Cysteine and Methionine Biosynthetic Enzymes Have Distinct Effects on Seed Nutritional Quality and on Molecular Phenotypes Associated With Accumulation of a Methionine-Rich Seed Storage Protein in Rice.

Staple crops in human and livestock diets suffer from deficiencies in certain "essential" amino acids including methionine. With the goal of increasing methionine in rice seed, we generated a pair of "Push × Pull" double transgenic lines, each containing a methionine-dense seed storage protein (2S albumin from sunflower, HaSSA) and an exogenous enzyme for either methionine (feedback desensitized cystathionine gamma synthase from Arabidopsis, AtD-CGS) or cysteine (serine acetyltransferase from E. coli, EcSAT) biosynthesis. In both double transgenic lines, the total seed methionine content was approximately 50% higher than in their untransformed parental line, Oryza sativa ssp. japonica cv. Taipei 309. HaSSA-containing rice seeds were reported to display an altered seed protein profile, speculatively due to insufficient sulfur amino acid content. However, here we present data suggesting that this may result from an overloaded protein folding machinery in the endoplasmic reticulum rather than primarily from redistribution of limited methionine from endogenous seed proteins to HaSSA. We hypothesize that HaSSA-associated endoplasmic reticulum stress results in redox perturbations that negatively impact sulfate reduction to cysteine, and we speculate that this is mitigated by EcSAT-associated increased sulfur import into the seed, which facilitates additional synthesis of cysteine and glutathione. The data presented here reveal challenges associated with increasing the methionine content in rice seed, including what may be relatively low protein folding capacity in the endoplasmic reticulum and an insufficient pool of sulfate available for additional cysteine and methionine synthesis. We propose that future approaches to further improve the methionine content in rice should focus on increasing seed sulfur loading and avoiding the accumulation of unfolded proteins in the endoplasmic reticulum. Oryza sativa ssp. japonica: urn:lsid:ipni.org:names:60471378-2.

Calcium ameliorates the toxicity of sulfate salinity in Brassica rapa.

Salinity stress in Brassica, often only associated with osmotic effects and the toxicity of Na+, was more severe when applied as Na2SO4 than as NaCl, indicating that SO42- ions had toxic effects as well. Application of 10 mM calcium in the form of CaCl2 in the growth medium of plants only slightly ameliorated growth impairment by NaCl and KCl, but almost completely prevented negative effects of Na2SO4 and K2SO4 on plant biomass production. This effect was calcium specific, as MgCl2 ameliorated sulfate toxicity to a much lower extent. This sulfate toxicity coincided with a strong decrease in the plant content of calcium and manganese upon sulfate salinity. Application of CaCl2 largely alleviated this decrease, however, it did not prevent the higher tissue concentration of sulfate. CaCl2 prevented the increase in organic sulfur compounds presumably by reducing of relative gene expression of ATP-sulfurylase (ATPS) and adenosine 5'-phosphosulfate reductase (APR) indicating a possible regulation of sulfate assimilation by calcium. The upregulation of the genes encoding for Group 4 sulfate transporters (Sultr4;1 and 4;2) upon sulfate salinity, was absent in the presence of CaCl2. Therefore, additional calcium may facilitate an increased vacuolar capacity for sulfate accumulation.

Chloride and sulfate salinity differently affect biomass, mineral nutrient composition and expression of sulfate transport and assimilation genes in Brassica rapa.

