μ M ]. The enzyme exhibited a high specificity for β-alanine [K_m(app.) 1.34 m M ] compared to glycine [K_m(app.) 98 m M ]. Chloroplasts, isolated from the leaves of Phaseolus coccineus , contained about 17% of the hGSH synthetase activity in the leaf cells.     

Homoglutathione: isolation, quantification and occurrence in legumes

Homoglutathione (hGSH: γ-glutamyl-eysteinyl-β-alanine) was purified from seeds of Phaseolus coccineus L. cv. Preisgewinner, using anion-exchange chromatography and Cu₂O precipitation. Quantitative and specific determination of this thiol is possible by high-performance liquid chromatography (HPLC) after monobromobimane derivatization. The enzymatic recycling assay based on yeast glutathione reductase (EC 1.6.4.2) can also be applied, but only to samples containing either hGSH or glutathione (GSH), since enzyme reaction with hGSH is 2.7 times faster than with GSH. Using the very sensitive HPLC method, the thiol content of leaves, roots and seeds of several legumes was investigated. Although GSH and hGSH were found in all plants analysed, the GSH/hGSH ratio varied greatly within the different tribes as well as within the different organs of plants of one species. In seeds and leaves of Vicieae, only traces of hGSH were found beside the main thiol GSH, whereas in roots the hGSH content exceeded the GSH content. The Trifolieae contained both tripeptides and in the tribe Phaseoleae, hGSH predominated by far.     

Effects of water table depth and calcium perioxide application on cowpea (Vigna unguiculata) and soybean (Glycine max)

Summary The effects of three water table (WT) depths (0, 15 and 40 cm) and calcium peroxide (Calper) on the growth and yield of cowpea (Vigna unguiculata, L.) and soybean (Glycine max) were investigated in field lysimeters for a sandy loam soil. Cowpea growth was the best at 40 cm WT depth. Leaf area, plant height, dry matter production, number of leaves and pods, grain yield and consumptive water use of cowpea increases with deeper (lower) WT depth. Application of calcium peroxide improved per cent emergence, leaf area, dry matter, number of leaves and pods, weight of 100 seeds, grain yield and water use in cowpea. The optimum WT depth for vegetative growth of soybean was 15 cm, although the highest grain yield was obtained at 40 cm WT depth. Number of pods, grain yield and water use efficiency of soybean increased with deeper water table depth. Application of calcium peroxide to soybean increased number of leaves and pods per plant, and grain yield for the 15 cm WT depth treatment.     

Preferential Loss of an Abundant Storage Protein from Soybean Pods during Seed Development

A temporary vegetative storage protein, composed of similar 25 kilodalton and 27 kilodalton subunits, was found to be abundant in soybean (Glycine max (L.) Herr. var Hobbit) leaves, stems, pods, flower petals, germinated cotyledons, and less abundant in roots, nodules and seeds. Total pod protein was highest at 3 weeks after flowering and declined by 37% within 3 weeks during seed development. During this time the vegetative storage protein declined from 18% to 1.5% of the total pod protein and accounted for 45% of the protein lost from pods. This indicates that the vegetative storage protein makes a significant contribution to the pool of nutrients mobilized from pods for transport to developing seeds.     

Leaf age-dependent differences in sulphur assimilation and allocation in poplar (Populus tremula x P. alba) leaves

35S-sulphate was flap-fed to poplar leaves of different leaf development stages - young developing, expanding, mature, and old mature poplar leaves. (35)S-sulphate was taken up independent of the leaf development stage. Whereas young development leaves did not export the (35)S taken up, export increased with increasing leaf development stage. Expanding leaves allocated the exported (35)S mainly into apical tree parts (73-87%) and only to a minor extent (13-27%) in basipetal direction. Neither lower trunk sections nor the roots were sinks for the exported (35)S. Expanding and developing leaves, but not the shoot apex, were the main sinks for the (35)S allocated in apical direction. In contrast, mature and old mature leaves exported the (35)S taken up mainly in basipetal direction (65-82%) with the roots constituting the main sinks. The (35)S allocated into apical tree parts was found in expanding and developing leaves, but only to a minor extent in the shoot apex. Apical allocated (35)S was identified as sulphate. Apparently the demand of young developing leaves for reduced sulphur was not fulfilled by mature leaves. Therefore, reduced sulphur for growth and development of young developing leaves must be supplied from other sources. In vitro activity of enzymes involved in assimilatory sulphate reduction was measured to investigate whether demand for reduced sulphur by young leaves is met by their own sulphate reduction. ATP sulphurylase and APS reductase activities were not significantly lower in developing than in mature leaves. Sulphite reductase and serine acetyltransferase activities were highest in developing leaves; O:-acetylserine (thiol) lyase activity was similar in all leaf developing stages. Apparently, young developing poplar leaves are able to produce their own reduced sulphur for growth and development. Whether other sources such as storage tissues and/or roots are involved in reduced sulphur supply to developing leaves remains to be elucidated.     

