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.

Phylogeny and gene expression of the complete NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY in Triticum aestivum.

NPF genes encode membrane transporters involved in the transport of a large variety of substrates including nitrate and peptides. The NPF gene family has been described for many plants, but the whole NPF gene family for wheat has not been completely identified. The release of the wheat reference genome has enabled the identification of the entire wheat NPF gene family. A systematic analysis of the whole wheat NPF gene family was performed, including responses of specific gene expression to development and nitrogen supply. A total of 331 NPF genes (113 homoeologous groups) have been identified in wheat. The chromosomal location of the NPF genes is unevenly distributed, with predominant occurrence in the long arms of the chromosomes. The phylogenetic analysis indicated that wheat NPF genes are closely clustered with Arabidopsis, Brachypodium, and rice orthologues, and subdivided into eight subfamilies. The expression profiles of wheat NPF genes were examined using RNA-seq data, and a subset of 44 NPF genes (homoeologous groups) with contrasting expression responses to nitrogen and/or development in different tissues were identified. The systematic identification of gene composition, chromosomal locations, evolutionary relationships, and expression profiles contributes to a better understanding of the roles of the wheat NPF genes and lays the foundation for further functional analysis in wheat.

Functional QTL mapping and genomic prediction of canopy height in wheat measured using a robotic field phenotyping platform.

Genetic studies increasingly rely on high-throughput phenotyping, but the resulting longitudinal data pose analytical challenges. We used canopy height data from an automated field phenotyping platform to compare several approaches to scanning for quantitative trait loci (QTLs) and performing genomic prediction in a wheat recombinant inbred line mapping population based on up to 26 sampled time points (TPs). We detected four persistent QTLs (i.e. expressed for most of the growing season), with both empirical and simulation analyses demonstrating superior statistical power of detecting such QTLs through functional mapping approaches compared with conventional individual TP analyses. In contrast, even very simple individual TP approaches (e.g. interval mapping) had superior detection power for transient QTLs (i.e. expressed during very short periods). Using spline-smoothed phenotypic data resulted in improved genomic predictive abilities (5-8% higher than individual TP prediction), while the effect of including significant QTLs in prediction models was relatively minor (<1-4% improvement). Finally, although QTL detection power and predictive ability generally increased with the number of TPs analysed, gains beyond five or 10 TPs chosen based on phenological information had little practical significance. These results will inform the development of an integrated, semi-automated analytical pipeline, which will be more broadly applicable to similar data sets in wheat and other crops.

A breeding strategy targeting the secondary gene pool of bread wheat: introgression from a synthetic hexaploid wheat.

KEY MESSAGE: Introgressing one-eighth of synthetic hexaploid wheat genome through a double top-cross plus a two-phase selection is an effective strategy to develop high-yielding wheat varieties. The continued expansion of the world population and the likely onset of climate change combine to form a major crop breeding challenge. Genetic advances in most crop species to date have largely relied on recombination and reassortment within a relatively narrow gene pool. Here, we demonstrate an efficient wheat breeding strategy for improving yield potentials by introgression of multiple genomic regions of de novo synthesized wheat. The method relies on an initial double top-cross (DTC), in which one parent is synthetic hexaploid wheat (SHW), followed by a two-phase selection procedure. A genotypic analysis of three varieties (Shumai 580, Shumai 969 and Shumai 830) released from this program showed that each harbors a unique set of genomic regions inherited from the SHW parent. The first two varieties were generated from very small populations, whereas the third used a more conventional scale of selection since one of bread wheat parents was a pre-breeding material. The three varieties had remarkably enhanced yield potential compared to those developed by conventional breeding. A widely accepted consensus among crop breeders holds that introducing unadapted germplasm, such as landraces, as parents into a breeding program is a risky proposition, since the size of the breeding population required to overcome linkage drag becomes too daunting. However, the success of the proposed DTC strategy has demonstrated that novel variation harbored by SHWs can be accessed in a straightforward, effective manner. The strategy is in principle generalizable to any allopolyploid crop species where the identity of the progenitor species is known.

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.

The role of ZIP transporters and group F bZIP transcription factors in the Zn-deficiency response of wheat (Triticum aestivum).

