Organic Acid Synthesis Research Articles

Ecological functions of uncultured microorganisms in the cobalt-rich ferromanganese crust of a seamount in the central Pacific are elucidated by fosmid sequencing

Cobalt-rich ferromanganese is an important seafloor mineral and is abundantly present in the seamount crusts. Such crusts form potential hotspots for biogeochemical activity and microbial diversity, yet our understanding of their microbial communities is lacking. In this study, a cultivation-independent approach was used to recover genomic information and derive ecological functions of the microbes in a sediment sample collected from the cobalt-rich ferromanganese crust of a seamount region in the central Pacific. A total of 78 distinct clones were obtained by fosmid library screening with a 16S rRNA based PCR method. Proteobacteria and MGI Thaumarchaeota dominated the bacterial and archaeal 16S rRNA gene sequence results in the microbial community. Nine fosmid clones were sequenced and annotated. Numerous genes encoding proteins involved in metabolic functions and heavy metal resistance were identified, suggesting alternative metabolic pathways and stress responses that are essential for microbial survival in the cobalt-rich ferromanganese crust. In addition, genes that participate in the synthesis of organic acids and exoploymers were discovered. Reconstruction of the metabolic pathways revealed that the nitrogen cycle is an important biogeochemical process in the cobalt-rich ferromanganese crust. In addition, horizontal gene transfer (HGT) events have been observed, and most of them came from bacteria, with some occurring in archaea and plants. Clone W4-93a, belonging to MGI Thaumarchaeota, contained a region of gene synteny. Comparative analyses suggested that a high frequency of HGT events as well as genomic divergence play important roles in the microbial adaption to the deep-sea environment.

Water and carbon dioxide as the main precursors of organic matter on Earth and in space

The article considers possible ways of the origin of organic matter from inorganic components on the planetary and space scales. A special attention is given to practical possibility of the synthesis of organic acids, amino acids, nucleic bases from carbon dioxide and water under the effects of ultraviolet radiation under conditions of the Earth and other celestial bodies. The obtained data make it possible to put forward the concept of the origin of life in the Universe based on the presence in the outer space of water molecules, carbon dioxide and the external source of energy in the form of space radiation.

Use of probiotics to reduce faecal shedding of Shiga toxin-producing Escherichia coli in sheep.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic, foodborne pathogens of humans. Ruminants, including sheep, are the primary reservoirs of STEC and there is a need to develop intervention strategies to reduce the entry of STEC into the food chain. The initiation of the majority of bacterial, enteric infections involves colonisation of the gut mucosal surface by the pathogen. However, probiotic bacteria can serve to decrease the severity of infection via a number of mechanisms including competition for receptors and nutrients, and/or the synthesis of organic acids and bacteriocins that create an environment unfavourable for pathogen development. The aim of the current study was to determine whether the administration of a probiotic mixture to sheep experimentally infected with a non-O157 STEC strain, carrying stx1, stx2 and eae genes, was able to decrease faecal shedding of the pathogen. The probiotic mixture contained Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Streptococcus thermophilus and Enterococcus faecium. The numbers of non-O157 STEC in faecal samples collected from sheep receiving daily doses of the probiotic mixture were significantly lower at the 3rd, 5th and 6th week post-inoculation when compared to the levels recorded in untreated animals. It was concluded that administration of the probiotic mixture reduced faecal shedding of non-O157 STEC in sheep, and holds potential as a pre-harvest intervention method to reduce transmission to humans.

