Unveiling the dark side of guard cell metabolism.

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a13C-HCO3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.

Phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase isoenzymes play an important role in the filling and quality of Arabidopsis thaliana seed.

Three plant-type phosphoenolpyruvate carboxylase (PPC1 to PPC3) and two phosphoenolpyruvate carboxylase kinase (PPCKs: PPCK1 and 2) genes are present in the Arabidopsis thaliana genome. In seeds, all PPC genes were found to be expressed. Examination of individual ppc mutants showed little reduction of PEPC protein and global activity, with the notable exception of PPC2 which represent the most abundant PEPC in dry seeds. Ppc mutants exhibited moderately lower seed parameters (weight, area, yield, germination kinetics) than wild type. In contrast, ppck1-had much altered (decreased) yield. At the molecular level, ppc3-was found to be significantly deficient in global seed nitrogen (nitrate, amino-acids, and soluble protein pools). Also, N-deficiency was much more marked in ppck1-, which exhibited a tremendous loss of 95% and 90% in nitrate and proteins, respectively. The line ppck2-had accumulated amino-acids but lower levels of soluble proteins. Regarding carboxylic acid pools, Krebs cycle intermediates were found to be diminished in all mutants; this was accompanied by a consistent decrease in ATP. Lipids were stable in ppc mutants, however ppck1-seeds accumulated more lipids while ppck2-seeds showed high level of polyunsaturated fatty acid oleic and linolenic (omega 3). Altogether, the results indicate that the complete PEPC and PPCK family are needed for normal C/N metabolism ratio, growth, development, yield and quality of the seed.

Autophosphorylation Inhibits RcCDPK1, a Dual-Specificity Kinase that Phosphorylates Bacterial-Type Phosphoenolpyruvate Carboxylase in Castor Oil Seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme that plays a crucial anaplerotic role in central plant metabolism. Bacterial-type PEPC (BTPC) of developing castor oil seeds (COS) is highly expressed as a catalytic and regulatory subunit of a novel Class-2 PEPC heteromeric complex. Ricinus communis Ca2+-dependent protein kinase-1 (RcCDPK1) catalyzes in vivo inhibitory phosphorylation of COS BTPC at Ser451. Autokinase activity of recombinant RcCDPK1 was detected and 42 autophosphorylated Ser, Thr or Tyr residues were mapped via liquid chromatography-tandem mass spectrometry. Prior autophosphorylation markedly attenuated the ability of RcCDPK1 to transphosphorylate its BTPC substrate at Ser451. However, fully dephosphorylated RcCDPK1 rapidly autophosphorylated during the initial stages of a BTPC transphosphorylation assay. This suggests that Ca2+-dependent binding of dephospho-RcCDPK1 to BTPC may trigger a structural change that leads to rapid autophosphorylation and subsequent substrate transphosphorylation. Tyr30 was identified as an autophosphorylation site via LC-MS/MS and immunoblotting with a phosphosite-specific antibody. Tyr30 occurs at the junction of RcCDPK1's N-terminal variable (NTVD) and catalytic domains and is widely conserved in plant and protist CDPKs. Interestingly, a reduced rate and extent of BTPC transphosphorylation occurred with a RcCDPK1Y30F mutant. Prior research demonstrated that RcCDPK1's NTVD is essential for its Ca2+-dependent autophosphorylation or BTPC transphosphorylation activities but plays no role in target recognition. We propose that Tyr30 autophosphorylation facilitates a Ca2+-dependent interaction between the NTVD and Ca2+-activation domain that primes RcCDPK1 for transphosphorylating BTPC at Ser451. Our results provide insights into links between the post-translational control of COS anaplerosis, Ca2+-dependent signaling and the biological significance of RcCDPK1 autophosphorylation.

A conserved C-terminal peptide of sorghum phosphoenolpyruvate carboxylase promotes its proteolysis, which is prevented by Glc-6P or the phosphorylation state of the enzyme.

MAIN CONCLUSION: A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.