Understanding the molecular basis of zinc (Zn) uptake and transport in staple cereal crops is critical for improving both Zn content and tolerance to low-Zn soils. This study demonstrates the importance of group F bZIP transcription factors and ZIP transporters in responses to Zn deficiency in wheat (Triticum aestivum). Seven group F TabZIP genes and 14 ZIPs with homeologs were identified in hexaploid wheat. Promoter analysis revealed the presence of Zn-deficiency-response elements (ZDREs) in a number of the ZIPs. Functional complementation of the zrt1/zrt2 yeast mutant by TaZIP3, -6, -7, -9 and -13 supported an ability to transport Zn. Group F TabZIPs contain the group-defining cysteine-histidine-rich motifs, which are the predicted binding site of Zn2+ in the Zn-deficiency response. Conservation of these motifs varied between the TabZIPs suggesting that individual TabZIPs may have specific roles in the wheat Zn-homeostatic network. Increased expression in response to low Zn levels was observed for several of the wheat ZIPs and bZIPs; this varied temporally and spatially suggesting specific functions in the response mechanism. The ability of the group F TabZIPs to bind to specific ZDREs in the promoters of TaZIPs indicates a conserved mechanism in monocots and dicots in responding to Zn deficiency. In support of this, TabZIPF1-7DL and TabZIPF4-7AL afforded a strong level of rescue to the Arabidopsis hypersensitive bzip19 bzip23 double mutant under Zn deficiency. These results provide a greater understanding of Zn-homeostatic mechanisms in wheat, demonstrating an expanded repertoire of group F bZIP transcription factors, adding to the complexity of Zn homeostasis.

System analysis of metabolism and the transcriptome in Arabidopsis thaliana roots reveals differential co-regulation upon iron, sulfur and potassium deficiency.

Deprivation of mineral nutrients causes significant retardation of plant growth. This retardation is associated with nutrient-specific and general stress-induced transcriptional responses. In this study, we adjusted the external supply of iron, potassium and sulfur to cause the same retardation of shoot growth. Nevertheless, limitation by individual nutrients resulted in specific morphological adaptations and distinct shifts within the root metabolite fingerprint. The metabolic shifts affected key metabolites of primary metabolism and the stress-related phytohormones, jasmonic, salicylic and abscisic acid. These phytohormone signatures contributed to specific nutrient deficiency-induced transcriptional regulation. Limitation by the micronutrient iron caused the strongest regulation and affected 18% of the root transcriptome. Only 130 genes were regulated by all nutrients. Specific co-regulation between the iron and sulfur metabolic routes upon iron or sulfur deficiency was observed. Interestingly, iron deficiency caused regulation of a different set of genes of the sulfur assimilation pathway compared with sulfur deficiency itself, which demonstrates the presence of specific signal-transduction systems for the cross-regulation of the pathways. Combined iron and sulfur starvation experiments demonstrated that a requirement for a specific nutrient can overrule this cross-regulation. The comparative metabolomics and transcriptomics approach used dissected general stress from nutrient-specific regulation in roots of Arabidopsis.

Iron deprivation results in a rapid but not sustained increase of the expression of genes involved in iron metabolism and sulfate uptake in tomato (Solanum lycopersicum L.) seedlings.

Characterization of the relationship between sulfur and iron in both Strategy I and Strategy II plants, has proven that low sulfur availability often limits plant capability to cope with iron shortage. Here it was investigated whether the adaptation to iron deficiency in tomato (Solanum lycopersicum L.) plants was associated with an increased root sulfate uptake and translocation capacity, and modified dynamics of total sulfur and thiols accumulation between roots and shoots. Most of the tomato sulfate transporter genes belonging to Groups 1, 2, and 4 were significantly upregulated in iron-deficient roots, as it commonly occurs under S-deficient conditions. The upregulation of the two high affinity sulfate transporter genes, SlST1.1 and SlST1.2, by iron deprivation clearly suggests an increased root capability to take up sulfate. Furthermore, the upregulation of the two low affinity sulfate transporter genes SlST2.1 and SlST4.1 in iron-deficient roots, accompanied by a substantial accumulation of total sulfur and thiols in shoots of iron-starved plants, likely supports an increased root-to-shoot translocation of sulfate. Results suggest that tomato plants exposed to iron-deficiency are able to change sulfur metabolic balance mimicking sulfur starvation responses to meet the increased demand for methionine and its derivatives, allowing them to cope with this stress.

