Vulgarin, a Sesquiterpene Lactone from Artemisia judaica, Improves the Antidiabetic Effectiveness of Glibenclamide in Streptozotocin-Induced Diabetic Rats via Modulation of PEPCK and G6Pase Genes Expression.

The current investigation assessed the effect of the eudesmanolid, Vulgarin (VGN), obtained from Artemisia judaica (A. judaica), on the antidiabetic potential of glibenclamide (GLB) using streptozotocin (STZ) to induce diabetes. Seven groups of rats were used in the study; the first group received the vehicle and served as normal control. The diabetic rats of the second to the fifth groups were treated with the vehicle (negative control), GLB at 5 mg/kg (positive control), VGN at 10 mg/kg (VGN-10) and VGN at 20 mg/kg (VGN-20), respectively. The diabetic rats of the sixth and seventh groups were administered combinations of GLB plus VGN-10 and GLB plus VGN-20, respectively. The diabetic rats treated with GLB plus VGN-20 combination showed marked improvement in the fasting blood glucose (FBG), insulin and glycated hemoglobin (HbA1c), as well as the lipid profile, compared with those treated with GLB alone. Further, the pancreatic tissues of the diabetic rats that received the GLB+VGN-20 combination showed superior improvements in lipid peroxidation and antioxidant parameters than those of GLB monotherapy. The insulin content of the ß-cells was restored in all treatments, while the levels of glucagon and somatostatin of the α- and δ-endocrine cells were reduced in the pancreatic islets. In addition, the concurrent administration of GLB+VGN-20 was the most effective in restoring PEPCK and G6Pase mRNA expression in the liver. In conclusion, the results demonstrated that the GLB+VGN-20 combination led to greater glycemic improvement in diabetic rats compared with GLB monotherapy through its antioxidant effect and capability to modulate PEPCK and G6Pase gene expression in their livers.

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.

Pattern analysis suggests that phosphoenolpyruvate carboxylase in maturing soybean seeds promotes the accumulation of protein.

The protein and oil contents in soybean seeds are major factors in seed quality. Seed proteins and oils are synthesized from sucrose and nitrogenous compounds transported into maturing seeds. In this study, we compared changes in the activity of phosphoenolpyruvate carboxylase (PEPC) and the accumulation profiles of protein and oil in maturing seeds of two soybean cultivars, which exhibit different protein and oil contents in seeds, to determine the interrelationships of them. A principal component analysis indicated a concordance of seed PEPC activity with the protein content, but did not with the oil content. PEPC activity per seed was highest in the late maturation stage, when the physiological status of the vegetative organs drastically changed. The high-protein cultivar had higher PEPC activity compared to the low-protein cultivar. These results highlight the biological role of PEPC in the synthesis of protein, therefore it was implied that PEPC could be a biomarker in soybean breeding.Abbreviations: ANOVA: analysis of variance; DS: developmental stage; DW: dry weight; FW: fresh weight; NIR: near infrared; PEP(C): phosphoenolpyruvate (carboxylase); PC(A): principal component (analysis); S.E.: standard error; WC: water content.

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.

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.

Cloning and expression profiling of the PacSnRK2 and PacPP2C gene families during fruit development, ABA treatment, and dehydration stress in sweet cherry.