An RNA-Sequencing Study of the Genes and Metabolic Pathways Involved in Aspergillus niger Weathering of Potassium Feldspar

Microbial transformation of potassium feldspar to produce organic composite potassium fertilizer is recognized to be an important method of effective use of the huge reserves of low grade K+-bearing rock in China. The mechanism underlying microbial weathering of silicate minerals is still unclear, and this is an obstacle to practical methods of application. To thoroughly understand the molecular mechanism responsible for the weathering of potassium feldspar by Aspergillus niger at a molecular level, high-throughput RNA-sequencing (RNA-seq) and treatment with different potassium sources (cultured in Czapek medium with soluble K+ or potassium feldspar) were used to investigate the differentially expressed genes of A. niger associated with potassium feldspar weathering and the related metabolic pathways. A series of differentially expressed genes related to the synthesis and transportation of organic acids, polysaccharides, and proteins (enzymes) were found to be closely associated with the K+ released from minerals through bioinformatic analysis. In addition, 12 genes that showed apparent expression differences by RNA-seq analysis and are relevant to organic acid synthesis, protein modification, maintenance of cellular homeostasis, and material transportation, were selected to be further verified using RT-qPCR. Compared to the fungal samples cultured with soluble K+, those with potassium feldspar have certain genes that are more up-regulated, such as the genes for Na+,K+-ATPase (447.6 multiples), cystathionine beta-synthase (5.6 multiples), cysteine synthase (9 multiples), and glutathione synthase (3.5 multiples). The analysis indicates that A. niger weathering of potassium feldspar is due to the synergistic effect of many factors including the up-regulation of certain genes and activation of related metabolite pathways. The research improves our understanding of the mechanisms of microbial weathering of silicate minerals.

Gene co-expression network and function modules in three types of glioma.

The aim of the present study was to identify the disease‑associated genes and their functions involved in the development of three types of glioma (astrocytoma, glioblastoma and oligodendroglioma) with DNA microarray technology, and to analyze their differences and correlations. First, the gene expression profile GSE4290 was downloaded from the Gene Expression Omnibus database, then the probe‑level data were pre‑processed and the differentially expressed genes (DEGs) were identified with limma package in R language. Gene functions of the selected DEGs were further analyzed with the Database for Annotation, Visualization and Integrated Discovery. After the co‑expression network of DEGs was constructed by Cytoscape, the functional modules were mined and enrichment analysis was performed, and then the similarities and differences between any two types of glioma were compared. A total of 1151 genes between normal and astrocytoma tissues, 684 genes between normal and malignant glioma tissues, and 551 genes between normal and oligodendroglioma tissues were filtered as DEGs, respectively. By constructing co‑expression networks of DEGs, a total of 77232, 455 and 987 interactions were involved in the differentially co‑expressed networks of astrocytoma, oligodendroglioma and glioblastoma, respectively. The functions of DEGs were consistent with the modules in astrocytoma, glioblastoma and oligodendroglioma, which were mainly enriched in neuron signal transmission, immune responses and synthesis of organic acids, respectively. Model functions of astrocytoma and glioblastoma were similar (mainly related with immune response), while the model functions of oligodendroglioma differed markedly from that of the other two types. The identification of the associations among these three types of glioma has potential clinical utility for improving the diagnosis of different types of glioma in the future. In addition, these results have marked significance in studying the underlying mechanisms, distinguishing between normal and cancer tissues, and examining novel therapeutic strategies for patients with glioma.

Effect of physicochemical factors on glycerol production by simultaneous cultures of wine micro-organisms using the response surface method.

To evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc2, Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). Multifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6 days yeasts prevailed, especially S. cerevisiae (10(9) CFU ml(-1)), while O. oeni reached 10(7) CFU ml(-1). At initial fixed pH 5·5, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26°C, 50 mg l(-1) SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26°C, 60·24 mg l(-1) SO2 and pH 5·5 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. We propose a predictive condition to improve the performance of mixed cultures for must fermentations. To optimize the culture conditions to design mixed starters containing autochthonous yeasts and O. oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics.

