Supply of sulphur to S-deficient young barley seedlings restores their capability to cope with iron shortage

The effect of the S nutritional status on a plant's capability to cope with Fe shortage was studied in solution cultivation experiments in barley (Hordeum vulgare L. cv. Europa). Barley is a Strategy II plant and responds to Fe deficiency by secretion of chelating compounds, phytosiderophores (PS). All PS are derived from nicotianamine whose precursor is methionine. This suggests that a long-term supply of an inadequate amount of S could reduce a plant's capability to respond to Fe deficiency by limiting the rate of PS biosynthesis. The responses of barley (Hordeum vulgare L. cv. Europa) plants grown for 12 d on Fe-free nutrient solutions (NS) containing 0 or 1.2 mM SO42-, was examined after 24 h or 48 h from transfer to NS containing 1.2 mM SO42-. After the supply of S was restored to S-deprived plants, an increase in PS release in root exudates was evident after 24 h of growth in S-sufficient NS and the increment reached values up to 4-fold higher than the control 48 h after S resupply. When S was supplied to S-deficient plants, leaf ATPS (EC 2.7.7.4) and OASTL (EC 4.2.99.8) activities exhibited a progressive recovery. Furthermore, root HvST1 transcript abundance remained high for 48 h following S resupply and a significant increase in the level of root HvYS1 transcripts was also found after only 24 h of S resupply. Data support the idea that the extent to which the plant is able to cope with Fe starvation is strongly associated with its S nutritional status. In particular, our results are indicative that barley plants fully recover their capability to cope with Fe shortage after the supply of S is restored to S-deficient plants

Photosynthetic Adaptation to Length of Day Is Dependent on S-Sulfocysteine Synthase Activity in the Thylakoid Lumen

Abstract Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants under short-day growth conditions (SD) and long-day growth conditions (LD). Under LD, the photosynthetic characterization, which was based on substomatal CO2 concentrations and CO2 concentration in the chloroplast curves, revealed significant reductions in most of the photosynthetic parameters for cs26, which were unchanged under SD. These parameters included net CO2 assimilation rate, mesophyll conductance, and mitochondrial respiration at darkness. The analysis also showed that cs26 under LD required more absorbed quanta per driven electron flux and fixed CO2. The nonphotochemical quenching values suggested that in cs26 plants, the excess electrons that are not used in photochemical reactions may form reactive oxygen species. A photoinhibitory effect was confirmed by the background fluorescence signal values under LD and SD, which were higher in young leaves compared with mature ones under SD. To hypothesize the role of CS26 in relation to the photosynthetic machinery, we addressed its location inside of the chloroplast. The activity determination and localization analyses that were performed using immunoblotting indicated the presence of an active CS26 enzyme exclusively in the thylakoid lumen. This finding was reinforced by the observation of marked alterations in many lumenal proteins in the cs26 mutant compared with the wild type.</jats:p

Investigation into the relationship between ethylene and sulfur assimilation in Arabidopsis thaliana and onion (Allium cepa L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (with Honours) in Biochemistry at Massey University

