O-acetylserine Thiol Lyase Research Articles

Characterization of the O-acetylserine(thiol)lyase gene family in Solanum lycopersicum L.

This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L. Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a β-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.

Impact of overexpression of cytosolic isoform of O-acetylserine sulfhydrylase on soybean nodulation and nodule metabolome

Nitrogen-fixing nodules, which are also major sites of sulfur assimilation, contribute significantly to the sulfur needs of whole soybean plants. Nodules are the predominant sites for cysteine accumulation and the activity of O-acetylserine(thiol)lyase (OASS) is central to the sulfur assimilation process in plants. Here, we examined the impact of overexpressing OASS on soybean nodulation and nodule metabolome. Overexpression of OASS did not affect the nodule number, but negatively impacted plant growth. HPLC measurement of antioxidant metabolites demonstrated that levels of cysteine, glutathione, and homoglutathione nearly doubled in OASS overexpressing nodules when compared to control nodules. Metabolite profiling by LC-MS and GC-MS demonstrated that several metabolites related to serine, aspartate, glutamate, and branched-chain amino acid pathways were significantly elevated in OASS overexpressing nodules. Striking differences were also observed in the flavonoid levels between the OASS overexpressing and control soybean nodules. Our results suggest that OASS overexpressing plants compensate for the increase in carbon requirement for sulfur assimilation by reducing the biosynthesis of some amino acids, and by replenishing the TCA cycle through fatty acid hydrolysis. These data may indicate that in OASS overexpressing soybean nodules there is a moderate decease in the supply of energy metabolites to the nodule, which is then compensated by the degradation of cellular components to meet the needs of the nodule energy metabolism.

Ascorbic acid improves the tolerance of wheat plants to lead toxicity

ABSTRACTAmong the heavy metals (HMs), lead (Pb) is considered as a toxic HM which adversely affects growth and development of crop plants. The present experiment was aimed to investigate the potential role of ascorbic acid (ASC) in the reversal of Pb-inhibited nitrogen and sulfur assimilation enzymes activity and activity of photosynthesis enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and growth response in wheat plants. Wheat seedlings were subjected to 0 mM (control) and 0.2 mM and 0.6 mM of ASC with and without 2 mM of Pb. Plants treated with Pb exhibited the following reduced growth characteristics (root length, shoot length, root fresh weight (FW), shoot FW, root dry weight (DW) and shoot DW). A decrease was also observed in the activity of Rubisco and ATP sulfurylase (ATP-S), relative water content (RWC), accumulation of total chlorophyll (Total Chl) and content of nutrients [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg)] in Pb-treated plants. However, an increase in Chl degradation and in the activity of O-acetylserine(thiol)lyase (OAS-TL) and accumulation of cysteine (Cys), malondialdehyde (MDA) and hydrogen peroxide (H2O2) was observed in plants under Pb stress. On the contrary, exogenous application of ASC mitigated the Pb-toxicity-induced oxidative damage by enhancing the activities of antioxidant enzymes, such as superoxide dismutase, catalase and glutathione reductase. Improved activity of antioxidant enzymes suppressed the formation of MDA and H2O2, which was reflected in the form of improved growth characteristics. Moreover, ASC induced improvement in plants defense systems by reduced Chl degradation and improved the content of essential nutrients (N, P, K, Ca and Mg) and Cys, RWC and the activity of Rubisco, ATP-S, NR and OAS-TL.