PEPC of sugarcane regulated glutathione S-transferase and altered carbon-nitrogen metabolism under different N source concentrations in Oryza sativa.

BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) plays an important role in the primary metabolism of higher plants. Several studies have revealed the critical importance of PEPC in the interaction of carbon and nitrogen metabolism. However, the function mechanism of PEPC in nitrogen metabolism is unclear and needs further investigation. RESULTS: This study indicates that transgenic rice expressing the sugarcane C4-PEPC gene displayed shorter primary roots and fewer crown roots at the seedling stage. However, total nitrogen content was significantly higher in transgenic rice than in wild type (WT) plants. Proteomic analysis revealed that there were more differentially expressed proteins (DEPs) responding to nitrogen changes in transgenic rice. In particular, the most enriched pathway "glutathione (GSH) metabolism", which mainly contains GSH S-transferase (GST), was identified in transgenic rice. The expression of endogenous PEPC, GST and several genes involved in the TCA cycle, glycolysis and nitrogen assimilation changed in transgenic rice. Correspondingly, the activity of enzymes including GST, citrate synthase, 6-phosphofructokinase, pyruvate kinase and ferredoxin-dependent glutamate synthase significantly changed. In addition, the levels of organic acids in the TCA cycle and carbohydrates including sucrose, starch and soluble sugar altered in transgenic rice under different nitrogen source concentrations. GSH that the substrate of GST and its components including glutamic acid, cysteine and glycine accumulated in transgenic rice. Moreover, the levels of phytohormones including indoleacetic acid (IAA), zeatin (ZT) and isopentenyladenosine (2ip) were lower in the roots of transgenic rice under total nutrients. Taken together, the phenotype, physiological and biochemical characteristics of transgenic rice expressing C4-PEPC were different from WT under different nitrogen levels. CONCLUSIONS: Our results revealed the possibility that PEPC affects nitrogen metabolism through regulating GST, which provide a new direction and concepts for the further study of the PEPC functional mechanism in nitrogen metabolism.

Overexpression of an Agave Phosphoenolpyruvate Carboxylase Improves Plant Growth and Stress Tolerance.

It has been challenging to simultaneously improve photosynthesis and stress tolerance in plants. Crassulacean acid metabolism (CAM) is a CO2-concentrating mechanism that facilitates plant adaptation to water-limited environments. We hypothesized that the ectopic expression of a CAM-specific phosphoenolpyruvate carboxylase (PEPC), an enzyme that catalyzes primary CO2 fixation in CAM plants, would enhance both photosynthesis and abiotic stress tolerance. To test this hypothesis, we engineered a CAM-specific PEPC gene (named AaPEPC1) from Agave americana into tobacco. In comparison with wild-type and empty vector controls, transgenic tobacco plants constitutively expressing AaPEPC1 showed a higher photosynthetic rate and biomass production under normal conditions, along with significant carbon metabolism changes in malate accumulation, the carbon isotope ratio δ13C, and the expression of multiple orthologs of CAM-related genes. Furthermore, AaPEPC1 overexpression enhanced proline biosynthesis, and improved salt and drought tolerance in the transgenic plants. Under salt and drought stress conditions, the dry weight of transgenic tobacco plants overexpressing AaPEPC1 was increased by up to 81.8% and 37.2%, respectively, in comparison with wild-type plants. Our findings open a new door to the simultaneous improvement of photosynthesis and stress tolerance in plants.

Differential expression in leaves of Saccharum genotypes contrasting in biomass production provides evidence of genes involved in carbon partitioning.