Synthesis of high-value organic acids from sugars promoted by hydrothermally loaded Cu oxide species on magnesia

Design of a suitable catalyst for the conversion of inedible-biomass, a renewable resource, into high-value chemicals is an immense and important area of research in an era of energy crisis. This paper demonstrates batch conversion of sugars into lactic acid (LA) and formic acid (FA) employing a supported copper catalyst. A magnesia-supported copper catalyst was synthesized by a hydrothermal methodology using CTAB as the capping agent (denoted as CuCTAB/MgO). We found that the CuCTAB/MgO not only dramatically boosted the yields of LA and FA from sugars but also decreased the energy demand of the process by decreasing the reaction temperature from 523K to 393K. The high yields of LA (70%) in the presence of NaOH and of FA (65%) in the presence of H2O2 were achieved from glucose at 393K in water using a CuCTAB/MgO catalyst, which could be recycled without any significant loss of activity. The copper catalyst was also found to exhibit excellent activity for the transformation of other sugars. The catalyst was characterized using PXRD, H2-TPR, N2 adsorption–desorption, and other analytical techniques to investigate the active Cu species and propose a plausible mechanistic pathway to LA.

Short-term supply of elevated phosphate alters the belowground carbon allocation costs and functions of lupin cluster roots and nodules

The legume Lupinus albus is able to survive under low nutrient conditions due to the presence of two specialized below ground organs for the acquisition of nitrogen and phosphate, respectively.In this regard, cluster roots increase phosphate uptake and root nodules acquire atmospheric N₂via biological nitrogen fixation(BNF). Although these organs normally tolerate low phosphate conditions, very little is known about their physiological and metabolic flexibility during short-term changes in phosphate supply. The aim of this investigation was therefore to determine the physiological and metabolic flexibility of these organs during short-term supply of elevated phosphate nutrition. L. albus was cultivated in sand culture for 4 weeks at 0.1 mM phosphate supply, and then supplied with 2 mM phosphate for 2 weeks. Short-term elevated phosphate supply caused increased allocation of carbon and respiratory costs to nodules, at the expense of cluster root function. This alteration was also reflected in the increase in nodule enzyme activities related to organic acid synthesis, such as Phosphoenol-pyruvate Carboxylase (PEPC), Pyruvate Kinase (PK), Malate Dehydrogenase(NADH-MDH) and Malic Enzyme (ME). In cluster roots, elevated phosphate conditions caused a decline in these organic acid synthesizing enzymes. Phosphate recycling via Acid Phosphatase (APase),declined in nodules with elevated phosphate supply, but increased in cluster roots. Our findings suggest that during short-term elevated phosphate supply, there is a great degree of physiological and metabolic flexibility in lupin nutrient acquiring structures, and that these changes are related to the altered physiology of these organs [corrected].

Synthesis and Characterization of Ultraviolet Light-Emitting Organic Acids

Three ultraviolet light-emitting organic acids of 3,3'-(4-phenyl-4H-1,2,4-triazole-3,5-diyl)dibenzoic acid (Tz-1), 4,4',4″-(4H-1,2,4-triazole-3,4,5-triyl)tribenzoic acid (Tz-2), and 4,4'-(4-(4'-carboxy-[1,1'-biphenyl]-4-yl)-4H-1,2,4-triazole-3,5-diyl)dibenzoic acid (Tz-3) were successfully synthesized and fully characterized by the (1)H NMR, the IR absorption spectra, and the X-ray single crystal diffraction. It was found that Tz-1, Tz-2, and Tz-3 could give out the ultraviolet photoluminescent spectra centered at 369 nm, 365 nm and 350 nm, respectively. The luminescence quantum yields of Tz-1 and Tz-2 were measured to be 0.20 and 0.14, respectively. Additionally, the density functional theory (DFT) and the time-dependent DFT calculations were also carried out for Tz-1, Tz-2, and Tz-3.

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis.Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04CmolOA/Cmollac) and a higher P(3HB) production (18.88gP(3HB)/L) were achieved, comparing to the original recombinant strain (0.11CmolOA/Cmollac and 7.8gP(3HB)/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28CmolP(3HB)/Cmollac vs 0.10CmolP(3HB)/Cmollac of the original strain).