BACKGROUND AND AIMS: It remains uncertain whether a higher toxicity of either NaCl or Na2SO4 in plants is due to an altered toxicity of sodium or a different toxicity of the anions. The aim of this study was to determine the contributions of sodium and the two anions to the different toxicities of chloride and sulfate salinity. The effects of the different salts on physiological parameters, mineral nutrient composition and expression of genes of sulfate transport and assimilation were studied. METHODS: Seedlings of Brassica rapa L. have been exposed to NaCl, Na2SO4, KCl and K2SO4 to assess the potential synergistic effect of the anions with the toxic cation sodium, as well as their separate toxicities if accompanied by the non-toxic cation potassium. Biomass production, stomatal resistance and Fv/fm were measured to determine differences in ionic and osmotic stress caused by the salts. Anion content (HPLC), mineral nutrient composition (ICP-AES) and gene expression of sulfate transporters and sulfur assimilatory enzymes (real-time qPCR) were analyzed. RESULTS: Na2SO4 impeded growth to a higher extent than NaCl and was the only salt to decrease Fv/fm. K2SO4 reduced plant growth more than NaCl. Analysis of mineral nutrient contents of plant tissue revealed that differences in sodium accumulation could not explain the increased toxicity of sulfate over chloride salts. Shoot contents of calcium, manganese and phosphorus were decreased more strongly by exposure to Na2SO4 than by NaCl. The expression levels of genes encoding proteins for sulfate transport and assimilation were differently affected by the different salts. While gene expression of primary sulfate uptake at roots was down-regulated upon exposure to sulfate salts, presumably to prevent an excessive uptake, genes encoding for the vacuolar sulfate transporter Sultr4;1 were upregulated. Gene expression of ATP sulfurylase was hardly affected by salinity in shoot and roots, the transcript level of 5'-adenylylsulfate reductase (APR) was decreased upon exposure to sulfate salts in roots. Sulfite reductase was decreased in the shoot by all salts similarly and remained unaffected in roots. CONCLUSIONS: The higher toxicity of Na2SO4 over NaCl in B. rapa seemed to be due to an increased toxicity of sulfate over chloride, as indicated by the higher toxicity of K2SO4 over KCl. Thus, toxicity of sodium was not promoted by sulfate. The observed stronger negative effect on the tissue contents of calcium, manganese and phosphorus could contribute to the increased toxicity of sulfate over chloride. The upregulation of Sultr4;1 and 4;2 under sulfate salinity might lead to a detrimental efflux of stored sulfate from the vacuole into the cytosol and the chloroplasts. It remains unclear why expression of Sultr4;1 and 4;2 was upregulated. A possible explanation is a control of the gene expression of these transporters by the sulfate gradient across the tonoplast.

Interactions of Sulfate with Other Nutrients As Revealed by H2S Fumigation of Chinese Cabbage.

Sulfur deficiency in plants has severe impacts on both growth and nutrient composition. Fumigation with sub-lethal concentrations of H2S facilitates the supply of reduced sulfur via the leaves while sulfate is depleted from the roots. This restores growth while sulfate levels in the plant tissue remain low. In the present study this system was used to reveal interactions of sulfur with other nutrients in the plant and to ascertain whether these changes are due to the absence or presence of sulfate or rather to changes in growth and organic sulfur. There was a complex reaction of the mineral composition to sulfur deficiency, however, the changes in content of many nutrients were prevented by H2S fumigation. Under sulfur deficiency these nutrients accumulated on a fresh weight basis but were diluted on a dry weight basis, presumably due to a higher dry matter content. The pattern differed, however, between leaves and roots which led to changes in shoot to root partitioning. Only the potassium, molybdenum and zinc contents were strongly linked to the sulfate supply. Potassium was the only nutrient amongst those measured which showed a positive correlation with sulfur content in shoots, highlighting a role as a counter cation for sulfate during xylem loading and vacuolar storage in leaves. This was supported by an accumulation of potassium in roots of the sulfur-deprived plants. Molybdenum and zinc increased substantially under sulfur deficiency, which was only partly prevented by H2S fumigation. While the causes of increased molybdenum under sulfur deficiency have been previously studied, the relation between sulfate and zinc uptake needs further clarification.

Mineral composition analysis: measuring anion uptake and anion concentrations in plant tissues.

This chapter describes two basic complementary methods relevant to at least three major macronutrients in plants: NO(3)(-), SO(4)(2-), and phosphate. The first method is the simultaneous determination of tissue content of the oxyanions, NO(3)(-), SO(4)(2-), and phosphate by HPLC, and the second is the determination of tissue uptake (transport) capacity for these same oxyanions. NO(3)(-), phosphate, and SO(4)(2-) , as well as other anions including chloride, malate, and nitrite are extracted from milligram quantities of plant tissue and are separated and quantified in a single chromatographic (HPLC) run. Information on uptake (flux) of these same anions through the roots may be obtained using isotopically labeled elements, enabling transport capacity of roots and subsequent translocation to shoot tissues to be determined.

Influence of sulfur deficiency on the expression of specific sulfate transporters and the distribution of sulfur, selenium, and molybdenum in wheat.