Natural variation in grain composition of wheat and related cereals.

The wheat grain comprises three groups of major components, starch, protein, and cell wall polysaccharides (dietary fiber), and a range of minor components that may confer benefits to human health. Detailed analyses of dietary fiber and other bioactive components were carried out under the EU FP6 HEALTHGRAIN program on 150 bread wheat lines grown on a single site, 50 lines of other wheat species and other cereals grown on the same site, and 23-26 bread wheat lines grown in six environments. Principal component analysis allowed the 150 bread wheat lines to be classified on the basis of differences in their contents of bioactive components and wheat species (bread, durum, spelt, emmer, and einkorn wheats) to be clearly separated from related cereals (barley, rye, and oats). Such multivariate analyses could be used to define substantial equivalence when novel (including transgenic) cereals are considered.

Comparative spatiotemporal analysis of root aerenchyma formation processes in maize due to sulphate, nitrate or phosphate deprivation.

Nitrate (N), phosphate (P) or sulphate (S) deprivation causes aerenchyma formation in maize (Zea mays L.) nodal roots. The exact mechanisms that trigger the formation of aerenchyma under these circumstances are unclear. We have compared aerenchyma distribution across the nodal roots of first whorl (just emerging in 10-day-old seedlings), which were subject to S, N or P deprivation over a period of 10 days in connection with oxygen consumption, ATP concentration, cellulase and polygalacturonase activity in the whole root. The effect of deprivation on aerenchyma formation was examined using light and electron microscopy, along with in situ detection of calcium and of reactive oxygen species (ROS) by fluorescence microscopy. Aerenchyma was not found in the root base regardless of the deprivation. Programmed cell death (PCD) was observed near the root tip, either within the first two days (-N) or a few days later (-S, -P) of the treatment. Roots at day 6 under all three nutrient-deprived conditions showed signs of PCD 1 cm behind the cap, whereas only N-deprived root cells 0.5 cm behind the cap showed severe ultrastructural alterations, due to advanced PCD. The lower ATP concentration and the higher oxygen consumptions observed at day 2 in N-, P- and S-deprived roots compared to the control indicated that PCD may be triggered by perturbations in energy status of the root. The peaks of cellulase activity located between days 3 (-N) and 6 (-P), along with the respective alterations in polygalacturonase activity, indicated a coordination which preceded aerenchyma formation. ROS and calcium seemed to contribute to PCD initiation, with ROS possessing dual roles as signals and eliminators. All the examined parameters presented both common features and characteristic variations among the deprivations.

Regulation of sulfate uptake, expression of the sulfate transporters Sultr1;1 and Sultr1;2, and APS reductase in Chinese cabbage (Brassica pekinensis) as affected by atmospheric H2S nutrition and sulfate deprivation.

The activity and expression of sulfate transporters and adenosine 5'-phosphosulfate (APS) reductase (APR) in plants are modulated by the plant sulfur status and the demand for growth. To elucidate regulatory mechanisms in Chinese cabbage [Brassica pekinensis (Lour.) Rupr.], the interactions between atmospheric H2S and sulfate nutrition and the impact on the activity and expression of the Group 1 sulfate transporters and APR were studied. At an ample sulfate supply, H2S exposure of Chinese cabbage resulted in a partial decrease of the sulfate uptake capacity, and at concentrations ≥0.25 µL L-1 a decreased expression of Sultr1;2 in the root and APR in the root and shoot. Upon sulfate deprivation there was a more than 3-fold increase in the sulfate uptake capacity of the root, accompanied by an induced expression of Sultr1;1 and an enhanced expression of Sultr1;2 in the root, along with an induction of Sultr1;2 in the shoot. The enhanced sulfate uptake capacity, the expression of the sulfate transporters in the root and the altered shoot-to-root partitioning appearing during sulfate deprivation were not alleviated upon H2S exposure and not rapidly affected by sulfate re-supply. Expression of APR was strongly enhanced in the root and shoot of sulfate-deprived plants and decreased again upon H2S exposure and sulfate re-supply. The significance of shoot-to-root interaction and sulfate and thiols as regulating signals in the activity and expression of Sultr1;1 and 1;2 is evaluated.

