Malate Synthase Research Articles

Germination and the Biochemical Response of Pumpkin Seeds to Different Concentrations of Humic Acid under Cadmium Stress

The poisoning of heavy metals and their accumulation in food chains are major environmental and health risks. There have been several reports that determined that pumpkins tend to collect small amounts of nitrate or heavy metals. Therefore, the aim of the present study is to investigate the effect of organic matter (humic acid) on the germination and activity of antioxidant enzymes, glycosylate cycle enzymes, and utilization of lipid and protein reserves of pumpkin seeds under cadmium stress conditions. An experiment was conducted to quantify the germination response and biochemical change of pumpkin seeds to the use of humic acid under cadmium stress conditions. The treatments were cadmium at three levels (0 (control), 100, and 200 mg.L−1) and humic acid at five levels (0 (control), 100, 200, 300, and 400 mg.L−1). Linear and sigmoidal models were used to investigate the trend of trait changes. The results show that changes in the germination percentage and seed vigor were affected by applying humic acid and cadmium stress. The highest germination percentage for pumpkins was observed without stress and cadmium stress at a concentration of 200 mg.L−1. The results of quantification for the germination and seed vigor also showed that the model of germination changes by the use of humic acid was sigmoidal in non-stress and cadmium stress conditions of 100 mg.L−1, but it was linear for seed vigor in the stress conditions of 200 mg.L−1. The activity of superoxide dismutase, catalase, peroxidase, isocitrate lyase, and malate synthase was also affected by the simultaneous use of humic acid and cadmium stress, and the trend of their changes was linear.

Insights Into the Peroxisomal Protein Inventory of Zebrafish.

Peroxisomes are ubiquitous, oxidative subcellular organelles with important functions in cellular lipid metabolism and redox homeostasis. Loss of peroxisomal functions causes severe disorders with developmental and neurological abnormalities. Zebrafish are emerging as an attractive vertebrate model to study peroxisomal disorders as well as cellular lipid metabolism. Here, we combined bioinformatics analyses with molecular cell biology and reveal the first comprehensive inventory of Danio rerio peroxisomal proteins, which we systematically compared with those of human peroxisomes. Through bioinformatics analysis of all PTS1-carrying proteins, we demonstrate that D. rerio lacks two well-known mammalian peroxisomal proteins (BAAT and ZADH2/PTGR3), but possesses a putative peroxisomal malate synthase (Mlsl) and verified differences in the presence of purine degrading enzymes. Furthermore, we revealed novel candidate peroxisomal proteins in D. rerio, whose function and localisation is discussed. Our findings confirm the suitability of zebrafish as a vertebrate model for peroxisome research and open possibilities for the study of novel peroxisomal candidate proteins in zebrafish and humans.

Unraveling the malate biosynthesis during development of Torreya grandis nuts

Torreya grandis is a characteristic rare economic tree species in subtropical mountainous areas. The kernels of T. grandis have rich content of organic acids, and malate is the predominant organic acid in T. grandis kernels. However, the contents, biosynthesis/metabolism pathway and transcriptional regulation of malate in developing T. grandis kernels remain completely unknown. Here, the organic acid composition in developing T. grandis kernels was first analyzed. The results showed that the content of malate was increased during the maturation T. grandis kernels. A malate synthase (TgMLS) gene might be involved in the accumulation of malate based on transcriptome data, gene expression and enzyme activity analysis. Transient expression of TgMLS in tobacco resulted in the high malate synthase activity and malate content. Furthermore, a basic helix-loop-helix transcription factor (bHLH), TgbHLH87 was identified to positively regulate the TgMLS expression via directly binding the TgMLS promoter. Our finding contributes to mechanism underlying malate accumulation in T. grandis kernels.

Transcriptional Analysis of Metalloprotease, Metallocarboxypeptidase, Isocitrate Lyase, Citrate Synthase, Malate Synthase and Dipeptidylpeptidase V of Trichophyton Rubrum Isolates from Dermatophytosis Patients

Transcriptional Analysis of Metalloprotease, Metallocarboxypeptidase, Isocitrate Lyase, Citrate Synthase, Malate Synthase and Dipeptidylpeptidase V of Trichophyton Rubrum Isolates from Dermatophytosis Patients

Transcriptome and proteome profiling revealed molecular mechanism of selenium responses in bread wheat (Triticum aestivum L.)

