Cysteine Synthase Research Articles

Comparative proteomics of oxidative stress response of Lactobacillus acidophilus NCFM reveals effects on DNA repair and cysteine de novo synthesis

Probiotic cultures encounter oxidative conditions during manufacturing, yet protein abundance changes induced by such stress have not been characterized for some of the most common probiotics and starters. This comparative proteomics investigation focuses on the response by Lactobacillus acidophilus NCFM to H2 O2, simulating an oxidative environment. Bacterial growth was monitored by BioScreen and batch cultures were harvested at exponential phase for protein profiling of stress responses by 2D gel based comparative proteomics. Proteins identified in 19 of 21 spots changing in abundance due to H2 O2 were typically related to carbohydrate and energy metabolism, cysteine biosynthesis, and stress. In particular, increased cysteine synthase activity may accumulate a cysteine pool relevant for protein stability, enzyme catalysis, and the disulfide-reducing pathway. The stress response further included elevated abundance of biomolecules reducing damage such as enzymes from DNA repair pathways and metabolic enzymes with active site cysteine residues. By contrast, a protein-refolding chaperone showed reduced abundance, possibly reflecting severe oxidative protein destruction that was not overcome by refolding. The proteome analysis provides novel insight into resistance mechanisms in lactic acid bacteria against reactive oxygen species and constitutes a valuable starting point for improving industrial processes, food design, or strain engineering preserving microorganism viability.

Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome

Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases—CysE, CysK, and CysM—from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34S-labeled L-cysteine from O-acetyl-L-serine and 34S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur (34S) and nitrogen (15N) atoms was also achieved by performing enzyme reactions with 15N-labeled L-serine, acetyl-CoA, and 34S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34S-labeled N-acetyl-L-cysteine (NAC) by incubating 34S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology.

Effect of N-acetyl cysteine and glycine supplementation on growth performance, glutathione synthesis, and antioxidative ability of grass carp, Ctenopharyngodon idella.

An 8-week feeding trial was conducted to evaluate the effect of N-acetyl cysteine (NAC) and glycine supplementation on growth performance, glutathione (GSH) synthesis, and antioxidative ability of grass carp, Ctenopharyngodon idella. Four practical diets were formulated: control, control+0.2% NAC, control+0.5% glycine, and control+0.2% NAC+0.5% glycine. Each diet was randomly assigned to quadruplicate groups of 30 fish (approximately 8.8g). Weight gain and specific growth rate were significantly increased with the supplementation of NAC. Supplementation of NAC plus glycine significantly increased the feed efficiency. Glutathione peroxidase (GPx) and γ-glutamine cysteine synthase (γ-GCS) in plasma were significantly increased with the supplementation of NAC plus glycine. GSH in plasma increased and malondialdehyde (MDA) decreased in fish fed diets supplemented with NAC. Respiratory burst, superoxide dismutase (SOD), and catalase (CAT) activity were not affected by NAC or glycine. These results clearly indicated that NAC improved the growth performance and restored GSH of grass carp, supplemented NAC together with glycine enhanced GSH synthesis, and improved the antioxidative ability of grass carp.

The effect of nuclear factor erythroid 2-related factor/antioxidant response element signalling pathway in the lanthanum chloride-induced impairment of learning and memory in rats.

Lanthanum exerts adverse effects on the central nervous system. However, the mechanism underlying these adverse effects has not been clarified. It is known that oxidative stress plays an important role in neurological injuries induced by harmful factors. Nuclear factor erythroid 2-related factor (Nrf2) is very important in the response to oxidative stress in tissues and cells. The purpose of this study was to explore the effect of lanthanum chloride (LaCl3 ) on the spatial learning and memory of rats and to determine whether the Nrf2/antioxidant response element pathway acts in the hippocampus. Four groups of Wistar rats were exposed to 0mM, 9mM, 18mM or 36mM LaCl3 through their drinking water from the day of birth to 2months after weaning. The results showed that LaCl3 impaired the spatial learning and memory of the rats, damaged the neuronal ultrastructure, increased reactive oxygen species levels and significantly down-regulated Nrf2 as well as the mRNA and protein expression of Nrf2-regulated genes, including NADP(H): dehydrogenase quinone 1, haeme oxygenase-1, superoxide dismutase 2, glutathione peroxidase 1, glutathione-S-transferase, γ-glutamine cysteine synthase and glutathione reductase, in the hippocampus. This study suggests that LaCl3 can impair the spatial learning and memory of rats, possibly by perturbing the Nrf2/antioxidant response element signalling pathway.