Plant SNF1-related protein kinase 2 (SnRK2) and protein phosphatase 2C (PP2C) family members are core components of the ABA signal transduction pathway. SnRK2 and PP2C proteins have been suggested to play crucial roles in fruit ripening and improving plant tolerance to drought stress, but supporting genetic information has been lacking in sweet cherry (Prunus avium L.). Here, we cloned six full-length SnRK2 genes and three full-length PP2C genes from sweet cherry cv. Hong Deng. Quantitative PCR analysis revealed that PacSnRK2.2, PacSnRK2.3, PacSnRK2.6, and PacPP2C1-3 were negatively regulated in fruits in response to exogenous ABA treatment, PacSnRK2.4 and PacSnRK2.5 were upregulated, and PacSnRK2.1 expression was not affected. The ABA treatment also significantly promoted the accumulation of anthocyanins in sweet cherry fruit. The expression of all PacSnRK2 and PacPP2C genes was induced by dehydration stress, which also promoted the accumulation of drought stress signaling molecules in the sweet cherry fruits, including ABA, soluble sugars, and anthocyanin. Furthermore, a yeast two-hybrid analysis demonstrated that PacPP2C1 interacts with all six PacSnRK2s, while PacPP2C3 does not interact with PacSnRK2.5. PacPP2C2 does not interact with PacSnRK2.1 or PacSnRK2.4. These results indicate that PacSnRK2s and PacPP2Cs may play a variety of roles in the sweet cherry ABA signaling pathway and the fruit response to drought stress.

The physiological and metabolic changes in sugar beet seedlings under different levels of salt stress.

Salinity stress is a major limitation to global crop production. Sugar beet, one of the world's leading sugar crops, has stronger salt tolerant characteristics than other crops. To investigate the response to different levels of salt stress, sugar beet was grown hydroponically under 3 (control), 70, 140, 210 and 280 mM NaCl conditions. We found no differences in dry weight of the aerial part and leaf area between 70 mM NaCl and control conditions, although dry weight of the root and whole plant treated with 70 mM NaCl was lower than control seedlings. As salt concentrations increased, degree of growth arrest became obvious In addition, under salt stress, the highest concentrations of Na+ and Cl- were detected in the tissue of petioles and old leaves. N and K contents in the tissue of leave, petiole and root decreased rapidly with the increase of NaCl concentrations. P content showed an increasing pattern in these tissues. The activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione peroxidase showed increasing patterns with increase in salt concentrations. Moreover, osmoprotectants such as free amino acids and betaine increased in concentration as the external salinity increased. Two organic acids (malate and citrate) involved in tricarboxylic acid (TCA)-cycle exhibited increasing contents under salt stress. Lastly, we found that Rubisco activity was inhibited under salt stress. The activity of NADP-malic enzyme, NADP-malate dehydrogenase and phosphoenolpyruvate carboxylase showed a trend that first increased and then decreased. Their activities were highest with salinity at 140 mM NaCl. Our study has contributed to the understanding of the sugar beet physiological and metabolic response mechanisms under different degrees of salt stress.

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.

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.

Interactions between light intensity and phosphorus nutrition affect the phosphate-mining capacity of white lupin (Lupinus albus L.).

Light intensity affects photosynthetic carbon (C) fixation and the supply of carbon to roots. To evaluate interactions between carbon supply and phosphorus (P) supply, effects of light intensity on sucrose accumulation, root growth, cluster root formation, carboxylate exudation, and P uptake capacity were studied in white lupin (Lupinus albus L.) grown hydroponically with either 200 µmol m(-2) s(-1) or 600 µmol m(-2) s(-1) light and a sufficient (50 µM P) or deficient (1 µM P) P supply. Plant biomass and root:shoot ratio increased with increasing light intensity, particularly when plants were supplied with sufficient P. Both low P supply and increasing light intensity increased the production of cluster roots and citrate exudation. Transcripts of a phosphoenol pyruvate carboxylase gene (LaPEPC3) in cluster roots (which is related to the exudation of citrate), transcripts of a phosphate transporter gene (LaPT1), and P uptake all increased with increasing light intensity, under both P-sufficient and P-deficient conditions. Across all four experimental treatments, increased cluster root formation and carboxylate exudation were associated with lower P concentration in the shoot and greater sucrose concentration in the roots. It is suggested that C in excess of shoot growth capabilities is translocated to the roots as sucrose, which serves as both a nutritional signal and a C-substrate for carboxylate exudation and cluster root formation.

Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase by a Ca2+-dependent protein kinase suggests a link between Ca2+ signalling and anaplerotic pathway control in developing castor oil seeds.