BackgroundAlthough selenium (Se) plays important roles in scavenging free radicals, alleviating oxidative stresses, and strengthening immune system, the knowledge about Se responses in bread wheat is still limited. In order to clarify the molecular mechanism of Se responses in bread wheat, 2-week-old wheat seedlings of cultivar ‘Jimai22’ treated with 10 μM disodium selenate (Na2SeO4) for 0, 3, and 24 h were collected and analyzed by transcriptional sequencing and tandem mass tag-based (TMT) quantitative proteomics.ResultsAt least 11,656 proteins and 133,911 genes were identified, and proteins including ATP sulfurylase (APS), cysteine synthase (CS), SeCys lyase, sulfate transporters, glutathione S-transferase (GSTs), glutathione peroxidase (GSH-Px), glutaredoxins (GRXs), superoxide dismutases (SODs), catalases (CATs), heat shock proteins (HSPs), UDP-glycose flavonoid glycosyltransferases (UFGTs), sucrose-6-phosphate hydrolases (Suc-6-PHs), archaeal phosphoglucose isomerases (APGIs), malate synthases (MSs), and endo-1,4-beta-xylanase (Xyn) in Se accumulation, ROS scavenging, secondary metabolism, and carbohydrate metabolism were significantly differently expressed.ConclusionsThis is the first complementary analyses of the transcriptome and proteome related with selenium responses in bread wheat. Our work enhances the understanding about the molecular mechanism of selenium responses in bread wheat.

Adaption and Degradation Strategies of Methylotrophic 1,4-Dioxane Degrading Strain Xanthobacter sp. YN2 Revealed by Transcriptome-Scale Analysis.

Biodegradation of 1,4-dioxane (dioxane) contamination has gained much attention for decades. In our previous work, we isolated a highly efficient dioxane degrader, Xanthobacter sp. YN2, but the underlying mechanisms of its extraordinary degradation performance remained unresolved. In this study, we performed a comparative transcriptome analysis of YN2 grown on dioxane and citrate to elucidate its genetic degradation mechanism and investigated the transcriptomes of different dioxane degradation stages (T0, T24, T48). We also analyzed the transcriptional response of YN2 over time during which the carbon source switched from citrate to dioxane. The results indicate that strain YN2 was a methylotroph, which provides YN2 a major advantage as a pollutant degrader. A large number of genes involved in dioxane metabolism were constitutively expressed prior to dioxane exposure. Multiple genes related to the catabolism of each intermediate were upregulated by treatment in response to dioxane. Glyoxylate metabolism was essential during dioxane degradation by YN2, and the key intermediate glyoxylate was metabolized through three routes: glyoxylate carboligase pathway, malate synthase pathway, and anaplerotic ethylmalonyl–CoA pathway. Genes related to quorum sensing and transporters were significantly upregulated during the early stages of degradation (T0, T24) prior to dioxane depletion, while the expression of genes encoding two-component systems was significantly increased at late degradation stages (T48) when total organic carbon in the culture was exhausted. This study is the first to report the participation of genes encoding glyoxalase, as well as methylotrophic genes xoxF and mox, in dioxane metabolism. The present study reveals multiple genetic and transcriptional strategies used by YN2 to rapidly increase biomass during growth on dioxane, achieve high degradation efficiency and tolerance, and adapt to dioxane exposure quickly, which provides useful information regarding the molecular basis for efficient dioxane biodegradation.

Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle ‘metabolon’ which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

A metabolic daylength measurement system mediates winter photoperiodism in plants.

