Orthophosphate Dikinase Research Articles

Adaptation responses in C4 photosynthesis of sweet maize (Zea mays L.) exposed to nicosulfuron

Nicosulfuron is an ingredient in photosynthesis-inhibiting herbicides and has been widely used in corn post-emergence weed control. In the current study, a pair of sister lines, HK301 (nicosulfuron-tolerence, NT) and HK320 (nicosulfuron-sensitive, NS), was used to study the effect of nicosulfuron in sweet maize seedlings on C4 photosynthetic enzymes and non-enzymatic substances, expression levels of key enzymes, and chloroplast structure. Nicosulfuron was sprayed at the four-leaf stage, and water was sprayed as a control. After nicosulfuron treatment, phosphoenolpyruvate carboxylase (PEPC), NADP-malic dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), pyruvate orthophosphate dikinase (PPDK), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities of NT were significantly higher than those of NS. Compared to NT, malate, oxaloacetic acid, and pyruvic acid significantly decreased as exposure time increased in NS. Compared to NS, nicosulfuron treatment significantly increased the expression levels of PEPC, NADP-MDH, NADP-ME, PPDK, and Rubisco genes in NT. Under nicosulfuron treatment, chloroplast ultrastructure of NS, compared to that of NT, nicosulfuron induced swelling of the chloroplast volume and reduced starch granules in NS. In general, our results indicate that in different resistant sweet maize, C4 photosynthetic enzymes activity and key genes expression play a critical role in enhancing the adaptability of plants to nicosulfuron stress at a photosynthetic physiological level.

Response of transgenic Arabidopsis expressing maize C4 photosynthetic enzyme genes to high light

ABSTRACT This study assessed the responses of wild-type (WT) and transgenic Arabidopsis expressing seven combinations of maize (Zea mays) genes phosphoenolpyruvate carboxylase (pepc), pyruvate phosphate dikinase (ppdk), and NADP-malic enzyme (nadp-me) to high light. Our results showed that the net CO2 assimilation rate (P n) and shoot dry weight of four of the transgenic Arabidopsis genotypes were significantly different from those of WT under high-light treatment, being in the order of Zmpepc+Zmppdk+Zmnadp-me (PC-K-M) > Zmpepc+Zmppdk (PC-K) > Zmpepc (PC), Zmpepc+Zmnadp-me (PC-M) > WT. The other genotypes did not differ from WT. This indicated that Zmpepc was essential for maintaining high photosynthetic performance under high light, Zmppdk had a positive synergistic effect on Zmpepc, and the combination of all three genes had the greatest synergistic effect. These four genotypes also maintained higher photosystem II (PSII) activity (K-phase, J-phase, RC/CSm), electron transfer capacity (J-phase), and photochemical efficiency (TRo/ABS), and accumulated less reactive oxygen species (O2·−, H2O2) and suffered less damage to the membrane system (MDA) than WT under high light. Collectively, PC, PC-K, PC-M, and PC-K-M used most of the absorbed energy for CO2 assimilation through a significantly higher P n, which reduced the generation of excess electrons in the photosynthetic apparatus, thereby reducing damage to the membrane system and PSII. This ultimately resulted in improved high-light tolerance. P n was the main reason for the significant difference in the high-light tolerance of the four genotypes. Joint expression of the three maize genes may be of great value in the genetic improvement of high-light tolerance in C3 crops.

Glucose Oxidation to Pyruvate Is Not Essential for Brucella suis Biovar 5 Virulence in the Mouse Model.

Brucella species cause brucellosis, a worldwide extended zoonosis. The brucellae are related to free-living and plant-associated α2-Proteobacteria and, since they multiply within host cells, their metabolism probably reflects this adaptation. To investigate this, we used the rodent-associated Brucella suis biovar 5, which in contrast to the ruminant-associated Brucella abortus and Brucella melitensis and other B. suis biovars, is fast-growing and conserves the ancestral Entner-Doudoroff pathway (EDP) present in the plant-associated relatives. We constructed mutants in Edd (glucose-6-phosphate dehydratase; first EDP step), PpdK (pyruvate phosphate dikinase; phosphoenolpyruvate ⇌ pyruvate), and Pyk (pyruvate kinase; phosphoenolpyruvate → pyruvate). In a chemically defined medium with glucose as the only C source, the Edd mutant showed reduced growth rates and the triple Edd-PpdK-Pyk mutant did not grow. Moreover, the triple mutant was also unable to grow on ribose or xylose. Therefore, B. suis biovar 5 sugar catabolism proceeds through both the Pentose Phosphate shunt and EDP, and EDP absence and exclusive use of the shunt could explain at least in part the comparatively reduced growth rates of B. melitensis and B. abortus. The triple Edd-PpdK-Pyk mutant was not attenuated in mice. Thus, although an anabolic use is likely, this suggests that hexose/pentose catabolism to pyruvate is not essential for B. suis biovar 5 multiplication within host cells, a hypothesis consistent with the lack of classical glycolysis in all Brucella species and of EDP in B. melitensis and B. abortus. These results and those of previous works suggest that within cells, the brucellae use mostly 3 and 4 C substrates fed into anaplerotic pathways and only a limited supply of 5 and 6 C sugars, thus favoring the EDP loss observed in some species.