Fermentation and proteome profiles of Lactobacillus plantarum strains during growth under food-like conditions

This study aimed at investigating the proteomic adaptation of Lactobacillus plantarum strains. Cultivation of L. plantarum strains under food-like conditions (wheat flour hydrolyzed, whey milk, tomato juice) affected some metabolic traits (e.g., consumption of carbohydrates and synthesis of organic acids) compared to de Man, Rogosa and Sharpe (MRS) broth. The analysis of the fermentation profile showed that the highest number of carbon sources metabolized by L. plantarum strains was found using cells cultivated in media containing low concentration of glucose or no glucose at all. The proteomic maps of the strains were comparatively determined after growth on MRS broth and under food-like conditions. The amount of proteins depended on strain and, especially, on culture conditions. Proteins showing decreased or increased amounts under food-like conditions were identified using MALDI-TOF-MS/MS or LC-nano-ESI-MS/MS. Changes of the proteome concerned proteins that are involved in carbohydrate transport and metabolism, energy metabolism, Sec-dependent secretion system, stress response, nucleotide metabolism, regulation of nitrogen metabolism, and protein biosynthesis. A catabolic repression by glucose on carbohydrate transport and metabolism was also found. The characterization of the proteomes in response to changing environmental conditions could be useful to get L. plantarum strains adapted for specific applications. Microbial cell performance during food biotechnological processes has become one of the greatest concerns all over the world. L. plantarum is a lactic acid bacterium with a large industrial application for fermented foods or functional foods (e.g., probiotics). The present study compared the fermentation and proteomic profiling of L. plantarum strains during growth under food-like conditions and under optimal laboratory conditions (MRS broth). This study provides specific mechanisms of proteomic adaptation involved in the microbial performances (carbohydrates utilization, energy metabolism, stress resistance, etc.) affecting the main biotechnological tracts of L. plantarum strains. The finding of this study provides evidences that may be exploited to get strains adapted for specific applications in food biotechnology.

Interactions between Bifidobacterium and Bacteroides Species in Cofermentations Are Affected by Carbon Sources, Including Exopolysaccharides Produced by Bifidobacteria

Cocultures of strains from two Bifidobacterium and two Bacteroides species were performed with exopolysaccharides (EPS) previously purified from bifidobacteria, with inulin, or with glucose as the carbon source. Bifidobacterium longum NB667 and Bifidobacterium breve IPLA20004 grew in glucose but showed poor or no growth in complex carbohydrates (inulin, EPS E44, and EPS R1), whereas Bacteroides grew well in the four carbon sources tested. In the presence of glucose, the growth of Bacteroides thetaiotaomicron DSM-2079 was inhibited by B. breve, whereas it remained unaffected in the presence of B. longum. Ba. fragilis DSM-2151 contributed to a greater survival of B. longum, promoting changes in the synthesis of short-chain fatty acids (SCFA) and organic acids in coculture with respect to monocultures. In complex carbohydrates, cocultures of bifidobacterium strains with Ba. thetaiotaomicron did not modify the behavior of Bacteroides nor improve the poor growth of bifidobacteria. The metabolic activity of Ba. fragilis in coculture with bifidobacteria was not affected by EPS, but greater survival of bifidobacteria at late stages of incubation occurred in cocultures than in monocultures, leading to a higher production of acetic acid than in monocultures. Therefore, cocultures of Bifidobacterium and Bacteroides can behave differently against fermentable carbohydrates as a function of the specific characteristics of the strains from each species. These results stress the importance of considering specific species and strain interactions and not simply higher taxonomic divisions in the relationship among intestinal microbial populations and their different responses to probiotics and prebiotics.