Salinity-induced accumulation of endogenous H2S and NO is associated with modulation of the antioxidant and redox defense systems in Nicotiana tabacum L. cv. Havana

Salinity is one of the abiotic factors that most affect crop growth and production. This study focused on the effect of high salinity on the endogenous levels of the signaling molecules hydrogen sulfite (H2S) and nitric oxide (NO) in Nicotiana tabacum leaves and the extent of these for the biochemically-driven plant tolerance to such abiotic stress. The NaCl treatment for 10days led to an expressive augment of H2S and NO levels. This increase was correlated with the raise of l-Cys and l-Arg and the induction of l-cysteine desulfhydrase, cyanoalanine synthase, cysteine synthase, nitrate reductase and arginase, enzymes known to be involved in the biosynthesis of H2S or NO. The enzymatic antioxidant system (superoxide dismutase and catalase activity) was boosted and the non-enzymatic antioxidant glutathione was intensively oxidized in leaves upon stress allowing plants to cope with oxidative stress. Lower stomatal conductance was observed in stressed plants in comparison with control ones. Moreover, the high activity of antioxidant enzymes and high rate of glutathione oxidation following salt stress were considerably decreased upon NO or H2S scavenging. Thus, increment in NO and H2S levels and their interplay, along with metabolic and physiological changes, contributed to tobacco survival to extreme salinity conditions.

Selenite modulates the level of phenolics and nutrient element to alleviate the toxicity of arsenite in rice (Oryza sativa L.)

Arsenic (As) contamination of paddy rice is a serious threat all over the world particularly in South East Asia. Selenium (Se) plays important role in protection of plants against various abiotic stresses including heavy metals. Moreover, arsenite (AsIII) and selenite (SeIV) can be biologically antagonistic due to similar electronic configuration and sharing the common transporter for their uptake in plant. In the present study, the response of oxidative stress, phenolic compounds and nutrient elements was analyzed to investigate Se mediated As tolerance in rice seedlings during AsIII and SeIV exposure in hydroponics. Selenite (25µM) significantly decreased As accumulation in plant than As (25µM) alone treated plants. Level of oxidative stress related parameters viz., reactive oxygen species (ROS), lipid peroxidation, electrical conductivity, nitric oxide and pro-oxidant enzyme (NADPH oxidase), were in the order of As>As+Se>control>Se. Selenium ameliorated As phytotoxicity by increased level of phenolic compounds particularly gallic acid, protocatechuic acid, ferulic acid and rutin and thiol metabolism related enzymes viz., serine acetyl transferase (SAT) and cysteine synthase (CS). Selenium supplementation enhanced the uptake of nutrient elements viz., Fe, Mn, Co, Cu, Zn, Mo, and improved plant growth. The results concluded that Se addition in As contaminated environment might be an important strategy to reduce As uptake and associated phytotoxicity in rice plant by modulation of phenolic compounds and increased uptake of nutrient elements.

Elucidation of Complex Nature of PEG Induced Drought-Stress Response in Rice Root Using Comparative Proteomics Approach.

Along with many adaptive strategies, dynamic changes in protein abundance seem to be the common strategy to cope up with abiotic stresses which can be best explored through proteomics. Understanding of drought response is the key to decipher regulatory mechanism of better adaptation. Rice (Oryza sativa L.) proteome represents a phenomenal source of proteins that govern traits of agronomic importance, such as drought tolerance. In this study, a comparison of root cytoplasmic proteome was done for a drought tolerant rice (Heena) cultivar in PEG induced drought conditions. A total of 510 protein spots were observed by PDQuest analysis and 125 differentially regulated spots were subjected for MALDI-TOF MS-MS analysis out of which 102 protein spots identified which further led to identification of 78 proteins with a significant score. These 78 differentially expressed proteins appeared to be involved in different biological pathways. The largest percentage of identified proteins was involved in bioenergy and metabolism (29%) and mainly consists of malate dehydrogenase, succinyl-CoA, putative acetyl-CoA synthetase, and pyruvate dehydrogenase etc. This was followed by proteins related to cell defense and rescue (22%) such as monodehydroascorbate reductase and stress-induced protein sti1, then by protein biogenesis and storage class (21%) e.g. putative thiamine biosynthesis protein, putative beta-alanine synthase, and cysteine synthase. Further, cell signaling (9%) proteins like actin and prolyl endopeptidase, and proteins with miscellaneous function (19%) like Sgt1 and some hypothetical proteins were also represented a large contribution toward drought regulatory mechanism in rice. We propose that protein biogenesis, cell defense, and superior homeostasis may render better drought-adaptation. These findings might expedite the functional determination of the drought-responsive proteins and their prioritization as potential molecular targets for perfect adaptation.