The phytohormone ethylene (C2H4) mediates the adaptive responses of plants to various nutrient deficiencies including iron (Fe)-deficiency, phosphorus (P)-deficiency and potassium (K)-deficiency. However, evidence for the involvement this hormone in the sulfur (S) deficiency response is limited to date. In this study, the effect of C2H4 treatment on the accumulation of the S-assimilation enzymes ATP sulfurylase (ATPS). adenosine-5 -phosphosulfate-reductase (APR), O-acetylserine-(thiol)-lyase (OASTL) and sulfite reductase (SiR) was examined in A. thaliana and onion (A. cepa). To complement this, the effect of short-term S-depletion on the expression of the 12-member gene family of the C2H4 biosynthetic enzyme, l-amino-cyclopropane-l-carboxylic acid (ACC) synthase (ACS) from A. thaliana, designated AtACS1-12, was also examined. Western analyses were used to show that plants of A. thaliana pre-treated with the C2H4-signalling inhibitor 1-MCP, had elevated levels of ATPS, APR and OASTL protein in leaf tissue at all time points examined, suggesting that C2H4 has an inhibitory effect on the accumulation of these enzymes. However, SiR appeared to be under dual regulation by C2H4: under S-sufficient conditions C2H4 appears to prevent the unnecessary accumulation of SiR and conversely promote the fast accumulation of SiR under S-depleted conditions. The changes in AtACS1-12 expression in the root and leaf tissues of S-sufficient and S-depleted plants of A. thaliana were examined by RT-PCR using gene-specific, exon-spanning primers. The expression patterns of AtACS2, AtACS6 and AtACS7 were comparable regardless of S availability and may therefore be housekeeping genes. In contrast, the expression of AtACS5 in leaf, and AtACS8 and AtACS9 in roots was repressed under S-depleted conditions, although the mechanism of this repression cannot be elucidated from this study. The protein products of these closely-related genes are believed to be phosphorylated and stabilised by a CDPK whose activity may be compromised by S-depletion. The inhibition of AtACS5, AtACS8 and AtACS9 expression, and the decrease in AtACS5, AtACS8 and AtACS9 accumulation, and hence less C2H4 production, may be part of the plant adaptive response to S-depletion, as the C2H4 -mediated repression of root growth is alleviated to allow the plant to better seek out the lacking nutrient. The expression of the MPK-stabilised genes AtACS2 and AtACS6 appeared to be similar regardless of S availability, although this may merely be a consequence of the scoring method used in this study, which cannot determine whether there was any difference in the level of expression of these genes. The expression of AtACS10 and AtACS12 was repressed in S-deficient plants. Although both AtACS10 and AtACS12 isozymes posses the hallmark seven conserved regions found in the ACSes of other plant species, they are also phylogenetically related to alanine and aspartate aminotransferases, and are known to encode aspartate (AtACS10) and aromatic amino acid transaminases (AtACS12). Therefore, the apparent downregulation of these genes suggests that the downregulation of amino acid metabolism may be part of the plant adaptive response to S-depletion. The downregulation of several AtACS genes, and therefore possibly also C2H4 biosynthesis, in S-deficient plants was accompanied by an accumulation of APR protein. The increase in APR protein that also occurred in 1-MCP-treated plants indicates that C2H4 may be involved in the plant response to S-depletion, because in both cases the upregulation of the S-assimilation pathway, as manifested by the accumulation of APR protein, occurred when C2H4 biosynthesis and signalling was repressed. However, the possible role of other phytohormoes in the plant response to S-depletion cannot be excluded, as there is evidence for crosstalk between the C2H4 signalling pathway and those of auxin, abscisic acid (ABA), cytokinins and jasmonic acid (JA). Furthermore, because C2H4 has been implicated in the response of various plants to Fe-deficiency, P-deficiency, and K-deficiency, in addition to S-deficiency, it may be a regulator of the plant adaptive response to nutrient stresses in general

Affinity purification of O-acetylserine(thiol)lyase from chlorella sorokinianaby recombinant proteins from Arabidopsis thaliana

In the unicellular green alga Chlorella sorokiniana (211/8 k), the protein O-acetylserine(thiol)lyase (OASTL), representing the key-enzyme in the biosynthetic cysteine pathway, was isolated and purified to apparent homogeneity. The purification was carried out in cells grown in the presence of all nutrients or in sulphate (S) deprived cells. After 24 h of S-starvation, a 17-fold increase in the specific activity of OASTL was measured. In order to enable the identification of OASTL proteins from non-model organisms such as C. sorokiniana, the recombinant his-tagged SAT5 protein from Arabidopsis thaliana was immobilized by metal chelate chromatography. OASTL proteins from C. sorokiniana were affinity purified in one step and activities were enhanced 29- and 41-fold, from S-sufficient and S-starved (24 h) cells, respectively. The successful application of SAT/OASTL interaction for purification confirms for the first time the existence of the cysteine synthase complexes in microalgae. The purified proteins have apparent molecular masses between 32–34 kDa and are thus slightly larger compared to those found in other vascular plants. The enhanced OASTL activity in S-starved cells can be attributed to increased amounts of plastidic and the emergence of cytosolic OASTL isoforms. The results provide proof-of-concept for the biochemical analysis of the cysteine synthase complex in diverse microalgal species

Leaves play a central role in the adaptation of nitrogen and sulfur metabolism to ammonium nutrition in oilseed rape (Brassica napus)

Background: The coordination between nitrogen (N) and sulfur (S) assimilation is required to suitably provide plants with organic compounds essential for their development and growth. The N source induces the adaptation of many metabolic processes in plants; however, there is scarce information about the influence that it may exert on the functioning of S metabolism. The aim of this work was to provide an overview of N and S metabolism in oilseed rape (Brassica napus) when exposed to different N sources. To do so, plants were grown in hydroponic conditions with nitrate or ammonium as N source at two concentrations (0.5 and 1 mM).Results: Metabolic changes mainly occurred in leaves, where ammonium caused the up-regulation of enzymes involved in the primary assimilation of N and a general increase in the concentration of N-compounds (NH4 +, amino acids and proteins). Similarly, the activity of key enzymes of primary S assimilation and the content of S-compounds (glutathione and glucosinolates) were also higher in leaves of ammonium-fed plants. Interestingly, sulfate level was lower in leaves of ammonium-fed plants, which was accompanied by the down-regulation of SULTR1 transporters gene expression. Conclusions: The results highlight the impact of the N source on different steps of N and S metabolism in oilseed rape, notably inducing N and S assimilation in leaves, and put forward the potential of N source management to modulate the synthesis of compounds with biotechnological interest, such as glucosinolates