Managing sulphur metabolism in plants.

Resolution and analysis of genes encoding components of the pathways of primary sulphur assimilation have provided the potential to elucidate how sulphur is managed by plants. Individual roles for members of gene families and regulatory mechanisms operating at gene, cellular and whole plant levels have been recognized. Sulphur is taken up and transported around the plant principally as sulphate, catalysed for the most part by a single gene family of highly regulated transporters. Additional regulation occurs in the pathway of reduction of sulphate to sulphide and its incorporation into cysteine, which occurs principally within the plastid. Cellular and whole-plant regulation of uptake, and the assimilatory pathway attempt to balance supply with demand for growth and include mechanisms for re-mobilization and redistribution of sulphur. Furthermore, optimization of sulphur assimilation requires coordination with carbon and nitrogen pathways, and multiple processes have been proposed to contribute to this balance. Present studies on cis and trans elements are focusing on transcriptional regulation, but this regulation still needs to be linked to apparent metabolite sensing. Whilst the components of the assimilatory pathways have been resolved after many years of controversy, uncertainties remain concerning roles of individual genes in gene families, their sub-cellular localization and their significance in balancing sulphur flux to sulphur demand of the plant for growth under variable environmental conditions.

Biofortification of UK food crops with selenium.

Se is an essential element for animals. In man low dietary Se intakes are associated with health disorders including oxidative stress-related conditions, reduced fertility and immune functions and an increased risk of cancers. Although the reference nutrient intakes for adult females and males in the UK are 60 and 75 microg Se/d respectively, dietary Se intakes in the UK have declined from >60 microg Se/d in the 1970s to 35 microg Se/d in the 1990s, with a concomitant decline in human Se status. This decline in Se intake and status has been attributed primarily to the replacement of milling wheat having high levels of grain Se and grown on high-Se soils in North America with UK-sourced wheat having low levels of grain Se and grown on low-Se soils. An immediate solution to low dietary Se intake and status is to enrich UK-grown food crops using Se fertilisers (agronomic biofortification). Such a strategy has been adopted with success in Finland. It may also be possible to enrich food crops in the longer term by selecting or breeding crop varieties with enhanced Se-accumulation characteristics (genetic biofortification). The present paper will review the potential for biofortification of UK food crops with Se.

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.

Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants.

Sulfur is an essential macro-element in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.

Using a suppression subtractive library-based approach to identify tobacco genes regulated in response to short-term sulphur deficit.

Monitoring expression at the transcriptional level is an essential first step for the functional analysis of plant genes. Genes encoding proteins directly involved in sulphur metabolism constitute only a small fraction of all the genes affected by sulphur deficiency stress. Transcriptional responses to various periods of sulphur deprivation have been extensively studied in Arabidopsis thaliana; however, no corresponding data are available for Solanaceae sp. To address this problem, a subtractive library-based approach to search for tobacco genes regulated by a short-term sulphur starvation has been adopted. In this work, 38 genes were identified, of which 22 were regulated positively and 16 were regulated negatively. The transcript levels of the representative genes were monitored in four parts of the plants (mature and immature leaves, stems, and roots), which exhibited differential sulphur deficiency. Interestingly, some genes exhibit different regulation of expression in different parts of the plants. Database analysis allowed assignment of the potential function for many of the identified genes; however, the functions of a small number of genes strongly regulated by sulphur starvation remain unknown. The genes were grouped into nine functional categories, each including both up- and down-regulated genes. The possible links between the identified regulated genes and sulphur metabolism are considered, and compared where possible with expression patterns in Arabidopsis thaliana. Although no obvious regulatory genes were identified, the genes encoding proteins of unknown function remain as potential components of the regulatory processes.

Phylogeny and expression of paralogous and orthologous sulphate transporter genes in diploid and hexaploid wheats.