Influence of salt stress on C4 photosynthesis in Miscanthus sinensis Anderss.

Miscanthus sinensis Anderss. is a good candidate for C4 bioenergy crop development for marginal lands. As one of the characteristics of marginal lands, salinization is a major limitation to agricultural production. The present work aimed to investigate the possible factors involved in the tolerance of M. sinensis C4 photosynthesis to salinity stress. Seedlings of two accessions (salt-tolerant 'JM0119' and salt-sensitive 'JM0099') were subjected to 0mm NaCl (control) or 250mm NaCl (salt stress treatment) for 2weeks. The chlorophyll content, parameters of photosynthesis and chlorophyll a fluorescence, activity of C4 enzymes and expression of C4 genes were measured. The results showed that photosynthesis rate, transpiration rate, chlorophyll content, PSII operating efficiency, coefficient of photochemical quenching, activity of phosphoenolpyruvate carboxylase (PEPC) and pyruvate, orthophosphate dikinase (PPDK) and gene expression of PEPC and PPDK under salinity were higher after long-term salinity exposure in 'JM0119' than in 'JM0099', while activity of NADP-malate dehydrogenase (NADP-MDH) and NADP-malic enzyme (NADP-ME), together with expression of NADP-MDH and NADP-ME, were much higher in 'JM0099' than in 'JM0119'. In conclusion, the increased photosynthetic capacity under long-term salt stress in the salt-tolerant relative to the salt-sensitive M. sinensis accession was mainly associated with non-stomatal factors, such as reduced chlorophyll loss, higher PSII operating efficiency, enhanced activity of PEPC and PPDK and relatively lower activity of NADP-ME.

Joint expression of Zmpepc, Zmppdk, and Zmnadp-me is more efficient than expression of one or two of those genes in improving the photosynthesis of Arabidopsis

This study assessed the effects of seven combinations of maize (Zea mays) genes phosphoenolpyruvate carboxylase (pepc), pyruvate phosphate dikinase (ppdk), and NADP-malic enzyme (nadp-me), on the photosynthesis of Arabidopsis. The photosynthetic rate, carboxylation efficiency, and shoot-dry-weight of Zmpepc (PC), Zmpepc + Zmppdk (PCK), Zmpepc + Zmnadp-me (PCM), and Zmpepc + Zmppdk + Zmnadp-me (PCKM) were significantly higher than those of the control wild-type (WT), with a trends to be PCKM > PCK > PC and PCM > WT. This indicated that Zmpepc was a prerequisite for improved photosynthetic performance; Zmppdk had a positive effect on Zmpepc, and the triple gene combination had the most significant synergistic effects. PCKM significantly enhanced activity of photosystem (PS)II (K, J phase) and PSI, light energy absorption (ABS/CSm) and conversion (TRo/ABS), and electron transfer (ETo/TRo). PCKM up-regulated 18 photosynthesis-related proteins, among which, 11 were involved in light reaction resulting in improved light-energy absorption and conversion efficiency, electron transfer, activity and stability of PSII and PSI, and the ATP and NADPH production. The remaining seven proteins were involved in dark reaction. The up-regulation of these proteins in PCKM improved the coordinated operation of light and dark reaction, increasing the photosynthesis and dry weight ultimately. These results also provide a promising strategy for the genetic improvement of the photosynthetic performance of C3 crops by inserting major C4 photosynthetic genes.

Installation of C4 photosynthetic pathway enzymes in rice using a single construct.

SummaryIntroduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two‐cell metabolic prototype for an NADP‐malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP‐malate dehydrogenase, pyruvate orthophosphate dikinase and NADP‐malic enzyme from Zea mays, driven by cell‐preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13CO2 labelling demonstrated a 10‐fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP‐malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.