Plants have served as a preeminent study system for photoperiodism due to their propensity to flower in concordance with the seasons. A nearly singular focus on understanding photoperiodic flowering has prevented the discovery of other photoperiod measuring systems necessary for vegetative health. Here, we use bioinformatics to identify photoperiod-induced genes in Arabidopsis. We show that one, PP2-A13, is expressed exclusively in, and required for, plant fitness in short, winter-like photoperiods. We create a real-time photoperiod reporter, using the PP2-A13 promoter driving luciferase, and show that photoperiodic regulation is independent of the canonical CO/FT mechanism for photoperiodic flowering. We then reveal that photosynthesis combines with circadian-clock-controlled starch production to regulate cellular sucrose levels to control photoperiodic expression of PP2-A13. This work demonstrates the existence of a photoperiod measuring system housed in the metabolic network of plants that functions to control seasonal cellular health.

Free Amino Acids Profile and Expression Analysis of Core Genes Involved in Branched-Chain Amino Acids Metabolism during Fruit Development of Longan (Dimocarpus longan Lour.) Cultivars with Different Aroma Types.

Simple SummaryIn this study, three longan cultivars, including non-aroma types ‘Shixia’ (SX), ‘Lidongben’ (LDB), and strong aroma type ‘Xiangcui’ (XC), were selected to analyze free amino acids (FAAs) variations at six distinct growth stages. The genome-wide identification and expression analysis of genes related to the branched-chain amino acids (BCAA) synthesis pathway were carried out. Results showed that thirty-six FAAs were identified, which increased drastically with fruit development until ripening. During the period of rapid fruit expansion, the aroma of XC changed from light to strong, and the contents of L-alanine and L-leucine were significantly higher than those of SX and LDB. The content of Leu was negatively correlated with the expression of DilBCAT1, -6, and -9 in three varieties, but positively correlated with DilBCAT16, indicating that these four genes may be responsible for the different synthesis and degradation of Leu among cultivars.Amino acids are important component of fruit nutrition and quality. In this study, three longan cultivars, including non-aroma types ‘Shixia’ (SX), ‘Lidongben’ (LDB), and strong aroma type ‘Xiangcui’ (XC), were selected to analyze free amino acids (FAAs) variations at six distinct growth stages (S1–S6). The genome-wide identification and expression analysis of genes related to the branched-chain amino acids (BCAA) synthesis pathway were carried out. Results showed that 36 FAAs were identified, and the total FAAs content ranged from 2601.0 to 9073.5 mg/kg, which increased drastically with fruit development until ripening. L-glutamic acid (Glu), L-alanine (Ala), L-arginine (Arg), γ-Aminobutyric acid (GABA), L-aspartic acid (Asp), L-leucine (Leu), hydroxyl-proline (Hypro), and L-serine (Ser) were the predominant FAAs (1619.9–7213.9 mg/kg) in pulp, accounting for 62.28–92.05% of the total amino acids. During the period of rapid fruit expansion (S2–S4), the aroma of XC changed from light to strong, and the contents of L-alanine (Ala) and L-leucine (Leu) were significantly higher than those of SX and LDB. Furthermore, a total of two 2-isopropyl malate synthase (IPMS), two 3-isopropyl malate dehydrogenase (IPMD), and 16 BCAA transferase (BCAT) genes were identified. The expression levels of DilBCAT1, -6, and -9 genes in XC were significantly higher than those in SX and LDB, while DilBCAT16 in XC was lower. The content of Leu was negatively correlated with the expression of DilBCAT1, -6, and -9 in three varieties, but positively correlated with DilBCAT16, indicating that these four genes may be responsible for the different synthesis and degradation of Leu among cultivars.

Progesterone Promotes Mitochondrial Respiration at the Biochemical and Molecular Level in Germinating Maize Seeds