Twelve genes encoding two closely related subtypes (ST1.1a and ST1.1b) of a sulphate transporter have been identified in the diploid wheats Aegilops tauschii, Triticum urartu, and Aegilops speltoides, as well as the hexaploid Triticum aestivum. Based on phylogenetic comparisons with other plant sulphate transporters, the ST1.1a and 1.1b subtypes aligned with group 1 of the plant sulphate transporter gene family. The exon-intron structure was conserved within the ST1.1a or ST1.1b genes; however, substantial variability in intron sequences existed between the two types. The high overall sequence similarity indicated that ST1.1b represented a duplication of the ST1.1a gene, which must have occurred before the evolution of the ancestral diploid wheat progenitor. In contrast with the close relationship of the T. urartu and Ae. tauschii sequences to the corresponding A and D genome sequences of T. aestivum, the divergence between the Ae. speltoides sequences and the B genome sequences suggested that the B genome ST1.1a gene has been modified by recombination. Transcript analysis revealed predominant expression of the ST1.1a type and an influence of sulphur availability on the level of expression.

Influence of iron status on cadmium and zinc uptake by different ecotypes of the hyperaccumulator Thlaspi caerulescens.

We have previously identified an ecotype of the hyperaccumulator Thlaspi caerulescens (Ganges), which is far superior to other ecotypes (including Prayon) in Cd uptake. In this study, we investigated the effect of Fe status on the uptake of Cd and Zn in the Ganges and Prayon ecotypes, and the kinetics of Cd and Zn influx using radioisotopes. Furthermore, the T. caerulescens ZIP (Zn-regulated transporter/Fe-regulated transporter-like protein) genes TcZNT1-G and TcIRT1-G were cloned from the Ganges ecotype and their expression under Fe-sufficient and -deficient conditions was analyzed. Both short- and long-term studies revealed that Cd uptake was significantly enhanced by Fe deficiency only in the Ganges ecotype. The concentration-dependent kinetics of Cd influx showed that the V(max) of Cd was 3 times greater in Fe-deficient Ganges plants compared with Fe-sufficient plants. In Prayon, Fe deficiency did not induce a significant increase in V(max) for Cd. Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes. These results are in agreement with the gene expression study. The abundance of ZNT1-G mRNA was similar between the Fe treatments and between the two ecotypes. In contrast, abundance of the TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions. The present results indicate that the stimulatory effect of Fe deficiency on Cd uptake in Ganges may be related to an up-regulation in the expression of genes encoding for Fe(2+) uptake, possibly TcIRT1-G.

Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors.

The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.

Elemental sulfur and thiol accumulation in tomato and defense against a fungal vascular pathogen.

The occurrence of fungicidal, elemental S is well documented in certain specialized prokaryotes, but has rarely been detected in eukaryotes. Elemental S was first identified in this laboratory as a novel phytoalexin in the xylem of resistant genotypes of Theobroma cacao, after infection by the vascular, fungal pathogen Verticillium dahliae. In the current work, this phenomenon is demonstrated in a resistant line of tomato, Lycopersicon esculentum, in response to V. dahliae. A novel gas chromatography-mass spectroscopy method using isotope dilution analysis with 34S internal standard was developed to identify unambiguously and quantify 32S in samples of excised xylem. Accumulation of S in vascular tissue was more rapid and much greater in the disease-resistant than in the disease-susceptible line. Levels of S detected in the resistant variety (approximately 10 microg g-1 fresh weight excised xylem) were fungitoxic to V. dahliae (spore germination was inhibited >90% at approximately 3 microg mL-1). Scanning electron microscopy-energy dispersive x-ray microanalysis confirmed accumulation of S in vascular but not in pith cells and in greater amounts and frequency in the Verticillium spp.-resistant genotype. More intensive localizations of S were occasionally detected in xylem parenchyma cells, vessel walls, vascular gels, and tyloses, structures in potential contact with and linked with defense to V. dahliae. Transient increases in concentrations of sulfate, glutathione, and Cys of vascular tissues from resistant but not susceptible lines after infection may indicate a perturbation of S metabolism induced by elemental S formation; this is discussed in terms of possible S biogenesis.