Nitric oxide-induced synthesis of hydrogen sulfide alleviates osmotic stress in wheat seedlings through sustaining antioxidant enzymes, osmolyte accumulation and cysteine homeostasis

Nitric oxide (NO) and hydrogen sulfide (H2S) have been shown to act as signaling molecules in various physiological processes, play significant roles in plant cellular processes, and also mediate responses to both biotic and abiotic stresses in plants. The present investigation was carried out to test the effect of exogenous NO on endogenous synthesis of H2S in osmotic-stressed wheat (Triticum aestivum L.) seedlings. The results show that application of NO to wheat seedlings, suffered from PEG8000-induced osmotic stress, considerably enhanced the activities of H2S-synthesizing enzymes l-cysteine desulfhydrase (LCD) and d-cysteine desulfhydrase (DCD) leading to enhanced level of endogenous H2S content. At the same time exogenous NO also enhanced the activity of cysteine (Cys)-synthesizing enzyme O-acetylserine(thiol)lyase (OAS-TL) and maintained Cys homeostasis under osmotic stress. NO and H2S together markedly improved the activities of antioxidant enzymes viz. ascorbate peroxidase (APX), glutathione reductase (GR), peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT). Furthermore, NO and H2S caused additional accumulation of osmolytes proline (Pro) and glycine betaine (GB), all these collectively resulted in the protection of plants against osmotic stress-induced oxidative stress. On the other hand, NO scavenger cPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] and H2S scavenger HT (hypotaurine) invalidated the effect of NO on endogenous H2S levels and Cys homeostasis which resulted in weak protection against osmotic stress. Application of N-ethylmaleimide (NEM) suppressed GR activity and caused an increase in oxidative stress. We concluded that NO in association with endogenous H2S activates the defense system to the level required to counter osmotic stress and maintains normal functioning of cellular machinery.

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).

Sulphur alters chromium (VI) toxicity in Solanum melongena seedlings: Role of sulphur assimilation and sulphur-containing antioxidants

The present study investigates modulation in hexavalent chromium [Cr(VI) 25 μM] toxicity by sulphur (S; 0.5, 1.0 and 1.5 mM S as low (LS), medium (MS) and high sulphur (HS), respectively) in Solanum melongena (eggplant) seedlings. Biomass accumulation (fresh and dry weights), photosynthetic pigments, photosynthetic oxygen evolution and S content were declined by Cr(VI) toxicity. Furthermore, fluorescence characteristics (JIP-test) were also affected by Cr(VI), but Cr(VI) toxicity on photosystem II photochemistry was ameliorated by HS treatment via reducing damaging effect on PS II reaction centre and its reduction side. Enhanced respiration, Cr content and oxidative biomarkers: superoxide radical, hydrogen peroxide, lipid peroxidation and membrane damage were observed under Cr(VI) stress. Though Cr(VI) enhanced adenosine triphasphate sulfurylase (ATPS) and o-acetylserine(thiol)lyase (OASTL), glutathione-S-transferase (GST), glutathione reductase (GR) and ascorbate peroxidase (APX) activity, and content of total glutathione, cysteine and NP-SH, however, their levels/activity were further enhanced by S being maximum with HS treatment. The results show that Cr(VI) toxicity does increase under LS treatment while HS protected Cr(VI)-induced damaging effects in brinjal seedlings. Under HS treatment, in mitigating Cr(VI) toxicity, S assimilation and its associated metabolites such as cysteine, glutathione and NP-SH play crucial role.

Impact of Sulfur Starvation in Autotrophic and Heterotrophic Cultures of the Extremophilic Microalga Galdieria phlegrea (Cyanidiophyceae).

In plants and algae, sulfate assimilation and cysteine synthesis are regulated by sulfur (S) accessibility from the environment. This study reports the effects of S deprivation in autotrophic and heterotrophic cultures of Galdieria phlegrea (Cyanidiophyceae), a unicellular red alga isolated in the Solfatara crater located in Campi Flegrei (Naples, Italy), where H2S is the prevalent form of gaseous S in the fumarolic fluids and S is widespread in the soils near the fumaroles. This is the first report on the effects of S deprivation on a sulfurous microalga that is also able to grow heterotrophically in the dark. The removal of S from the culture medium of illuminated cells caused a decrease in the soluble protein content and a significant decrease in the intracellular levels of glutathione. Cells from heterotrophic cultures of G. phlegrea exhibited high levels of internal proteins and high glutathione content, which did not diminish during S starvation, but rather glutathione significantly increased. The activity of O-acetylserine(thiol)lyase (OASTL), the enzyme synthesizing cysteine, was enhanced under S deprivation in a time-dependent manner in autotrophic but not in heterotrophic cells. Analysis of the transcript abundance of the OASTL gene supports the OASTL activity increase in autotrophic cultures under S deprivation.