This research aimed to investigate the effects of progesterone, a mammalian steroid sex hormone, on the mitochondrial respiration in germinating maize seeds. For this purpose, maize seeds were divided into four different groups (control, 10−6, 10−8, and 10−10 mol·L−1 progesterone) and were grown in a germination cabinet in the dark at 24.5 ± 0.5 °C for 4 d. The changes in gene expression levels of citrate synthase (CS), cytochrome oxidase (COX19), pyruvate dehydrogenase (Pdh1), and ATP synthase (ATP6), which is involved in mitochondrial respiration, were studied in root and cotyledon tissues. Significant increases were recorded in the gene expression levels of all studied enzymes. In addition, progesterone applications stimulated activities of malate synthase (MS), isocitrate lyase (ICL), and alpha-amylase, which are important enzymes of the germination step. The changes in gene expression levels of mas1 and icl1 were found parallel to the rise in these enzymes’ activities. It was determined similar increases in root and coleoptile lengths and total soluble protein and total carbohydrate contents. The most remarkable changes were detected in 10−8 mol·L−1 progesterone-treated seedlings. These results clearly indicate that progesterone stimulates mitochondrial respiration by inducing biochemical and molecular parameters and thus accelerates seed germination thanks to the activation of other pathways related to mitochondrial respiration.

Overall Retention of Methyl Stereochemistry during B12-Dependent Radical SAM Methyl Transfer in Fosfomycin Biosynthesis.

Methylcobalamin-dependent radical S-adenosylmethionine (SAM) enzymes methylate non-nucleophilic atoms in a range of substrates. The mechanism of the methyl transfer from cobalt to the receiving atom is still mostly unresolved. Here we determine the stereochemical course of this process at the methyl group during the biosynthesis of the clinically used antibiotic fosfomycin. In vitro reaction of the methyltransferase Fom3 using SAM labeled with 1H, 2H, and 3H in a stereochemically defined manner, followed by chemoenzymatic conversion of the Fom3 product to acetate and subsequent stereochemical analysis, shows that the overall reaction occurs with retention of configuration. This outcome is consistent with a double-inversion process, first in the SN2 reaction of cob(I)alamin with SAM to form methylcobalamin and again in a radical transfer of the methyl group from methylcobalamin to the substrate. The methods developed during this study allow high-yield in situ generation of labeled SAM and recombinant expression and purification of the malate synthase needed for chiral methyl analysis. These methods facilitate the broader use of in vitro chiral methyl analysis techniques to investigate the mechanisms of other novel enzymes.

Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

BackgroundCyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium.ResultsWe were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium.ConclusionsHeterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Cloning and Characterization of Three Sugar Metabolism Genes (LBGAE, LBGALA, and LBMS) Regulated in Response to Elevated CO2 in Goji Berry (Lycium barbarum L.).

The composition and content of sugar play a pivotal role in goji berry (Lycium barbarum L.) fruits, determining fruit quality. Long-term exposure of goji berry to elevated CO2 (eCO2) was frequently demonstrated to reduce sugar content and secondary metabolites. In order to understand the regulatory mechanisms and improve the quality of fruit in the changing climate, it is essential to characterize sugar metabolism genes that respond to eCO2. The objectives of this study were to clone full-length cDNA of three sugar metabolism genes—LBGAE (Lycium barbarum UDP-glucuronate 4-epimerase), LBGALA (Lycium barbarum alpha-galactosidase), and LBMS (Lycium barbarum malate synthase)—that were previously identified responding to eCO2, and to analyze sequence characteristics and expression regulation patterns. Sugar metabolism enzymes regulated by these genes were also estimated along with various carbohydrates from goji berry fruits grown under ambient (400 μmol mol−1) and elevated (700 μmol mol−1) CO2 for 90 and 120 days. Homology-based sequence analysis revealed that the protein-contained functional domains are similar to sugar transport regulation and had a high sequence homology with other Solanaceae species. The sucrose metabolism-related enzyme’s activity varied significantly from ambient to eCO2 in 90-day and 120-day samples along with sugars. This study provides fundamental information on sugar metabolism genes to eCO2 in goji berry to enhance fruit quality to climate change.