EFFECT OF ALKALINE POTASSIUM AND SODIUM SALTS ON GROWTH, PHOTOSYNTHESIS, IONS ABSORPTION AND SOLUTES SYNTHESIS OF WHEAT SEEDLINGS

SUMMARYPotassium (K) is an essential nutrient and abundant cation in plant cells. The application of K+could alleviate abiotic stress. However, it was reported that the alleviation of K+on salt-stressed plants only happened when K+concentration was low. Most studies were focused on effects of sodium salts on plants in salty soils, and little information was reported about potassium salts, especially a higher level of potassium in alkaline salts. To explore the effects of K+in alkaline salts on plant growth, and whether it had a same destructive impact as Na+, we mixed two alkaline sodium salts (ASS) (NaHCO3:Na2CO3= 9:1) and two alkaline potassium salts (APS) (KHCO3:K2CO3= 9:1) to treat 10-day-old wheat seedlings. Effects of ASS and APS on growth, photosynthesis, ions absorption and solutes accumulation were compared. Results indicated that effects of potassium salts in soil on plants growth were related to K+concentration. Both growth and photosynthesis of wheat seedlings decreased, and the reduction was higher in APS treatment than in ASS treatment at 40 mM alkalinity. ASS treatment absorbed Na+, competing with K+and free Ca2+, and inhibited the absorption of inorganic anions. APS treatments accumulated K+and reduced the absorption of anions, with no competition with other cations. Both APS and ASS treatments promoted free Mg2+accumulation and inhibited H2PO4−uptake. The reduction of H2PO4−promoted organic acid synthesis indirectly. Soluble sugar and proline accumulation were also related to the alkaline condition and extra K+addition. In conclusion, excess potassium ions in soil, especially in alkaline soils, were harmful to plants. APS was another severe salt stress, intensity of which was higher than ASS. The growth and physiological response mechanisms of wheat seedlings to APS were similar to ASS. Both inorganic ions and organic solutes took part in the osmotic adjustment. Differences for APS depended on K+, but ASS on Na+.

Carbonylation of halides using catalysts based on cobalt carbonyl: a promising approach to the synthesis of organic acids as intermediates in drug production

A universal method for the synthesis of fatty aromatic, aromatic, and heteroaromatic acids that was based on carbonylation under mild conditions using catalysts based on modified cobalt carbonyl was developed and could be used for drug production.

Evaluation of Two Transgenes for Aluminum Tolerance in Alfalfa

ABSTRACTAlfalfa (Medicago sativa L.) production is dramatically reduced in acidic, Al‐rich soil. Transgenic plants of several species overexpressing organic acid synthesis and/or organic acid transporter genes have shown enhanced tolerance to Al. The objective of this research was to evaluate the effect of the citrate synthase (CS) and the Daucus carota L. plasma membrane H+‐transporting adenosine triphosphatase (H+ATPase) (DcPA1) transgenes on Al tolerance in alfalfa. Stem cuttings from a full‐sib T2 population including four isogenic groups (plants with neither transgene, CS only, DcPA1 only, and both transgenes) together with nontransformed check genotypes were evaluated for Al and acid soil tolerance in a greenhouse assay in limed and unlimed soil. Aluminum and acid soil tolerance was assessed by measuring the ratios of root and shoot dry weight in unlimed soil compared to limed soil. The three transgenic populations, CS, DcPA1, and CS plus DcPA1, all showed higher Al and acid soil tolerance and lower levels of Al in shoot tissue than the nontransgenic isogenic population or the nontransgenic parental genotypes. This suggests that an Al‐exclusion mechanism could be driving Al and acid soil tolerance in this study. We observed no advantage of combining both transgenes in the same genetic background. These transgenes offer an efficient method to achieve enhanced Al and acid soil tolerant alfalfa cultivars, but more information is needed on their stability across generations and genetic backgrounds and their performance under field conditions.