A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms

An enzyme, O-acetylserine(thiol)lyase (OASTL), also known as O-acetylserine sulfhydrylase or cysteine synthase (CSase), catalyses the incorporation of sulfide into O-acetylserine and produces cysteine. We previously identified a cDNA encoding an OASTL-like protein from Spinacia oleracea, (SoCSaseLP), but a recombinant SoCSaseLP produced in Escherichia coli did not show OASTL activity. The exon-intron structure of the SoCSaseLP gene shared conserved structures with other spinach OASTL genes. The SoCSaseLP and a Beta vulgaris homologue protein, KMT13462, comprise a unique clade in the phylogenetic tree of the OASTL family. Interestingly, when the SoCSaseLP gene was expressed in tobacco plants, total OASTL activity in tobacco leaves was reduced. This reduction in total OASTL activity was most likely caused by interference by SoCSaseLP with cytosolic OASTL. To investigate the possible interaction of SoCSaseLP with a spinach cytosolic OASTL isoform SoCSaseA, a pull-down assay was carried out. The recombinant glutathione S-transferase (GST)-SoCSaseLP fusion protein was expressed in E. coli together with the histidine-tagged SoCSaseA protein, and the protein extract was subjected to glutathione affinity chromatography. The histidine-tagged SoCSaseA was co-purified with the GST-SoCSaseLP fusion protein, indicating the binding of SoCSaseLP to SoCSaseA. Consistent with this interaction, the OASTL activity of the co-purified SoCSaseA was reduced compared with the activity of SoCSaseA that was purified on its own. These results strongly suggest that SoCSaseLP negatively regulates the activity of other cytosolic OASTL family members by direct interaction.

β-Substituting alanine synthases: roles in cysteine metabolism and abiotic and biotic stress signalling in plants.

Cysteine is required for the synthesis of proteins and metabolites, and is therefore an indispensable compound for growth and development. The β-substituting alanine synthase (BSAS) gene family encodes enzymes known as O-acetylserine thiol lyases (OASTLs), which carry out cysteine biosynthesis in plants. The functions of the BSAS isoforms have been reported to be crucial in assimilation of S and cysteine biosynthesis, and homeostasis in plants. In this review we explore the functional variation in this classic pyridoxal-phosphate-dependent enzyme family of BSAS isoforms. We discuss how specialisation and divergence in BSAS catalytic activities makes a more dynamic set of biological routers that integrate cysteine metabolism and abiotic and biotic stress signalling in Arabidopsis thaliana (L.) Heynh. and also other species. Our review presents a universal scenario in which enzymes modulating cysteine metabolism promote survival and fitness of the species by counteracting internal and external stress factors.

Sulfur decreases cadmium translocation and enhances cadmium tolerance by promoting sulfur assimilation and glutathione metabolism in Brassica chinensis L.

We investigated the ameliorative role of sulfur (S) in protecting plants against cadmium (Cd) toxicity by using two pakchoi (Brassica chinensis L.) cultivars with different Cd tolerance levels. The exposure of pakchoi seedlings to 100μM Cd inhibited plant growth, increased superoxide content, enhanced membrane lipid peroxidation, and induced Cd accumulation in the roots and shoots. Application of S to Cd-stressed plants alleviated Cd-induced oxidative stress by promoting the capacity of the ascorbate (AsA)–glutathione (GSH) cycle, enhanced S assimilation by increasing the activity of ATP sulfurylase (ATPS) and o-acetylserine(thiol)lyase (OASTL), and decreased Cd translocation from the roots to the shoots by enhancing phytochelatins (PCs) biosynthesis. Results suggested that S reversed Cd-induced growth inhibition and oxidative stress by restraining Cd translocation from the roots to the shoots and upregulating S assimilation and GSH metabolism, including the AsA–GSH cycle and PCs synthesis.