Increasing glycolysis by deletion of kcs1 and arg82 improved S-adenosyl-l-methionine production in Saccharomyces cerevisiae

Reprogramming glycolysis for directing glycolytic metabolites to a specific metabolic pathway is expected to be useful for increasing microbial production of certain metabolites, such as amino acids, lipids or considerable secondary metabolites. In this report, a strategy of increasing glycolysis by altering the metabolism of inositol pyrophosphates (IPs) for improving the production of S-adenosyl-l-methionine (SAM) for diverse pharmaceutical applications in yeast is presented. The genes associated with the metabolism of IPs, arg82, ipk1 and kcs1, were deleted, respectively, in the yeast strain Saccharomyces cerevisiae CGMCC 2842. It was observed that the deletions of kcs1 and arg82 increased SAM by 83.3 % and 31.8 %, respectively, compared to that of the control. In addition to the improved transcription levels of various glycolytic genes and activities of the relative enzymes, the levels of glycolytic intermediates and ATP were also enhanced. To further confirm the feasibility, the kcs1 was deleted in the high SAM-producing strain Ymls1ΔGAPmK which was deleted malate synthase gene mls1 and co-expressed the Acetyl-CoA synthase gene acs2 and the SAM synthase gene metK1 from Leishmania infantum, to obtain the recombinant strain Ymls1Δkcs1ΔGAPmK. The level of SAM in Ymls1Δkcs1ΔGAPmK reached 2.89 g L−1 in a 250-mL flask and 8.86 g L−1 in a 10-L fermentation tank, increasing 30.2 % and 46.2 %, respectively, compared to those levels in Ymls1ΔGAPmK. The strategy of increasing glycolysis by deletion of kcs1 and arg82 improved SAM production in yeast.

The effects of illumination and trophic strategy on gene expression in Chlamydomonas reinhardtii

Microalgae are of substantial biotechnological interest due their polyphyletic nature which grants them access to a wide array of high-value metabolites. The inherent genetic diversity of microalgae combined with their metabolic plasticity when grown using different trophic and illumination strategies necessitate the establishment of a reference knowledge base. In the present study we present a detailed characterisation of the combined effects of wavelength selection and trophic strategy on the growth kinetics and gene expression profile of the model microalgae Chlamydomonas reinhardtii grown under moderate to high light intensity (400 μmolph m−2·s−1). The aim is twofold: (a) to establish a list of reliable housekeeping genes valid for quantitative comparisons across several combinations of different wavelengths and trophic strategies and (b) to enable the investigation of the response of central carbon metabolic pathways under these process conditions. White, blue and red light emitting diodes (LEDs) were used to grow pH controlled photo- and mixo-trophic cultures over a period of 136 h in batch mode. A panel of 10 candidate genes, along with biomass growth rate and pigment content were dynamically monitored across all conditions. Statistical analysis identified genes (acetyl-CoA carboxylase subunit α and photosystem I reaction centre subunit II) with less variability observed in their expression levels across the entirety of conditions evaluated compared to housekeeping genes often referred to in literature (receptor of activated protein kinase C and ribosomal protein (large subunit) 19). Further analysis of gene expression profiles revealed substantial differences in response to changes in both wavelength selection (upregulation of ribulose bisphosphate carboxylase small subunit under red phototrophic growth) and trophic strategy (upregulation of malate synthase from phototrophic to mixotrophic conditions). The systematic approach used to establish reliable reference genes presented herein enables robust comparisons of cellular responses across different conditions to better understand algal metabolism and improve process performance.

Transcriptomic and proteomic profiling revealed reprogramming of carbon metabolism in acetate-grown human pathogen Candida glabrata

BackgroundEmergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection.MethodsIn this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches.ResultsCollectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages.ConclusionHere, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.

Hot air treatment inhibits chilling injury in peaches by regulating metabolism of sugar and organic acid

ABSTRACT Hot air (HA) treatment has been demonstrated to control chilling injury (CI) in peaches effectively, however, little is known about the effects of HA on the changes of sugar and organic acid. In order to explore the relationship among CI susceptibility, soluble sugar and organic acid metabolism, peach fruit (Prunus persica) were treated with HA (40°C, 4 h) and then stored at 1°C for 35 days plus 3 days at 20°C. Results showed that HA avoided or decreased the development of CI symptoms and maintained a better quality of peaches. HA suppressed sucrose degradation by inhibiting expression of invertase as well as enhancing the relative expressions of sucrose synthase and sucrose phosphate synthase. The low degradation of sucrose contributed to low contents of glucose and fructose in the treated groups. Besides, HA significantly increased contents of citric and malic acids during the whole storage by enhancing the expressions of citrate synthase and malate dehydrogenase, but inhibiting malate synthase. These results suggest that HA suppressed chilling injury by regulating sucrose degradation and organic acid metabolism. The high contents of sucrose, citric and malic acids contributed to enhancement of CI tolerance in peaches.