Interactions between sulfur (S) nutritional status and sulfate transporter expression in field-grown wheat (Triticum aestivum) were investigated using Broadbalk +S and -S treatments (S fertilizer withheld) at Rothamsted, United Kingdom. In 2008, S, sulfate, selenium (Se), and molybdenum (Mo) concentrations and sulfate transporter gene expression were analyzed throughout development. Total S concentrations were lower in all tissues of -S plants, principally as a result of decreased sulfate pools. S, Se, and Mo concentrations increased in vegetative tissues until anthesis, and thereafter, with the exception of Mo, decreased until maturity. At maturity, most of the S and Se were localized in the grain, indicating efficient remobilization from vegetative tissues, whereas less Mo was remobilized. At maturity, Se and Mo were enhanced 7- and 3.7-fold, respectively, in -S compared with +S grain, while grain total S was not significantly reduced. Enhanced expression of sulfate transporters, for example Sultr1;1 and Sultr4;1, in -S plants explains the much increased accumulation of Se and Mo (7- and 3.7-fold compared with +S in grain, respectively). Sultr5;2 (mot1), thought to be involved in Mo accumulation in Arabidopsis (Arabidopsis thaliana), did not fully explain patterns of Mo distribution; it was expressed in all tissues, decreasing in leaf and increasing in roots under -S conditions, and was expressed in florets at anthesis but not in grain at any other time. In conclusion, S fertilizer application has a marked impact on Mo and Se distribution and accumulation, which is at least partially a result of altered gene expression of the sulfate transporter family.

The sulfate transporter family in wheat: tissue-specific gene expression in relation to nutrition.

Sulfate uptake and distribution in plants are managed by the differential expression of a family of transporters, developmentally, spatially, and in response to sulfur nutrition. Elucidation of the signaling pathways involved requires a knowledge of the component parts and their interactions. Here, the expression patterns of the full complement of sulfate transporters in wheat, as influenced by development and sulfur nutrition, are described. The 10 wheat sulfate transporters characterized here are compared to the gene families for both rice and Brachypodium, for whom full genome information is available. Expression is reported in young seedlings with a focus on roles in uptake from nutrient solution and differential expression in relation to sulfate deprivation. In addition, patterns of expression in all organs at the grain filling stage are reported and indicate differential responses to nutritional signals of the individual transporters in specific tissues and an overall coordination of uptake, storage, and remobilization to deliver sulfur to the developing grain.

Copper exposure interferes with the regulation of the uptake, distribution and metabolism of sulfate in Chinese cabbage.

Exposure of Chinese cabbage (Brassica pekinensis) to enhanced Cu(2+) concentrations (1-10 microM) resulted in leaf chlorosis, a loss of photosynthetic capacity and lower biomass production at > or = 5 microM. The decrease in pigment content was likely not the consequence of degradation, but due to hindered chloroplast development upon Cu exposure. The Cu content of the root increased with the Cu(2+) concentration (up to 40-fold), though only a minor proportion (4%) was transferred to the shoot. The nitrate uptake by the root was substantially reduced at > or = 5 microM Cu(2+). The nitrogen content of the root was affected little at lower Cu(2+) levels, whereas that in the shoot was decreased at > or = 5 microM Cu(2+). Cu affected the uptake, distribution and metabolism of sulfate in Chinese cabbage. The total sulfur content of the shoot was increased at > or = 2 microM Cu(2+), which could be attributed mainly to an increase in sulfate content. Moreover, there was a strong increase in water-soluble non-protein thiol content in the root and, to a lesser extent, in the shoot at > or = 1 microM, which could only partially be ascribed to a Cu-induced enhancement of the phytochelatin content. The nitrate uptake by the root was substantially reduced at > or = 5 microM Cu(2+), coinciding with a decrease in biomass production. However, the activity of the sulfate transporters in the root was slightly enhanced at 2 and 5 microM Cu(2+), accompanied by enhanced expression of the Group 1 high affinity transporter Sultr1;2, and the Group 4 transporters Sultr4;1 and Sultr4;2. In the shoot, there was an induction of expression of Sultr4;2 at 5 and 10 microM Cu(2+). The expression of APS reductase was affected little in the root and shoot up to 10 microM Cu(2+). The upregulation of the sulfate transporters may be due not only to greater sulfur demand at higher Cu levels, but also the consequence of interference by Cu with the signal transduction pathway regulating the expression and activity of the sulfate transporters.

A sulphur deficiency-induced gene, sdi1, involved in the utilization of stored sulphate pools under sulphur-limiting conditions has potential as a diagnostic indicator of sulphur nutritional status.