Oligo-carrageenan kappa increases C, N and S assimilation, auxin and gibberellin contents, and growth in Pinus radiata trees

Oligo-carrageenans (OCs) obtained from pure carrageenans extracted from marine red algae stimulate growth by enhancing photosynthesis and basal metabolism in tobacco plants and Eucalyptus trees. In addition, OCs stimulate secondary metabolism, increasing the level of metabolites involved in defense against pathogens. In this work, we analyzed the effect of OC kappa on the increase in height, in activities of basal metabolism enzymes involved in carbon, nitrogen and sulphur assimilation, ribulose 1,5 biphosphate carboxylase/oxygenase (rubisco), glutamate dehydrogenase (GDH) and O-acetylserine thiol-lyase (OASTL), and in the level of growth-promoting hormones, the auxin indole acetic acid (IAA) and the gibberellin GA3, in pine (Pinus radiata) trees treated with OC kappa at concentrations of 1 and 5 mg mL−1 and cultivated for 9 months without additional treatment. Pines treated with OC kappa at 1 mg mL−1 showed a similar increase in height but displayed a higher increased in total chlorophyll, activities of rubisco, GDH and OASTL and level of IAA and GA3 than those treated with OC kappa at 5 mg mL−1. Thus, OC kappa stimulates growth and basal metabolism and increases the level of growth-promoting hormones in pine trees, mainly at 1 mg mL−1.

Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants.

In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family of the crop plant Vitis vinifera. The identified four members of the VvSERAT protein family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labeled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine). Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription.

Differential expression of sulfur assimilation pathway genes in Acidithiobacillus ferrooxidans under Cd²⁺ stress: evidence from transcriptional, enzymatic, and metabolic profiles.

Acidithiobacillus ferrooxidans is a heavy metal-tolerant acidophilic chemolithotroph found in acidic mine effluent and is used commercially in the bioleaching of sulfide ores. In this work, we investigated the interplay between divalent cadmium (Cd(2+)) resistance and expression of genes involved in the sulfur assimilation pathway (SAP). We also investigated the response of the thiol-containing metal-chelating metabolites, cysteine and glutathione(GSH), to increasing Cd(2+) concentrations. During growth in the presence of 30 mM Cd(2+), the concentrations of mRNA for 5 genes in the SAP pathway increased more than fourfold: these encode ATP sulfurylase (ATPS), adenosine 5'-phosphosulfate (APS) reductase, sulfite reductase (SiR), serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Increased transcription was also reflected in increased enzyme activities: those of SAT and adenosylphosphosulfate reductase (APR) reached a peak of 26- and 15.8-fold, respectively, compared to the control culture in the presence of 15 mM Cd(2+). In contrast, the activity of OAS-TL, which is responsible for the biosynthesis of cysteine, was diminished. At the metabolite level, the intracellular cysteine and GSH contents nearly doubled. These results suggested that Cd(2+) induced transcription of SAP genes, while directly inhibiting the activities of some enzymes (e.g., OAS-TL). Overall, these results are consistent with a detoxification/resistance mechanism involving enhanced sulfur uptake and sequestration of Cd(2+) by cysteine and glutathione.

Cross-Effects of Nitrogen and Sulphur Starvation in <i>Chlorella sorokiniana</i> 211/8K