The aim of the present study was to characterize the native protein kinase [BTPC (bacterial-type phosphoenolpyruvate carboxylase)-K (BTPC Ser451 kinase)] that in vivo phosphorylates Ser451 of the BTPC subunits of an unusual Class-2 PEP (phosphoenolpyruvate) carboxylase hetero-octameric complex of developing COS (castor oil seeds). COS BTPC-K was highly purified by PEG fractionation and hydrophobic size-exclusion anion-exchange and affinity chromatographies. BTPC-K phosphorylated BTPC strictly at Ser451 (Km=1.0 µM; pH optimum=7.3), a conserved target residue occurring within an intrinsically disordered region, as well as the protein histone III-S (Km=1.7 µM), but not a COS plant-type PEP carboxylase or sucrose synthase or α-casein. Its activity was Ca2+- (K0.5=2.7 µM) and ATP- (Km=6.6 µM) dependent, and markedly inhibited by trifluoperazine, 3-phosphoglycerate and PEP, but insensitive to calmodulin or 14-3-3 proteins. BTPC-K exhibited a native molecular mass of ~63 kDa and was soluble rather than membrane-bound. Inactivation and reactivation occurred upon BTPC-K's incubation with GSSG and then DTT respectively. Ser451 phosphorylation by BTPC-K inhibited BTPC activity by ~50% when assayed under suboptimal conditions (pH 7.3, 1 mM PEP and 10 mM L-malate). Our collective results indicate a possible link between cytosolic Ca2+ signalling and anaplerotic flux control in developing COS.

Effects of granulation on organic acid metabolism and its relation to mineral elements in Citrus grandis juice sacs.

We investigated the effects of granulation on organic acid metabolism and its relation to mineral elements in 'Guanximiyou' pummelo (Citrus grandis) juice sacs. Granulated juice sacs had decreased concentrations of citrate and isocitrate, thus lowering juice sac acidity. By contrast, malate concentration was higher in granulated juice sacs than in normal ones. The reduction in citrate concentration might be caused by increased degradation, as indicated by enhanced aconitase activity, whilst the increase in malate concentration might be caused by increased biosynthesis, as indicated by enhanced phosphoenolpyruvate carboxylase (PEPC). Real time quantitative reverse transcription PCR (qRT-PCR) analysis showed that the activities of most acid-metabolizing enzymes were regulated at the transcriptional level, whilst post-translational modifications might influence the PEPC activity. Granulation led to increased accumulation of mineral elements (especially phosphorus, magnesium, sulphur, zinc and copper) in juice sacs, which might be involved in the incidence of granulation in pummelo fruits.

Thiourea, a ROS scavenger, regulates source-to-sink relationship to enhance crop yield and oil content in Brassica juncea (L.).

In the present agricultural scenario, the major thrust is to increase crop productivity so as to ensure sustainability. In an earlier study, foliar application of thiourea (TU; a non physiological thiol based ROS scavenger) has been demonstrated to enhance the stress tolerance and yield of different crops under field condition. Towards this endeavor, present work deals with the effect of TU on photosynthetic efficiency and source-to-sink relationship of Indian mustard (Brassica juncea) for understanding its mode of action. The application of TU increased the efficiency of both PSI and PSII photosystems and vegetative growth of plant. The comparative analysis of sucrose to starch ratio and expression level of sugar transporters confirmed the higher source and sink strength in response to TU treatment. The biochemical evidence in support of this was derived from higher activities of sucrose phosphate synthase and fructose-1,6-bis-phosphatase at source; and sucrose synthase and different classes of invertases at both source and sink. This indicated an overall increase in photoassimilate level at sink. An additional contribution through pod photosynthesis was confirmed through the analysis of phosphoenol pyruvate carboxylase enzyme activity and level of organic acids. The increased photoassimilate level was also co-ordinated with acetyl coA carboxylase mediated oil biosynthesis. All these changes were ultimately reflected in the form of 10 and 20% increase in total yield and oil content, respectively under TU treatment as compared to control. Additionally, no change was observed in oil composition of seeds derived from TU treated plants. The study thus signifies the co-ordinated regulation of key steps of photosynthesis and source-to-sink relationship through the external application of TU resulting in increased crop yield and oil content.