Root uptake, transport, and metabolism of externally applied glutathione in Phaseolus vulgaris seedlings

The most abundant thiol in beans (Phaseolus vulgaris L. cv. Saxa) is the tripeptide homoglutathione (hGSH) rather than glutathione (GSH). At the whole-plant level the GSH content is less than 0.5% of the hGSH content. In the present study GSH was supplied to the roots of bean seedlings to test whether GSH can be taken up by roots and transported to the shoot. Therefore, 12-day-old plants were exposed to 1 mmol/L GSH for 4, 8 and 24 h prior to harvest. In response to this GSH exposure, elevated GSH contents were found in all tissues. After 4 h the GSH content increased in the roots from 1 +/- 1 to 22 +/- 2 nmol GSH g(-1) fresh weight (FW), in the leaves from 2 +/- 1 to 9 +/- 4 nmol GSH g(-1) FW, and in the apex from 30 +/- 5 to 75 +/- 4 nmol GSH g(-1) FW. These data indicate that GSH is taken up by bean roots and is transported to above above-ground parts of the plants. Roots exposed to GSH for 24 h contained 2-fold higher cysteine (Cys) and hGSH contents than the controls. Apparently, GSH taken up by the roots is not only loaded into the xylem but also partially degraded and used for hGSH synthesis.     

Glutathione and homoglutathione synthesis in legume root nodules

High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and gamma-glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for gamma-glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants.     

Chloroplast pigments,proteins, lipid peroxidation and activities of antioxidative enzymes during maturation and senescence of leaves and reproductive organs of Cajanus cajan L.

A comparative investigation was undertaken with pigeon pea leaves and attached flower buds/flowers/pods during their developmental stages including senescence in a natural system in experimental plots. Alterations in chloroplast pigments, total soluble proteins, lipid peroxidation, malondialdehyde (MDA) content and activities of guaiacol peroxidase (POD, EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) were studied at 5-day interval from initial to 40-day stage. Chloroplast pigments and proteins of leaves increased upto 15 and 20-day stages respectively followed by a steady decline. Reproductive parts, however, exhibited rise in chloroplast pigments upto 25-day and protein till last stage as developing pods gain the amount from the senescing leaves which are nearest to them. Senescing leaves show very high POD activity than the developing and senescing pods and POD appears to be associated with chlorophyll degradation. Considerably higher activity and amount of LOX and MDA respectively have been noticed in senescing leaves than in flowers and pods. Increase in SOD activity during early stage of leaf growth and maturation indicates protective role that declined at senescent stages. Pods are unique in having very high SOD activity, only last stage of senescence does show a decline.     

Pathway and regulation of phosphate translocation to the pods of soybean explants

We investigated the degree to which developing fruit compete directly with leaves for mineral nutrients, e.g. phosphate coming up from the roots. When soybean ( Glycine max (L.) Merrill cv. Anoka) explants cut at mid-late podfill were given a 15-min pulse of ³²Pi via the cut stem and then transferred to distilled water, 75% of the ³²P accumulated in the leaves and 21% in stem and petiole during the first hour. The amount of ³²P entering the seeds was low (1%) initially, but thereafter increased to 30% in 48 h. An accumulation of ³²P in the seed coats preceded its entry into the embryos. Disruption (with hot steam) of the phloem between the leaf and the pods after pulse labelling indicated that more than 80% of the ³²Pi pulse moved to the leaf before redistribution to the pods. Increasing "sink" size by adjusting the pod load from 1 to 2–3 did not increase the ³²P accumulated by the pods proportionally. Conversely, excision of the seeds after pulse labelling did not prevent translocation of ³²P out of the leaves. These results suggest that the rate of transport of phosphate to the pods at mid-late podfill is controlled primarily by factors in the leaves. The results are consistent with the observation that the relative size of the sink (pod load) does not regulate leaf senescence.     