Nitrogen (N) and sulphur (S), being essential macronutrients, have important roles in microalgae metabolism. Effects of N- or S-shortage were investigated in the green microalgae Chlorella sorokiniana subjected to 24 h of starvation, by measuring the glutamine synthetase (GS) and O-ace- tylserine(thiol)lyase (OASTL) activities, proteins and amino acids levels. To test possible metabolic impact related to carbon (C) metabolism in response to N- or S-deprivation, starch and total C, N and S contents were also determined. The growth of C. sorokiniana cells was affected by N or S availability. The algae cultured for 24 h in a medium deprived of nitrogen or sulphur showed a decrease in the growth rate and changes in the average volume cell. Nitrogen starvation affected proteins level in the algae cells more than S-deprivation did. The decline in the protein levels observed under S-deficient conditions was coupled with the accumulation of the amide glutamine and with OASTL activity increase; additionally, N-deficiency promoted a decrease in cysteine (Cys) levels (50%) and an increase in GS activity. Nevertheless, S-deprivation had negligible effects on GS activity, while N-deprivation significantly affected OASTL activity. Total C was also estimated in cells N- or S-deprived; nitrogen deprivation strongly affected total C content more than S-deprivation, which in addition reduced the content of C and N, but leaves intact their ratios. Our results support the hypothesis that in Chlorella sorokiniana cells a reciprocal influence of N, S and C assimilation occurs.

Sulfate resupply accentuates protein synthesis in coordination with nitrogen metabolism in sulfur deprived Brassica napus

To investigate the regulatory interactions between S assimilation and N metabolism in Brassica napus, de novo synthesis of amino acids and proteins was quantified by 15N and 34S tracing, and the responses of transporter genes, assimilatory enzymes and metabolites pool involving in nitrate and sulfate metabolism were assessed under continuous sulfur supply, sulfur deprivation and sulfate resupply after 3 days of sulfur (S) deprivation. S-deprived plants were characterized by a strong induction of sulfate transporter genes, ATP sulfurylase (ATPS) and adenosine 5′-phosphosulfate reductase (APR), and by a repressed activity of nitrate reductase (NR) and glutamine synthetase (GS). Sulfate resupply to the S-deprived plants strongly increased cysteine, amino acids and proteins concentration. The increase in sulfate and cysteine concentration caused by sulfate resupply was not matched with the expression of sulfate transporters and the activity of ATPS and APR which were rapidly decreased by sulfate resupply. A strong induction of O-acetylserine(thiol)lyase (OASTL), NR and GS upon sulfate resupply was accompanied with the increase in cysteine, amino acids and proteins pool. Sulfate resupply resulted in a strong increase in de novo synthesis of amino acids and proteins, as evidenced by the increases in N and S incorporation into amino acids (1.8- and 2.4-fold increase) and proteins (2.2-and 6.3-fold increase) when compared to S-deprived plants. The results thus indicate that sulfate resupply followed by S-deprivation accelerates nitrate assimilation for protein synthesis.

The role of compartment-specific cysteine synthesis for sulfur homeostasis during H2S exposure in Arabidopsis.

Sulfide is the end-product of assimilatory sulfate reduction in chloroplasts. It is then used by O-acetylserine(thiol)lyase (OAS-TL) to produce cysteine, the source of reduced sulfur in plants. While its formation in chloroplasts is essential for plant metabolism, sulfide is also a potent toxin mainly targeting respiration in mitochondria. Here, the application of sublethal concentrations of sulfide to Arabidopsis thaliana was used to by-pass assimilatory sulfate reduction, resulting in down-regulation of most genes of the pathway. The dualism of sulfide as substrate and toxin was investigated using knock-out mutants of the chloroplast-, mitochondrion- and cytosol-targeted OAS-TL isoforms. Surprisingly, growth retardation due to intoxication by sulfide was independent of the presence or absence of the three OAS-TL isoforms, indicating rapid exchange towards sulfur homoeostasis between the compartments. Cysteine, glutathione and sulfate, and less so S-sulfocysteine, were identified as major sinks for excess sulfide in wild-type plants. Additionally, the concentration of thiosulfate increased 1,000-fold, pointing towards a significant function of thiosulfate formation during H2S exposure. Synthesis of cysteine in the cytosol was found to be particularly important for accumulation of sulfite, sulfate and thiosulfate, indicating an important role for cytosolic OAS-TL for the re-oxidation of sulfide. The results show that thiosulfate and sulfate accumulation is strongly linked to cytosolic cysteine synthesis and that scavenging of sulfide by cysteine synthesis enhances sulfur compound accumulation. However, lack of cysteine synthesis in a subcellular compartment has no crucial consequences for toxicity and subsequent growth retardation.