In silico structural characterization of protein targets for drug development against Trypanosoma cruzi.

Trypanosoma cruzi is the protozoan pathogen responsible for Chagas disease, which is a major public health problem in tropical and subtropical regions of developing countries and particularly in Brazil. Despite many studies, there is no efficient treatment against Chagas disease, and the search for new therapeutic targets specific to T. cruzi is critical for drug development. Here, we have revisited 41 protein sequences proposed by the analogous enzyme pipeline, and found that it is possible to provide structures for T. cruzi sequences with clear homologs or analogs in H. sapiens and likely associated with trypanothione reductase, cysteine synthase, and ATPase functions, and structures for sequences specific to T. cruzi and absent in H. sapiens associated with 2,4-dienoyl-CoA reductase, and leishmanolysin activities. The implications of our structures refined by atomistic molecular dynamics (monomer or dimer states) in their in vitro environments (aqueous solution or membrane bilayers) are discussed for drug development and suggest that all protein targets, except cysteine synthase, merit further investigation.

Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L.donovani. CS and SAT genes of L.donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a Km of 15.86mM and 0.17mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania.

Unraveling the essential role of CysK in CDI toxin activation

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI(+) bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT(EC536) deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT(EC536) binary complex and the neutralized ternary complex of CysK/CdiA-CT/CdiI(EC536) CdiA-CT(EC536) inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT(EC536) exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT(EC536) thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT(EC536), and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.

Inhibitors of the Cysteine Synthase CysM with Antibacterial Potency against Dormant Mycobacterium tuberculosis.

Cysteine is an important amino acid in the redox defense of Mycobacterium tuberculosis, primarily as a building block of mycothiol. Genetic studies have implicated de novo cysteine biosynthesis in pathogen survival in infected macrophages, in particular for persistent M. tuberculosis. Here, we report on the identification and characterization of potent inhibitors of CysM, a critical enzyme in cysteine biosynthesis during dormancy. A screening campaign of 17 312 compounds identified ligands that bind to the active site with micromolar affinity. These were characterized in terms of their inhibitory potencies and structure-activity relationships through hit expansion guided by three-dimensional structures of enzyme-inhibitor complexes. The top compound binds to CysM with 300 nM affinity and displays selectivity over the mycobacterial homologues CysK1 and CysK2. Notably, two inhibitors show significant potency in a nutrient-starvation model of dormancy of Mycobacterium tuberculosis, with little or no cytotoxicity toward mammalian cells.

Proteomic and Metabolomic Analyses of Leaf from Clematis terniflora DC. Exposed to High-Level Ultraviolet-B Irradiation with Dark Treatment.

Clematis terniflora DC. has potential pharmaceutical value; on the contrary, high-level UV-B irradiation with dark treatment led to the accumulation of secondary metabolites. Metabolomic and proteomic analyses of leaf of C. terniflora were performed to investigate the systematic response mechanisms to high-level UV-B irradiation with dark treatment. Metabolites related to carbohydrates, fatty acids, and amino acids and/or proteins related to stress, cell wall, and amino acid metabolism were gradually increased in response to high-level UV-B irradiation with dark treatment. On the basis of cluster analysis and mapping of proteins related to amino acid metabolism, the abundances of S-adenosylmethionine synthetase and cysteine synthase as well as 1,1-diphenyl-2-picrylhydrazyl scavenging activity were gradually increased in response to high-level UV-B irradiation with dark treatment. Furthermore, the abundance of dihydrolipoyl dehydrogenase/glutamate dehydrogenase and the content of γ-aminobutyric acid were also increased following high-level UV-B irradiation with dark treatment. Taken together, these results suggest that high-level UV-B irradiation with dark treatment induces the activation of reactive oxygen species scavenging system and γ-aminobutyric acid shunt pathway in leaf of C. terniflora.