A sulphate deficiency-induced gene, sdi1, has been identified by cDNA-amplified fragment length polymorphism (AFLP) analysis utilizing field-grown, nutrient-deficient wheat (Triticum aestivum var. Hereward). The expression of sdi1 was specifically induced in leaf and root tissues in response to sulphate deficiency, but was not induced by nitrogen, phosphorus, potassium or magnesium deficiency. Expression was also shown to increase in plant tissues as the external sulphate concentration in hydroponically grown plants was reduced from 1.0 to 0.0 mm. On this basis, sdi1 gene expression has potential as a sensitive indicator of sulphur nutritional status in wheat. Genome-walking techniques were used to clone the 2.7-kb region upstream of sdi1 from genomic DNA, revealing several cis-element motifs previously identified as being associated with sulphur responses in plants. The Arabidopsis thaliana gene most highly homologous to sdi1 is At5g48850, which was also demonstrated to be induced by sulphur deficiency, an observation confirmed by the analysis of microarray data available in the public domain. The expression of Atsdi1 was induced more rapidly than previously characterized sulphur-responsive genes in the period immediately following the transfer of plants to sulphur-deficient medium. Atsdi1 T-DNA 'knockout' mutants were shown to maintain higher tissue sulphate concentrations than wild-type plants under sulphur-limiting conditions, indicating a role in the utilization of stored sulphate under sulphur-deficient conditions. The structural features of the sdi1 gene and its application in the genetic determination of the sulphur nutritional status of wheat crops are discussed.

Co-ordinated expression of amino acid metabolism in response to N and S deficiency during wheat grain filling.

Increasing demands for productivity together with environmental concerns about fertilizer use dictate that the future sustainability of agricultural systems will depend on improving fertilizer use efficiency. Characterization of the biological processes responsible for efficient fertilizer use will provide tools for crop improvement under reduced inputs. Transcriptomic and metabolomic approaches were used to study the impact of nitrogen (N) and sulphur (S) deficiency on N and S remobilization from senescing canopy tissues during grain filling in winter wheat (Triticum aestivum). Canopy tissue N was remobilized effectively to the grain after anthesis. S was less readily remobilized. Nuclear magnetic resonance (NMR) metabolite profiling revealed significant effects of suboptimal N or S supply in leaves but not in developing grain. Analysis of amino acid pools in the grain and leaves revealed a strategy whereby amino acid biosynthesis switches to the production of glutamine during grain filling. Glutamine accumulated in the first 7 d of grain development, prior to conversion to other amino acids and protein in the subsequent 21 d. Transcriptome analysis indicated that a down-regulation of the terminal steps in many amino acid biosynthetic pathways occurs to control pools of amino acids during leaf senescence. Grain N and S contents increased in parallel after anthesis and were not significantly affected by S deficiency, despite a suboptimal N:S ratio at final harvest. N deficiency resulted in much slower accumulation of grain N and S and lower final concentrations, indicating that vegetative tissue N has a greater control of the timing and extent of nutrient remobilization than S.

O-acetylserine and the regulation of expression of genes encoding components for sulfate uptake and assimilation in potato.

cDNAs encoding a high-affinity sulfate transporter and an adenosine 5'-phosphosulfate reductase from potato (Solanum tuberosum L. cv Desiree) have been cloned and used to examine the hypothesis that sulfate uptake and assimilation is transcriptionally regulated and that this is mediated via intracellular O-acetylserine (OAS) pools. Gas chromotography coupled to mass spectrometry was used to quantify OAS and its derivative, N-acetylserine. Treatment with external OAS increased sulfate transporter and adenosine 5'-phosphosulfate reductase gene expression consistent with a model of transcriptional induction by OAS. To investigate this further, the Escherichia coli gene cysE (serine acetyltransferase EC 2.3.1.30), which synthesizes OAS, has been expressed in potato to modify internal metabolite pools. Transgenic lines, with increased cysteine and glutathione pools, particularly in the leaves, had increased sulfate transporter expression in the roots. However, the small increases in the OAS pools were not supportive of the hypothesis that this molecule is the signal of sulfur (S) nutritional status. In addition, although during S starvation the content of S-containing compounds decreased (consistent with derepression as a mechanism of regulation), OAS pools increased only following extended starvation, probably as a consequence of the S starvation. Taken together, expression of these genes may be induced by a demand-driven model, via a signal from the shoots, which is not OAS. Rather, the signal may be the depletion of intermediates of the sulfate assimilation pathway, such as sulfide, in the roots. Finally, sulfate transporter activity did not increase in parallel with transcript and protein abundance, indicating additional posttranslational regulatory mechanisms.