Synthesis of organic phosphinic acids and studies on the relationship between their structure and extraction–separation performance of heavy rare earths from HNO3 solutions

Efficient new extractant design relies on systematic structure–property studies. However, systematic structure–property research on extractants of organic phosphinic acids is far from sufficient. To make up for this deficiency, twelve organic phosphinic acid extractants (including nine dialkylphosphinic acids, two dicycloalkylphosphinic acids and one diarylphosphinic acid) were synthesized with free-radical-addition-reaction method and Grignard-reaction method. Reaction rules, advantages and disadvantages of these two methods were discussed respectively. The extraction ability and separation performance of the synthesized organic phosphinic acids for Tm, Yb and Lu from HNO3 solutions were evaluated and compared. For dialkylphosphinic acids the nearer the branched chain moved to P atom, the more reduction generated on the extraction ability. Dialkylphosphinic acids without branched chain in α-C or β-C (e.g. HYY-3, HYY-5, HYY-7 and HYY-9) exhibited strong extraction ability as P507. On the contrary, dialkylphosphinic acids with branched chain in α-C (e.g. HYY-4, HYY-8) and dicyclohexylphosphinic acids exhibited poor extraction ability as Cyanex 272. New compound Di-(2,3-dimethylbutyl)-phosphinic acid (HYY-2) exhibited good extraction ability and separation performance and its separation factors of Lu/Yb, Yb/Tm and Lu/Tm were 1.42, 3.10 and 4.40 respectively, apparently superior to Cyanex 272 (βLu/Yb=1.31, βYb/Tm=3.25, βLu/Tm=4.26).

Nickel-induced changes in carbon metabolism in wheat shoots

In this study, we analyzed the toxic effect of Ni during the development of wheat shoots. Typical developmental alterations in carbon metabolism-related parameters reflecting changes associated with the transition of the seedlings from heterotrophic to autotrophic metabolism were observed in the control shoots between the 1st and the 4th days. Adverse effects of 50 and 100μM Ni became evident starting from the 4th day of growth on the metal-containing media. We found that Ni-induced stimulation of phosphoenolpyruvate carboxylase (PEPC) activity coincided with decrease in the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) level and with declines in net photosynthetic rate (PN) and stomatal conductance (gs). Application of Ni resulted in increased activities of several dehydrogenases: glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (NADP-ICDH) and malate dehydrogenase (NADH-MDH). In contrast, the activities of malic enzymes (NADP-ME and NAD-ME) decreased due to Ni stress. Treatment with Ni led to accumulation of glucose and declined concentration of sucrose as well as considerable increases in concentrations of malic and citric acids. Our results indicate that Ni stress redirects the carbon metabolism of developing wheat shoots to provide carbon skeletons for synthesis of amino acids and organic acids as well as to supply reducing power to sustain normal metabolic processes and to support defense mechanisms against oxidative stress.

Bisimidazole and Bisimidazolium Cruciforms: Synthesis and Discrimination of Organic Acids

通过Sonogashira偶联反应、N-芳基化反应和季铵化反应, 我们构筑了一类基于咪唑（鎓）的十字架型荧光分子4和5, 研究了烷基链以及咪唑和咪唑鎓分别作为电子受体对十字架型分子荧光性质的影响. 其中, 分子4a具有较高的荧光量子产率和大的斯托克斯位移; 在不同极性的溶剂中表现出明显的溶致变色性质, 荧光颜色覆盖由深蓝色到黄色区域; 同时, 该分子还具有明显的聚集诱导发光特性. 我们采用数码照相的方法代替繁琐的荧光光谱仪, 仅使用单一该荧光分子在两种有机溶剂中即可通过荧光颜色有效区分15种结构类似或pKa相近的有机酸. 最后, 分子4a和5a固载于硅胶板上通过可逆的荧光颜色变化实现了对HCl/Et3N气体的识别.

Fumarate and cytosolic pH as modulators of the synthesis or consumption of C4 organic acids through NADP-malic enzyme in Arabidopsis thaliana

Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1-4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C(4) compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.

Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco

Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30 μM aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The 13C-NMR analysis with NaH13CO3 showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500 μM Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.