Expression analysis of ROS scavenging enzyme encoding genes in rubber tree infected by Microcyclus ulei

South American Leaf Blight (SALB), caused by the ascomycete Microcyclus ulei is responsible for the low productivity of rubber tree in Latin America and represents an important threat for rubber plantations in Asia and Africa, where production is derived from highly susceptible clones. In order to identify candidate genes involved in the resistance, an unigene set of 1623 ESTs were obtained from five SSH libraries. Arrays analyses were performed to identify genes differentially expressed in resistant and susceptible cultivars. Fourteen genes were selected for further gene expression analysis in three non-inoculated and inoculated genotypes: FX2784 (HR), MDF180 (partially resistant) and PB314 (susceptible) from 24 to 216 hpi. These genes are involved in ROS-scavenging systems (HbCuZnSOD, HbMnSOD, HbCat, HbAPX1, HbAPX2, HbMDHAR, HbGCL1, HbGCL2, HbOASTL) and leaf senescence (HbCASP, HbPCYST, HbWRKY2, HbPLY, HbKAT2). Six genes were differentially expressed in the different genotypes and/or during the infection. The average basal levels of HbCat, HbGCL1 and 2 expressions in MDF180 were respectively 55-fold and 2- to 3-fold lower than in the other genotypes. HbPCYST was both up-regulated 216 hpi in inoculated and non-inoculated MDF180 tissues. HbWRKY2 was up-regulated in inoculated leaves of MDF180 at 216 hpi while in PB314, the up-regulation started at 96 hpi with a peak of expression at 168 hpi. HbPLY was up-regulated in PB314 inoculated tissue at 168 hpi while in MDF180 and FX2784 a down-regulation was observed in inoculated tissues. The results were compared with histological observations. These genes can be considered as reporter genes of the oxidative status and senescence of the leaf during the infection. (Résumé d'auteur

Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana

O-acetylserine(thiol)lyase (OASTL) is an enzyme catalysing the reaction of inorganic sulphide with O-acetylserine to form the S-containing amino acid L-cysteine. Here we describe an improved protocol to evaluate the activity of this enzyme from the microalga Chlorella sorokiniana. It is a colorimetric assay based on the reaction between cysteine, the product of OASTL activity, and ninhydrin reagent, which forms a thiazolidine (Thz)

Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana

In non-cyanogenic species, the main source of cyanide derives from ethylene and camalexin biosyntheses. In mitochondria, cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolised by the β-Cyanoalanine synthase CYS-C1, catalysing the conversion of cysteine and cyanide to hydrogen sulfide and β- cyanoalanine. The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine(thiol)lyase mitochondrial isoform, OAS-C, which catalyses the incorporation of sulfide to O-acetylserine to produce cysteine, thus generating a cyclic pathway in the mitochondria. The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme, showing defects in root hair formation. Genetic complementation with the OAS-C gene rescues the impairment of root hair elongation restoring the wild type phenotype. The mitochondria compromise their capacity to proper detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant. Consequently, we observe an accumulation of sulfide and cyanide and of the alternative oxidase, which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules. Our results allow us to suggest that the significance of OAS-C is related with its role in the proper sulfide and cyanide detoxification in mitochondria.Ministerio de Ciencia e Innovación BIO2010-15201Junta de Andalucía BIO–27

Geminivirus C2 protein represses genes involved in sulphur assimilation and this effect can be counteracted by jasmonate treatment

Geminiviruses are plant viruses that infect a broad range of crops and cause extensive losses worldwide, having an important economic impact. C2, a multifunctional pathogenicity factor encoded by geminiviruses, has been recently shown to suppress the responses to jasmonates in the host plant, which might at least partially explain its well-established role in pathogenicity. Sulphur is one of the essential macro-elements for plant life, and is considered to have a role in plant defence, in a phenomenon named sulphur-induced resistance (SIR) or sulphur-enhanced defence (SED). In this work, we show that geminivirus C2 protein represses the expression of genes involved in the sulphur assimilation pathway in Arabidopsis, but, interestingly, this effect can be neutralized by exogenous jasmonate treatment. These preliminary results may raise the idea that geminiviruses might be affecting sulphur metabolism, and maybe counteracting SIR/SED, through the manipulation of the jasmonate signalling pathway, which would define a novel strategy in plant-virus interactions and may unveil SIR/SED as an important player in the plant defence against viruses.Ministerio de Ciencia y Innovación/FEDER AGL2007-66062-C02-02/AGR AGL2010-22287-CO2European Regional Development Fund (ERDF) BIO2010-15201Junta de Andalucía BIO­27