Membrane Processing and Steady-State Regulation of the Alternative Peroxisomal Import Receptor Pex9p.

Import of peroxisomal matrix proteins with a type 1 peroxisomal targeting signal (PTS1) in Saccharomyces cerevisiae is facilitated by cytosolic import receptors Pex5p and Pex9p. While Pex5p has a broad specificity for all PTS1 proteins independent of the growth conditions, Pex9p is only expressed in fatty-acid containing media to mediate peroxisomal import of the two malate synthases, Mls1p and Mls2p, as well as the glutathione transferase Gto1p. Pex5p-cargo complexes dock at the peroxisomal membrane, translocate their cargo-protein via a transient pore and are recycled into the cytosol for a further round of import. The processing of Pex5p has been shown to require a complex network of interactions with other membrane-bound peroxins, as well as decoration with ubiquitin as signal for its ATP-dependent release and recycling. Here, we show that the alternative receptor Pex9p requires the same set of interacting peroxins to mediate peroxisomal import of Mls1p. However, while Pex5p is rather stable, Pex9p is rapidly degraded during its normal life cycle. The steady-state regulation of Pex9p, combining oleate-induced expression with high turnover rates resembles that of Pex18p, one of the two co-receptors of the PTS2-dependent targeting pathway into peroxisomes. Both Pex9p- and Pex18p-dependent import routes serve the fast metabolic adaptation to changes of carbon sources in baker’s yeast. By sequence similarities, we identified another Pex9p homolog in the human pathogenic fungus Candida glabrata, in which similar metabolic reprogramming strategies are crucial for survival of the pathogen.

Combined Transcriptome and Proteome Analysis of Masson Pine (Pinus massoniana Lamb.) Seedling Root in Response to Nitrate and Ammonium Supplementations.

Nitrogen (N) is an essential nutrient for plant growth and development. Plant species respond to N fluctuations and N sources, i.e., ammonium or nitrate, differently. Masson pine (Pinus massoniana Lamb.) is one of the pioneer plants in the southern forests of China. It shows better growth when grown in medium containing ammonium as compared to nitrate. In this study, we had grown masson pine seedlings in medium containing ammonium, nitrate, and a mixture of both, and performed comparative transcriptome and proteome analyses to observe the differential signatures. Our transcriptome and proteome resulted in the identification of 1593 and 71 differentially expressed genes and proteins, respectively. Overall, the masson pine roots had better performance when fed with a mixture of ammonium and nitrate. The transcriptomic and proteomics results combined with the root morphological responses suggest that when ammonium is supplied as a sole N-source to masson pine seedlings, the expression of ammonium transporters and other non-specific NH4+-channels increased, resulting in higher NH4+ concentrations. This stimulates lateral roots branching as evidenced from increased number of root tips. We discussed the root performance in association with ethylene responsive transcription factors, WRKYs, and MADS-box transcription factors. The differential analysis data suggest that the adaptability of roots to ammonium is possibly through the promotion of TCA cycle, owing to the higher expression of malate synthase and malate dehydrogenase. Masson pine seedlings managed the increased NH4+ influx by rerouting N resources to asparagine production. Additionally, flavonoid biosynthesis and flavone and flavonol biosynthesis pathways were differentially regulated in response to increased ammonium influx. Finally, changes in the glutathione s-transferase genes suggested the role of glutathione cycle in scavenging the possible stress induced by excess NH4+. These results demonstrate that masson pine shows increased growth when grown under ammonium by increased N assimilation. Furthermore, it can tolerate high NH4+ content by involving asparagine biosynthesis and glutathione cycle.

Characterisation and structural analysis of glyoxylate cycle enzymes of Teladorsagia circumcincta

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89–92 %) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71–87 % similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min−1. mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01μM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60–90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.