Distribution and Redistribution of Sulfur Supplied as [35S]Sulfate to Roots during Vegetative Growth of Soybean

Soybean (Glycine max L.) plants were grown in nutrient solution containing 10 [mu]M sulfate and were treated at various times with [35S]sulfate for 48 h. Growth was then continued in unlabeled solution. The sulfur content of each leaf increased rapidly until it was about 40% expanded; small, additional increases occurred until the leaf was about 70% expanded after which the sulfur content decreased by about 50%. Leaves that were about 60 to 70% expanded during the pulse were strongly labeled but then underwent a significant loss of 35S label. Leaves that were in the early stages of expansion imported little 35S label during the pulse but acquired 35S label during the chase period as they expanded (i.e. redistribution). Most of the redistributed 35S label was derived from other leaves. The rates of both sulfur import and sulfur export by a leaf were greatest at about 70% expansion. Leaves that acquired 35S label during early development retained a much higher proportion of their label than leaves that were more developed, suggesting that the sulfur acquired by leaves during early development is preferentially incorporated into a pool that is less mobile than the sulfur acquired in the later stages of leaf growth.     

Basal Application of Zinc to Improve Mung Bean Yield and Zinc-Grains Biofortfication

Worldwide, the dietary deficiency of zinc (Zn) is prevailing in almost all arid and semi-arid regions. Zinc deficiency is not only the major constraint of lower yield, but also dietary Zn deficiency in cereals grains may cause increasing malnutrition and chronic health problems in human. Exogenous application of Zn through basal soil nutrition might be a useful option to recover Zn deficiency in mung bean. Therefore, field study was conducted to optimize the optimum level and method of Zn nutrition to enhance crop yield and Zn biofortification of mung bean through basal application. Zinc was applied at 0, 5, 10 and 15 kg/ha as basal application and side dressing, and in combination (50% basal application + 50% side dressing). The results highlighted that Zn nutrition prominently improved the mung bean yield as compared with control (no Zn applied). The maximum grains yield and Zn concentration in grains were obtained where Zn was applied at 15 kg/ha as basal application as compared with all other combinations. Better improvement in grain yield was due to significant increase in more number of pods and grain size owing to welldeveloped root system, improved leaf area index and high chlorophyll contents in mung beans leaves. Amongst all applied Zn nutrition’s the basal application of Zn (15 kg/ha) was a viable option to get higher yield and Zn biofortification of mung bean.     

Chloroplast Structure and Starch Grain Accumulation in Leaves That Received Different Red and Far-Red Levels during Development

An important step in understanding influence of growth environment on carbon metabolism in plants is to gain a better understanding of effects of light quality on the photosynthetic system. Electron microscopy was used to study chloroplast ultrastructure in developing and fully expanded leaves of tobacco (Nicotiana tabacum L. cv Burley 21). Brief exposures to red or far-red light at the end of each day during growth under controlled environments influenced granum size, granum number and starch grain accumulation in chloroplasts, and the concentration of sugars in leaf lamina. Far-red-treated leaves had chloroplasts with more but smaller grana than did red-treated leaves. Red light at the end of the photosynthetic period resulted in more and larger starch grains in the chloroplasts and a lower concentration of sugars in leaves. Chloroplast ultrastructure and starch grain accumulation patterns that were initiated in the expanding leaves were also evident in the fully expanded leaves that received the treatment during development. It appears that the phytochrome system in the developing leaves sensed the light environment and initiated events which influenced chloroplast development and partitioning of photosynthate to adapt the plant for better survival under those environmental conditions.     

Evidence for a role of phytochelatins in regulating arsenic accumulation in rice grain

Phytochelatins (PCs) play a crucial role in detoxifying cellular arsenic (As) through complexation of arsenite. Here, we investigated whether PCs influence As accumulation in rice grain by using six rice cultivars varying in grain As accumulation. The cultivars with low grain As had significantly higher PCs concentration in the shoots than the cultivars with high grain As, but lower glutathione concentration. Shoot PCs concentration correlated negatively with grain As accumulation. Foliar sprays with 0.5 mM l-buthionine-sulphoxime (BSO) on rice leaves at grain filling stage decreased GSH and PC accumulation in rice shoots by 40-63% and 20-55%, respectively, but did not significantly affect plant growth. Foliar sprays with BSO decreased shoot As concentration, while increased As concentrations in husk and brown rice significantly. These results suggest that PC complexation of arsenite in rice leaves reduces As translocation from leaves to grains, and implicate that manipulation of PC synthesis might mitigate As accumulation in rice grain.     