Pharmacologic Neuroprotection for Functional Outcomes After Traumatic Brain Injury: A Systematic Review of the Clinical Literature.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. The deleterious effects of secondary brain injury may be attenuated by early pharmacological therapy in the emergency room and intensive care unit (ICU). Current medical management of acute TBI is primarily supportive, aimed at reducing intracranial pressure (ICP) and optimizing cerebral perfusion. There are no pharmacological therapies to date that have been unequivocally demonstrated to improve neurological outcomes after TBI. The purpose of this systematic review was to evaluate the recent clinical studies from January 2013 through November 2015 that investigated neuroprotective functional outcomes of pharmacological agents after TBI. The following databases were searched for relevant studies: MEDLINE (OvidSP January Week 1, 2013-November Week 2 2015), Embase (OvidSP 2013 January 1-2015 November 24), and the unindexed material in PubMed (National Library of Medicine/National Institutes of Health [NLM/NIH]). This systematic review included only full-length clinical studies and case series that included at least five patients and were published in the English language. Only studies that examined functional clinical outcomes were included. Twenty-five of 527 studies met our inclusion criteria, which investigated 15 independent pharmacological therapies. Eight of these therapies demonstrated possible neuroprotective properties and improved functional outcomes, of which five were investigated with randomized clinical trials: statins, N-acetyl cysteine (NAC), Enzogenol, Cerebrolysin, and nitric oxide synthase inhibitor (VAS203). Three pharmacological agents did not demonstrate neuroprotective effects, and four agents had mixed results. While there is currently no single pharmacological therapy that will unequivocally improve clinical outcomes after TBI, several agents have demonstrated promising clinical benefits for specific TBI patients and should be investigated further.

Effect of N-acetyl cysteine and glycine supplementation on growth performance, glutathione synthesis, anti-oxidative and immune ability of Nile tilapia, Oreochromis niloticus.

An 8-week feeding trial was conducted to evaluate the effect of N-acetyl cysteine (NAC) and glycine supplementation on growth performance, glutathione (GSH) synthesis, anti-oxidative and immune ability of Nile tilapia, Oreochromis niloticus. Four practical diets were formulated, control, control+0.2% NAC, control+0.5% glycine, control+0.2% NAC+0.5% glycine. Each diet was randomly assigned to quadruplicate groups of 30 fish (approximately 9.5g). The weight gain and specific growth rate were significantly increased with the supplementation of NAC and glycine. While they had no effect on feed efficiency feed intake and survival. Glutathion peroxidase (GPx) was increased by NAC and γ-glutamine cysteine synthase (γ-GCS) in plasma were increased by glycine. After the feeding trail, fish were challenged by Streptococcus iniae, fish fed the diet supplemented with NAC obtained significantly higher survival rate after 72h challenge test. NAC also decreased malonaldehyde (MDA) in liver, increased glutathione S-transferase (GST) activity in plasma, up-regulated mRNA expression of Superoxide dismutase (SOD) and GPx in liver and headkidney. Dietary supplementation of glycine increased the anti-oxidative ability of tilapia through increase anti-oxidative enzyme activity (SOD, glutathione reductase, myeloperoxidase) and up-regulate anti-oxidative gene expression (SOD). Immune ability only enhanced by the supplementation of NAC through increased interleukin-1β (IL-1β) mRNA expression. These results clearly indicated that the supplementation of NAC and glycine can significantly improve the growth performance of tilapia, and NAC also enhance the anti-oxidative and immune capacity of tilapia, glycine could only enhance the anti-oxidative ability.

Proteome changes in banana fruit peel tissue in response to ethylene and high-temperature treatments.