BACKGROUND: The development of biomass crops aims to meet industrial yield demands, in order to optimize profitability and sustainability. Achieving these goals in an energy crop like sugarcane relies on breeding for sucrose accumulation, fiber content and stalk number. To expand the understanding of the biological pathways related to these traits, we evaluated gene expression of two groups of genotypes contrasting in biomass composition. RESULTS: First visible dewlap leaves were collected from 12 genotypes, six per group, to perform RNA-Seq. We found a high number of differentially expressed genes, showing how hybridization in a complex polyploid system caused extensive modifications in genome functioning. We found evidence that differences in transposition and defense related genes may arise due to the complex nature of the polyploid Saccharum genomes. Genotypes within both biomass groups showed substantial variability in genes involved in photosynthesis. However, most genes coding for photosystem components or those coding for phosphoenolpyruvate carboxylases (PEPCs) were upregulated in the high biomass group. Sucrose synthase (SuSy) coding genes were upregulated in the low biomass group, showing that this enzyme class can be involved with sucrose synthesis in leaves, similarly to sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP). Genes in pathways related to biosynthesis of cell wall components and expansins coding genes showed low average expression levels and were mostly upregulated in the high biomass group. CONCLUSIONS: Together, these results show differences in carbohydrate synthesis and carbon partitioning in the source tissue of distinct phenotypic groups. Our data from sugarcane leaves revealed how hybridization in a complex polyploid system resulted in noticeably different transcriptomic profiles between contrasting genotypes.

Genome-wide association study of the oil content in upland cotton (Gossypium hirsutum L.) and identification of GhPRXR1, a candidate gene for a stable QTLqOC-Dt5-1.

Cottonseed oil is one of the most important renewable resources for edible oil and biodiesel. To detect QTLs associated with cottonseed oil content (OC) and identify candidate genes that regulate oil biosynthesis, a panel of upland cotton germplasm lines was selected among those previously used to perform GWASs in China. In the present study, 13 QTLs associated with 53 common SNPs on 13 chromosomes were identified in multiple environments based on 15,369 polymorphic SNPs using the Cotton63 KSNP array. Of these, the OC QTL qOC-Dt5-1 delineated by nine SNPs occurred in a confidence interval of 4 SSRs with previously reported OC QTLs. A combined transcriptome and qRT-PCR analysis revealed that a peroxidase gene (GhPRXR1) was predominantly expressed during the middle-late stage (20-35 days post anthesis) of ovule development. The overexpression of GhPRXR1 in yeast significantly increased the OC by 20.01-37.25 %. Suppression of GhPRXR1 gene expression in the virus-induced gene-silenced cotton reduced the OC by 18.11%. Our results contribute to identifying more OC QTLs and verifying a candidate gene that influences cottonseed oil biosynthesis.

Physiological and biochemical responses to aluminum-induced oxidative stress in two cyanobacterial species.

Phycoremediation technologies significantly contribute to solving serious problems induced by heavy metals accumulation in the aquatic systems. Here we studied the mechanisms underlying Al stress tolerance in two diazotrophic cyanobacterial species, to identify suitable species for Al phycoremediation. Al uptake as well as the physiological and biochemical responses of Anabaena laxa and Nostoc muscorum to 7 days Al exposure at two different concentrations i.e., mild (100 µM) and high dose (200 µM), were investigated. Our results revealed that A. laxa accumulated more Al, and it could acclimatize to long-term exposure of Al stress. Al induced a dose-dependent decrease in photosynthesis and its related parameters e.g., chlorophyll content (Chl a), phosphoenolpyruvate carboxylase (PEPC) and Ribulose‒1,5‒bisphosphate carboxylase/oxygenase (RuBisCo) activities. The affect was less pronounced in A. laxa than N. muscorum. Moreover, Al stress significantly increased cellular membrane damage as indicated by induced H2O2, lipid peroxidation, protein oxidation, and NADPH oxidase activity. However, these increases were lower in A. laxa compared to N. muscorum. To mitigate the impact of Al stress, A. laxa induced its antioxidant defense system by increasing polyphenols, flavonoids, tocopherols and glutathione levels as well as peroxidase (POX), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX) enzymes activities. On the other hand, the antioxidant increases in N. muscorum were only limited to ascorbate (ASC) cycle. Overall, high biosorption/uptake capacity and efficient antioxidant defense system of A. laxa recommend its feasibility in the treatment of Al contaminated waters/soils.

Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid.

BACKGROUND: Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements. RESULTS: We found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export. CONCLUSIONS: Since more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.