Translocation of Sulfate in Soybean (Glycine max L. Merr)

Sulfate translocation in soybean (Glycine max L. Merr) was investigated. More than 90% of the sulfate entering the shoot system was recoverable in one or two developing trifoliate leaves. In young plants, the first trifoliate leaf contained between 10 to 20 times as much sulfate as the primary leaves, even though both types of leaf had similar rates of transpiration and photosynthesis. We conclude that most of the sulfate entering mature leaves is rapidly loaded into the phloem and translocated to sinks elsewhere in the plant. This loading was inhibited by carbonylcyanide m-chlorophenylhydrazone and selenate. At sulfate concentrations below 0.1 millimolar, more than 95% of the sulfate entering primary leaves was exported. At higher concentrations the rate of export increased but so did the amount of sulfate remaining in the leaves. Removal of the first trifoliate leaf increased two-fold the transport of sulfate to the apex, indicating that these are competing sinks for sulfate translocated from the primary leaves. The small amount of sulfate transported into the mesophyll cells of primary leaves is a result of feedback regulation by the intracellular sulfate pool, not a consequence of their metabolic inactivity. For example, treatment of plants with 2 millimolar aminotriazole caused a 700 nanomoles per gram fresh weight increase in the glutathione content of primary leaves, but had no effect on sulfate aquisition.     

S nutrition affects the pools of S available to developing grains of wheat

Developing grains of rain-fed wheat must obtain S from reserves that the plant accumulated during vegetative growth. The effect of S nutrition on the sources of S that were transported from vegetative tissues during grain development was examined. Wheat was grown in solution culture with 2 mM N and either 200 M S (high-S) or 50 M S (low-S). All nutrients were withdrawn either at booting, ear emergence, anthesis, or 8 d post anthesis. At 13 d post anthesis, high-S plants contained sulphate in roots and leaves, GSH (glutathione) in leaves, and S in leaf proteins. Between harvests, sulphate was lost from roots and GSH was lost from leaves. Sulphate and GSH were not detected in roots or leaves of low-S plants at 13 d post anthesis, and between harvests, S in leaf-proteins decreased During reproductive growth, redistribution of sulphate from roots was likely to be in response to S demand, redistribution of GSH was likely to result from the breakdown of chloroplasts, and in low-S plants, hydrolysis of leaf proteins in response to N demand was the most important source of S for grain development.     

Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx, and nitrate reductase activity, but does not affect grain yield

The manipulation of cytokinin levels by senescence-regulated expression of the Agrobacterium tumefaciens ipt gene through its control by the Arabidopsis SAG12 (senescence-associated gene 12) promoter is an efficient tool for the prolongation of leaf photosynthetic activity which potentially can affect plant productivity. In the present study, the efficiency of this approach was tested on wheat (Triticum aestivum L.)-a monocarpic plant characterized by a fast switch from vegetative to reproductive growth, and rapid translocation of metabolites from leaves to developing grains after anthesis. When compared with the wild-type (WT) control plants, the SAG12::ipt wheat plants exhibited delayed chlorophyll degradation only when grown under limited nitrogen (N) supply. Ten days after anthesis the content of chlorophyll and bioactive cytokinins of the first (flag) leaf of the transgenic plants was 32% and 65% higher, respectively, than that of the control. There was a progressive increase in nitrate influx and nitrate reductase activity. However, the SAG12::ipt and the WT plants did not show differences in yield-related parameters including number of grains and grain weight. These results suggest that the delay of leaf senescence in wheat also delays the translocation of metabolites from leaves to developing grains, as indicated by higher accumulation of ((15)N-labelled) N in spikes of control compared with transgenic plants prior to anthesis. This delay interferes with the wheat reproductive strategy that is based on a fast programmed translocation of metabolites from the senescing leaves to the reproductive sinks shortly after anthesis.     

Sulfur assimilation in developing lupin cotyledons could contribute significantly to the accumulation of organic sulfur reserves in the seed

It is currently assumed that the assimilation of sulfur into reduced forms occurs predominantly in the leaves of plants. However, developing seeds have a strong requirement for sulfur amino acids for storage protein synthesis. We have assessed the capacity of developing seeds of narrow-leaf lupin (Lupinus angustifolius) for sulfur assimilation. Cotyledons of developing lupin seeds were able to transfer the sulfur atom from 35S-labeled sulfate into seed proteins in vitro, demonstrating the ability of the developing cotyledons to perform all the steps of sulfur reduction and sulfur amino acid biosynthesis. Oxidized sulfur constituted approximately 30% of the sulfur in mature seeds of lupins grown in the field and almost all of the sulfur detected in phloem exuded from developing pods. The activities of three enzymes of the sulfur amino acid biosynthetic pathway were found in developing cotyledons in quantities theoretically sufficient to account for all of the sulfur amino acids that accumulate in the protein of mature lupin seeds. We conclude that sulfur assimilation by developing cotyledons is likely to be an important source of sulfur amino acids for the synthesis of storage proteins during lupin seed maturation.