Banana (Musa AAA group) is one of the most consumed fruits in the world due to its flavor and nutritional value. As a typical climacteric fruit, banana responds to ethylene treatment, which induces rapid changes of color, flavor (aroma and taste), sweetness and nutritional composition. It has also been reported that ripening bananas at temperatures above 24 °C inhibits chlorophyll breakdown and color formation but increases the rate of senescence. To gain fundamental knowledge about the effects of high temperature and ethylene on banana ripening, a quantitative proteomic study employing multiplex peptide stable isotope dimethyl labeling was conducted. In this study, green (immature) untreated banana fruit were subjected to treatment with 10 μL L−1 of ethylene for 24 h. After ethylene treatment, treated and untreated fruit were stored at 20 or 30 °C for 24 h. Fruit peel tissues were then sampled after 0 and 1 day of storage, and peel color and chlorophyll fluorescence were evaluated. Quantitative proteomic analysis was conducted on the fruit peels after 1 day of storage. In total, 413 common proteins were identified and quantified from two biological replicates. Among these proteins, 91 changed significantly in response to ethylene and high-temperature treatments. Cluster analysis on these 91 proteins identified 7 groups of changed proteins. Ethylene treatment and storage at 20 °C induced 40 proteins that are correlated with pathogen resistance, cell wall metabolism, ethylene biosynthesis, allergens and ribosomal proteins, and it repressed 36 proteins that are associated with fatty acid and lipid metabolism, redox–oxidative responses, and protein biosynthesis and modification. Ethylene treatment and storage at 30 °C induced 32 proteins, which were mainly similar to those in group 1 but also included 8 proteins in group 3 (identified as chitinase, cinnamyl alcohol dehydrogenase 1, cysteine synthase, villin-2, leucine-transfer RNA ligase, CP47 protein and calmodulin) and repressed 43 proteins in 4 groups (groups 4–7), of which 6 were associated with photosynthesis II oxygen-evolving protein, the photosynthesis I reaction center, sugar metabolism, the redox–oxidative system and fatty acid metabolism. Differences in the response to ethylene and holding temperature at 30 °C were also revealed and have been discussed. The identities and quantities of the proteins found were linked with quality changes. This study demonstrates that ethylene and high temperature influence banana fruit ripening and senescence at the proteomic level and reveals the mechanisms by which high temperature accelerates banana fruit ripening.

Deciphering the interplay between cysteine synthase and thiol cascade proteins in the survival of L. donovani under oxidative stress

Deciphering the interplay between cysteine synthase and thiol cascade proteins in the survival of L. donovani under oxidative stress

Integration of Metabolic Modeling with Gene Co-expression Reveals Transcriptionally Programmed Reactions Explaining Robustness in Mycobacterium tuberculosis.

Robustness of metabolic networks is accomplished by gene regulation, modularity, re-routing of metabolites and plasticity. Here, we probed robustness against perturbations of biochemical reactions of M. tuberculosis in the form of predicting compensatory trends. In order to investigate the transcriptional programming of genes associated with correlated fluxes, we integrated with gene co-expression network. Knock down of the reactions NADH2r and ATPS responsible for producing the hub metabolites, and Central carbon metabolism had the highest proportion of their associated genes under transcriptional co-expression with genes of their flux correlated reactions. Reciprocal gene expression correlations were observed among compensatory routes, fresh activation of alternative routes and in the multi-copy genes of Cysteine synthase and of Phosphate transporter. Knock down of 46 reactions caused the activation of Isocitrate lyase or Malate synthase or both reactions, which are central to the persistent state of M. tuberculosis. A total of 30 new freshly activated routes including Cytochrome c oxidase, Lactate dehydrogenase, and Glycine cleavage system were predicted, which could be responsible for switching into dormant or persistent state. Thus, our integrated approach of exploring transcriptional programming of flux correlated reactions has the potential to unravel features of system architecture conferring robustness.

Involvement of a universal amino acid synthesis impediment in cytoplasmic male sterility in pepper.

To explore the mechanisms of pepper (Capsicum annuum L.) cytoplasmic male sterility (CMS), we studied the different maturation processes of sterile and fertile pepper anthers. A paraffin section analysis of the sterile anthers indicated an abnormality of the tapetal layer and an over-vacuolization of the cells. The quantitative proteomics results showed that the expression of histidinol dehydrogenase (HDH), dihydroxy-acid dehydratase (DAD), aspartate aminotransferase (ATAAT), cysteine synthase (CS), delta-1-pyrroline-5-carboxylate synthase (P5CS), and glutamate synthetase (GS) in the amino acid synthesis pathway decreased by more than 1.5-fold. Furthermore, the mRNA and protein expression levels of DAD, ATAAT, CS and P5CS showed a 2- to 16-fold increase in the maintainer line anthers. We also found that most of the amino acid content levels decreased to varying degrees during the anther tapetum period of the sterile line, whereas these levels increased in the maintainer line. The results of our study indicate that during pepper anther development, changes in amino acid synthesis are significant and accompany abnormal tapetum maturity, which is most likely an important cause of male sterility in pepper.