Differential response of hexaploid and tetraploid wheat to interactive effects of elevated [CO2] and low phosphorus.

KEY MESSAGE: Hexaploid wheat is more responsive than tetraploid to the interactive effects of elevated [CO2] and low P in terms of carboxylate efflux, enzyme activity and gene expression (TaPT1 and TaPAP). Availability of mineral nutrients to plants under changing climate has become a serious challenge to food security and economic development. An understanding of how elevated [CO2] influences phosphorus (P) acquisition processes at the whole-plant level would be critical in selecting cultivars as well as to maintain optimum yield in limited-P conditions. Wheat (Triticum aestivum and T. durum) grown hydroponically with sufficient and low P concentration were exposed to elevated and ambient [CO2]. Improved dry matter partitioning towards root resulted in increased root-to-shoot ratio, root length, volume, surface area, root hair length and density at elevated [CO2] with low P. Interaction of low P and [CO2] induced activity of enzymes (phosphoenolpyruvate carboxylase, malate dehydrogenase and citrate synthase) in root tissue resulting in twofold increase in carboxylates and acid phosphatase exudation. Physiological absorption capacity of roots showed that plants alter their uptake kinetics by increasing affinity (low Km) in response to elevated [CO2] under low P supply. Increased relative expression of genes, purple acid phosphatase (TaPAP) and high-affinity Pi transporter (TaPT1) in roots induced by elevated [CO2] and low P supported our physiological observations. Hexaploid wheat (PBW-396) being more responsive to elevated [CO2] at low P supply as compared to tetraploid (PDW-233) necessitates the ploidy effect to be explored further which might be advantageous under changing climate.

Heterologous Expression of Key C and N Metabolic Enzymes Improves Re-assimilation of Photorespired CO2 and NH3, and Growth.

We investigated the effect of the heterologous expression of phosphoenolpyruvate carboxylase (ZmPepcase), aspartate aminotransferase (GmAspAT), and glutamine synthetase (NtGS) on carbon (C) and nitrogen (N) metabolism in Arabidopsis (Arabidopsis thaliana). These transgenes were expressed either separately or in different combinations. The highest gains in shoot dry weight were observed in transgenic lines coexpressing all three genes. Tracer experiments using NaH14CO3 suggested that the coexpression of ZmPepcase, GmAspAT, and NtGS resulted in a higher flux of assimilated CO2 toward sugars and amino acids. Upon feeding the leaf discs with glycine-1-14C, transgenic lines evolved significantly lower 14CO2 levels than the wild type, suggesting that a higher reassimilation of CO2 evolved during photorespiration. Leaves of transgenic plants accumulated significantly lower ammonium without any significant difference in the levels of photorespiratory ammonium relative to the wild type, suggesting a higher reassimilation of photorespired NH3 Transgenic lines also showed improved photosynthetic rates, higher shoot biomass accumulation, and improved seed yield in comparison with wild-type plants under both optimum and limiting N conditions. This work demonstrates that the heterologous coexpression of ZmPepcase, GmAspAT, and NtGS reduced the photorespiratory loss of C and N with concomitant enhancements in shoot biomass and seed yield.

Transcript profiling indicates a widespread role for bacterial-type phosphoenolpyruvate carboxylase in malate-accumulating sink tissues.