Antidiabetic effects of rice hull smoke extract on glucose-regulating mechanism in type 2 diabetic mice.

The aim of this study is to determine the protective effect of a liquid rice hull smoke extract (RHSE) against type 2 diabetes (T2D) in mice induced by a high-fat diet. As compared to the control group of mice on a high-fat diet (HFD), feeding the HFD supplemented with 0.5% or 1% RHSE for 7 weeks resulted in significantly reduced blood glucose and triglyceride and cholesterol concentrations, higher serum insulin levels, and improved glucose tolerance, as assessed by an oral glucose tolerance assay. The hypoglycemic effect of RHSE was accompanied by changes in enzyme activities and cognate gene expression assessed using RT-PCR. Among the glucose metabolism regulating genes evaluated, hepatic glucokinase (GCK), the glucose transporters GLUT2 and GLUT4, and peroxisome proliferator-activated receptor-γ (PPAR-γ) were up-regulated, whereas glucose-6-phosphatase (G6 Pase) and phosphoenolpyruvate carboxykinase (PEPCK) were down-regulated in the liver of mice with RHSE-supplementation. These changes resulted in restoration of glucose-regulating activities to normal control levels. Histopathology showed that a high-fat diet intake also induced liver necrosis and damage of the islet of Langerhans in the pancreas, whereas RHSE supplementation restored necrotic damage to normal levels. Immunohistochemistry showed that RHSE supplementation can restore the reduced insulin-producing ß-cell population in islet of Langerhans associated with a high-fat diet intake to nondiabetic normal control levels in a dose-dependent manner. RHSE-supplemented food could protect insulin-producing islet cells against damage triggered by oxidative stress and local inflammation associated with diabetes.

The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.

Constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase enhances stomatal opening and photosynthetic performance of Arabidopsis thaliana.

The effect of constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase (PEPC) on the opening state of stomata and photosynthetic performance in Arabidopsis thaliana plants was studied. Transcript accumulation analyses of the A. thaliana dark-induced (Din10 and Din6) and the Pisum sativum asparagine synthetase 2 promoters (Asn2) in transiently transformed tobacco leaves showed that Din10 promoter induced more DsRed accumulation in the dark compared to the other din genes. Overexpression of PEPC under the control of the constitutive enhanced CaMV 35S (p35SS) and dark-induced Din10 promoter in stably transformed A. thaliana plants increased the number of opened stomata in dark adapted leaves. Gas exchange measurements using A. thaliana plants transgenic for p35SS-PEPC and Din10-PEPC revealed a marked increase in stomatal conductance, transpiration, and dark respiration rates measured in the dark compared to wild-type plants. Moreover, measurement of CO(2) assimilation rates at different external CO(2) concentrations (C(a) ) and different light intensities shows an increase in the CO(2) assimilation rates in transgenic Arabidopsis lines compared to wild-type plants. This is considered as first step towards transferring the aspects of Crassulacean acid metabolism-like photosynthetic mechanism into C3 plants.

Inflammation inhibits the expression of phosphoenolpyruvate carboxykinase in liver and adipose tissue.