Promising Role of Moderate Soil Drying and Subsequent Recovery Through Moderate Wetting at Grain-Filling Stage for Rice Yield Enhancement

The study was conducted at the grain-filling stage to elucidate the physiological and molecular mechanisms of the root to enhance yield under alternate wetting and drying (AWD) compared with conventional irrigation. Measurements of root dry weight (RDW), seed setting rate, total kernel weight, and grain yield were determined along with 2D electrophoresis to detect altered protein expression in response to moderate soil drying (MD) and the subsequent recovery phase as moderate wetting (MW) under AWD compared with continuous wetting under CI. We found significant enhancement in RDW as well as 14.30 % increase in inferior spikelets, seed setting and 10.32 g m−2 increase in final yield. Among the total 55 differentially expressed proteins, 26 proteins were differentially expressed under both MD treatment and MW treatment, whereas 14 proteins under MD and 15 proteins under MW showed distinct expression. Differentially expressed proteins were involved in redox homeostasis, signaling, defense, energy, photoassimilate remobilization and included 14-3-3 proteins, cysteine-rich receptor-like protein kinase, monodehydroascorbate reductase, ascorbate peroxidase, glutathione S-transferases, translationally controlled tumor protein, remorin C-terminal domain containing protein, protein disulfide isomerase, DnaK family protein, cysteine synthase, aminotransferase, phosphoglycerate mutase, pyruvate phosphate dikinase, ATP synthase, and abscisic acid stress ripening (ASR1). The differential expression ratio of the signaling, redox, and defense group proteins was almost the same under MD and MW. ABA signaling, amino acid synthesis, and N remobilization were upregulated under MD, and the enzymes involved in carbohydrate, energy, and transportation metabolism were upregulated under MW. In conclusion, at the rice grain-filling stage, AWD is a potential technique to trigger signaling and the enzymatic protein network for systematic senescence initiation, root enlargement for maximum nutrient uptake, and maximize photoassimilate remobilization for yield enhancement.

Proteomic responses of drought-tolerant and drought-sensitive cotton varieties to drought stress.

Drought, one of the most widespread factors reducing agricultural crop productivity, affects biological processes such as development, architecture, flowering and senescence. Although protein analysis techniques and genome sequencing have made facilitated the proteomic study of cotton, information on genetic differences associated with proteomic changes in response to drought between different cotton genotypes is lacking. To determine the effects of drought stress on cotton seedlings, we used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to comparatively analyze proteome of drought-responsive proteins during the seedling stage in two cotton (Gossypium hirsutum L.) cultivars, drought-tolerant KK1543 and drought-sensitive Xinluzao26. A total of 110 protein spots were detected on 2-DE maps, of which 56 were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. The identified proteins were mainly associated with metabolism (46.4%), antioxidants (14.2%), and transport and cellular structure (23.2%). Some key proteins had significantly different expression patterns between the two genotypes. In particular, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, UDP-D-glucose pyrophosphorylase and ascorbate peroxidase were up-regulated in KK1543 compared with Xinluzao26. Under drought stress conditions, the vacuolar H(+)-ATPase catalytic subunit, a 14-3-3g protein, translation initiation factor 5A and pathogenesis-related protein 10 were up-regulated in KK1543, whereas ribosomal protein S12, actin, cytosolic copper/zinc superoxide dismutase, protein disulfide isomerase, S-adenosylmethionine synthase and cysteine synthase were down-regulated in Xinluzao26. This work represents the first characterization of proteomic changes that occur in response to drought in roots of cotton plants. These differentially expressed proteins may be related to biochemical pathways responsible for drought tolerance in KK1543. Although further studies are needed, this proteomic analysis underlines the role of post-translational events. The differentially expressed proteins and their corresponding genes may be used as markers for the breeding of drought tolerance in cotton.