Phosphoenolpyruvate carboxylase (PEPC) is an important regulatory enzyme situated at a key branch point of central plant metabolism. Plant genomes encode several plant-type PEPC (PTPC) isozymes, along with a distantly related bacterial-type PEPC (BTPC). BTPC is expressed at high levels in developing castor oil seeds where it tightly interacts with co-expressed PTPC polypeptides to form unusual hetero-octameric Class-2 PEPC complexes that are desensitized to allosteric inhibition by L-malate. Analysis of RNA-Seq and microarray transcriptome datasets revealed two distinct patterns of tissue-specific BTPC expression in vascular plants. Species such as Arabidopsis thaliana, strawberry, rice, maize, and poplar mainly exhibited pollen- or floral-specific BTPC expression. By contrast, BTPC transcripts were relatively abundant in developing castor, cotton, and soybean seeds, cassava tubers, as well as immature tomato, cucumber, grape, and avocado fruit. Immunoreactive 118 kDa BTPC polypeptides were detected on immunoblots of cucumber and tomato fruit extracts. Co-immunoprecipitation established that as in castor, BTPCs physically interact with endogenous PTPCs to form Class-2 PEPC complexes in tomato and cucumber fruit. We hypothesize that Class-2 PEPCs simultaneously maintain rapid anaplerotic PEP carboxylation and respiratory CO2 refixation in diverse, biosynthetically active sinks that accumulate high malate levels.

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 µM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKß are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS.

Physiological investigation of C4-phosphoenolpyruvate-carboxylase-introduced rice line shows that sucrose metabolism is involved in the improved drought tolerance.

We compared the drought tolerance of wild-type (WT) and transgenic rice plants (PC) over-expressing the maize C4PEPC gene, which encodes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) gene, and evaluated the roles of saccharide and sugar-related enzymes in the drought response. Pot-grown seedlings were subjected to real drought conditions outdoors, and the yield components were compared between PC and untransformed wild-type (WT) plants. The stable yield from PC plants was associated with higher net photosynthetic rate under the real drought treatment. The physiological characters of WT and PC seedlings under a simulated drought treatment (25% (w/v) polyethylene glycol-6000 for 3 h; PEG 6000 treatment) were analyzed in detail for the early response of drought. The relative water content was higher in PC than in WT, and PEPC activity and the C4-PEPC transcript level in PC were elevated under the simulated drought conditions. The endogenous saccharide responses also differed between PC and WT under simulated drought stress. The higher sugar decomposition rate in PC than in WT under drought analog stress was related to the increased activities of sucrose phosphate synthase, sucrose synthase, acid invertase, and neutral invertase, increased transcript levels of VIN1, CIN1, NIN1, SUT2, SUT4, and SUT5, and increased activities of superoxide dismutase and peroxidase in the leaves. The greater antioxidant defense capacity of PC and its relationship with saccharide metabolism was one of the reasons for the improved drought tolerance. In conclusion, PEPC effectively alleviated oxidative damage and enhanced the drought tolerance in rice plants, which were more related to the increase of the endogenous saccharide decomposition. These findings show that components of C4 photosynthesis can be used to increase the yield of rice under drought conditions.

Glucose deprivation induced upregulation of phosphoenolpyruvate carboxykinase modulates virulence in Leishmania donovani.

Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP+ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.

The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.

Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca2+-dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser11 in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca2+-dependent Ser11 phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca2+ ions [K0.5(Ca2+) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser11 phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser451, RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca2+-signaling and the control of photosynthate partitioning during COS development.

Glyoxylate cycle and metabolism of organic acids in the scutellum of barley seeds during germination.

During the developmental processes from dry seeds to seedling establishment, the glyoxylate cycle becomes active in the mobilization of stored oils in the scutellum of barley (Hordeum vulgare L.) seeds, as indicated by the activities of isocitrate lyase and malate synthase. The succinate produced is converted to carbohydrates via phosphoenolpyruvate carboxykinase and to amino acids via aminotransferases, while free organic acids may participate in acidifying the endosperm tissue, releasing stored starch into metabolism. The abundant organic acid in the scutellum was citrate, while malate concentration declined during the first three days of germination, and succinate concentration was low both in scutellum and endosperm. Malate was more abundant in endosperm tissue during the first three days of germination; before citrate became predominant, indicating that malate may be the main acid acidifying the endosperm. The operation of the glyoxylate cycle coincided with an increase in the ATP/ADP ratio, a buildup of H2O2 and changes in the redox state of ascorbate and glutathione. It is concluded that operation of the glyoxylate cycle in the scutellum of cereals may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis.

New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.

Establishment and characterisation of a novel bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line.

Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 µM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.