Inhibition of adipocyte triglyceride biosynthesis is required for fatty acid mobilization during inflammation. Triglyceride biosynthesis requires glycerol 3-phosphate and phosphoenolpyruvate carboxykinase (PEPCK) plays a key role. We demonstrate that LPS, zymosan, and TNF-α decrease PEPCK in liver and fat. Turpentine decreases PEPCK in liver, but not in fat. The LPS-induced decrease in PEPCK does not occur in TLR4 deficient animals, indicating that this receptor is required. The LPS-induced decrease in hepatic PEPCK does not occur in TNF receptor/IL-1 receptor knockout mice, but occurs in fat, indicating that TNF-α/IL-1 is essential for the decrease in liver but not fat. In 3T3-L1 adipocytes TNF-α, IL-1, IL-6, and IFNγ inhibit PEPCK indicating that there are multiple pathways by which PEPCK is decreased in adipocytes. The binding of PPARγ and RXRα to the PPARγ response element in the PEPCK promoter is markedly decreased in adipose tissue nuclear extracts from LPS treated animals. Lipopolysaccharide and zymosan reduce PPARγ and RXRα expression in fat, suggesting that a decrease in PPARγ and RXRα accounts for the decrease in PEPCK. Thus, there are multiple cytokine pathways by which inflammation inhibits PEPCK expression in adipose tissue which could contribute to the increased mobilization of fatty acids during inflammation.

Characterization of flavins in roots of Fe-deficient strategy I plants, with a focus on Medicago truncatula.

The root accumulation and excretion of riboflavin (Rbfl) and Rbfl derivatives have been studied in the model legume species Medicago truncatula, grown in hydroponics in two different Fe deficiency conditions, with and without CaCO(3). Using high resolution mass spectrometry techniques coupled to liquid chromatography, three different flavin derivatives not previously reported in plants, putatively identified as 7-hydroxy-Rbfl, 7α-hydroxy-Rbfl and 7-carboxy-Rbfl, were found along with Rbfl in Fe-deficient M. truncatula roots. In the presence of CaCO(3) most of the flavins were accumulated in the roots, whereas in the absence of CaCO(3) there was partial export to the nutrient solution. The major flavins in roots and nutrient solution were Rbfl and 7-hydroxy-Rbfl, respectively. Flavins were located in the root cortex and epidermal cells, preferentially in a root region near the apex that also exhibited increased ferric chelate reductase (FCR) activity. Six out of 15 different species of horticultural interest showed root increases in both Rbfl (four of them also having Rbfl derivatives) and FCR. No significant correlation was found between Rbfl and either phosphoenolpyruvate carboxylase or FCR activities, whereas the latter two showed a good correlation between them. The possible roles of Rbfl and Rbfl derivatives in roots and nutrient solutions are discussed. Medicago truncatula is proposed as a model system for flavin studies.

Root release and metabolism of organic acids in tea plants in response to phosphorus supply.

Self-rooted, 10-month-old, uniform tea [Camellia sinensis (L.) O. Kuntze cv. Huangguanyin] plants were supplied for 17 weeks with 0, 40, 80, 160, 400, or 1000µM phosphorus (P) to investigate the effects of P supply on root citrate and malate release, the concentrations of malate and citrate and the activities of acid-metabolizing enzymes in leaves and roots. Root malate release and accumulation was induced by both 0 and 40µM P, while root citrate release and accumulation was induced only by 0µM P. Phosphorus-deficiency-induced malate and citrate release coincided with higher concentrations of root malate and citrate. The higher concentrations of malate and citrate were accompanied by increased activities of phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate phosphatase (PEPP), citrate synthase (CS) and NAD-malic enzyme (NAD-ME) and decreased activities of pyruvate kinase (PK), NADP-ME and NADP-isocitrate dehydrogenase (NADP-IDH) in roots. In contrast to roots, malate accumulated in the leaves only in response to 0µM P, and no change was observed in citrate levels. The P-deficiency-induced leaf malate accumulation coincided with increased activities of NADP-ME, NAD-ME and PK. Overall, the P-deficiency-induced changes in organic acid (OA) metabolism differed between roots and leaves. The high tolerance of tea plants to P-deficiency might be involved in two major processes: (a) increasing the availability of P by inducing root release of OA anions; and (b) improving the ability to use P efficiently by inducing bypass enzymes involved in tissue P economy.