Affinity Purification of O-Acetylserine(thiol)lyase from Chlorella sorokiniana by 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.

The link between transcript regulation and de novo protein synthesis in the retrograde high light acclimation response of Arabidopsis thaliana.

BackgroundEfficient light acclimation of photosynthetic cells is a basic and important property of plants. The process of acclimation depends on transformation of retrograde signals in gene expression, transcript accumulation and de novo protein synthesis. While signalling cues, transcriptomes and some involved players have been characterized, an integrated view is only slowly emerging, and information on the translational level is missing. Transfer of low (8 μmol quanta.m-2.s-1) or normal light (80 μmol quanta.m-2.s-1) acclimated 30 d old Arabidopsis thaliana plants to high light (800 μmol quanta.m-2.s-1) triggers retrograde signals. Using this established approach, we sought to link transcriptome data with de novo synthesized proteins by in vivo labelling with 35S methionine and proteome composition.ResultsDe novo synthesized protein and proteome patterns could reliably be matched with newly annotated master gels. Each molecular level could be quantified for a set of 41 proteins. Among the proteins preferentially synthesized in plants transferred to high light were enzymes including carbonic anhydrase, fructose-1,6-bisphosphate aldolase, O-acetyl serine thiol lyase, and chaperones, while low rates upon transfer to high light were measured for e.g. dehydroascorbate reductase, glyceraldehyde-3-phosphate dehydrogenase and CuZn superoxide dismutase, and opposite responses between 10-fold and 100-fold light increment for e.g. glutamine synthetase and phosphoglycerate kinase.ConclusionsThe results prove the hypothesis that transcript abundance is poorly linked to de novo protein synthesis due to profound regulation at the level of translation. This vertical systems biology approach enables to quantitatively and kinetically link the molecular levels for scrutinizing signal processing and response generation.

An O-acetylserine (thiol) lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthase.

In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the Km was calculated to be 1,850±414 μM sulfide and the Vmax was 200.6±19.92 μM cysteine min(-1). The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.

Differential Response of Cysteine-deficient Lentil (<i>Lens culinaris</i> Medik.) Mutants Impaired in Foliar O-acetylserine(thiol)-lyase Expression

Lentil is a cool-season pulse crop, rich in protein but deficient in two sulphur-containing amino acids cysteine and methionine. Due to low genetic variability in existing germplasm, induced mutagenic technique has been adopted in lentil, and two mutant lines exhibiting poor growth and low dry weight were isolated in M 2 -mutagenized (0.10% and 0.15% EMS, 6 h) population of variety L 414. Further analysis revealed that plants from both mutant lines were highly deficient in seed cysteine (Cys) content, and thus, were tentatively designated as cysLc1 and cysLc2 mutants. Mutant plants were advanced to M 3 generation. Biochemical analysis through cysteine synthesizing pathway in leaves revealed that activity of serine acetyl transferase (SAT) was normal in both the mutant progenies but both were highly deficient in foliar O -acetylserine(thiol)-lyase (OAS-TL) activity. Transcriptomic analysis by qRT-PCR confirmed normal expression of SAT in both mutants but revealed differential expressions of two OAS-TL isoforms; OAS-TL 1 isoform was not detectable in cysLc1 mutant while expression of OAS-TL 2 isoform was totally repressed in leaves of cysLc2 mutant. Genetic studies and test of allelism pointed out that both the mutants were recessive and were complementing with each other to produce normal in F 1 and normal along with mutant plants in F 2 progeny. The progeny plants exhibiting normal phenotype showed normal mRNA transcripts of both OAS-TL isoforms. Being stable and self-fertile, the mutants will give vital clues in genetic basis of thiol-metabolic network of lentil crops.