Hysteresis of pyruvate phosphate dikinase from Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.

A Partial C4 Photosynthetic Biochemical Pathway in Rice

Introduction of a C4 photosynthetic pathway into C3 rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously accumulates four of the core C4 photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME), and pyruvate phosphate dikinase (ZmPPDK). This led to enhanced enzyme activity and mild phenotypic perturbations but was largely neutral in its effects on photosynthetic rate. Measurements of the flux of 13CO2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13C into malate, consistent with increased fixation of 13CO2 via PEP carboxylase in lines expressing the maize PEPC enzyme. However, there was no significant differences in labeling of 3-phosphoglycerate (3PGA) indicating that there was no carbon flux through NADP-ME into the Calvin-Benson cycle. There was also no significant difference in labeling of phosphoenolpyruvate (PEP) indicating that there was no carbon flux through PPDK. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labeling of malate, aspartate and citrate than in the quintuple line. There was evidence of 13C labeling of aspartate indicating 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C4 cycle, these results demonstrate for the first-time a partial flux through the carboxylation phase of NADP-ME C4 metabolism in transgenic rice containing two of the key metabolic steps in the C4 pathway.

Disruption of pyruvate phosphate dikinase in Brucella ovis PA CO2-dependent and independent strains generates attenuation in the mouse model

Brucella ovis is a non-zoonotic rough Brucella that causes genital lesions, abortions and increased perinatal mortality in sheep and is responsible for important economic losses worldwide. Research on virulence factors of B. ovis is necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specific vaccine, a need in areas where the cross-protecting ovine smooth B. melitensis Rev1 vaccine is banned. Although several B. ovis virulence factors have been identified, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of pyruvate phosphate dikinase (PpdK, catalyzing the bidirectional conversion pyruvate ⇌ phosphoenolpyruvate) in B. ovis PA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, this ppdK mutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in a B. ovis PA CO2-independent construct previously proposed for developing B. ovis vaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independent ppdK mutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis of B. ovis ovine brucellosis.

A Novel Vibriophage vB_VcaS_HC Containing Lysogeny-Related Gene Has Strong Lytic Ability against Pathogenic Bacteria.

To avoid the negative effects of antibiotics, using phage to prevent animal disease becomes a promising method in aquaculture. Here, a lytic phage provisionally named vB_VcaS_HC that can infect the pathogen (i.e., Vibrio campbellii 18) of prawn was isolated. The phage has an isometric head and a non-contractile tail. During phage infection, the induced host mortality in 5.5h reached ca. 96%, with a latent period of 1.5h and a burst size of 172 PFU/cell. It has an 81,566bp circular dsDNA genome containing 121 open reading frames (ORFs), and ca. 71% of the ORFs are functionally unknown. Comparative genomic and phylogenetic analysis revealed that it is a novel phage belonging to Delepquintavirus, Siphoviridae, Caudovirales. In the phage genome, besides the ordinary genes related to structure assembly and DNA metabolism, there are 10 auxiliary metabolic genes. For the first time, the pyruvate phosphate dikinase (PPDK) gene was found in phages whose product is a key rate-limiting enzyme involving Embden-Meyerhof-Parnas (EMP) reaction. Interestingly, although the phage has a strong bactericidal activity and contains a potential lysogeny related gene, i.e., the recombinase (RecA) gene, we did not find the phage turned into a lysogenic state. Meanwhile, the phage genome does not contain any bacterial virulence gene or antimicrobial resistance gene. This study represents the first comprehensive characterization of a lytic V. campbellii phage and indicates that it is a promising candidate for the treatment of V. campbellii infections.

Using Bioluminescence Assay to Detect Snps Cause Drug Resistant of Mycobacterium Tuberculosis in Iraq

In this search, a new bioluminescent technique was proved for pyrophosphate which was employed to single- nucleotide polymorphism (SNP) diagnosis using one-base extension reaction. Four Mycobacterium tuberculosis genes were chosen (Rpob, InhA, KatG, GyrA) genes. Fifty-four specimens were used in this study fifty-three proved as drug-resistant specimens by The Iraqi Institute of Chest and Respiratory Diseases in Baghdad., also one specimen was used as a negative control. The procedure of this assay was as follows. A specific primer within each aliquot owning a short 3-OH end of the base of the target gene was hybridized to the single-stranded DNA template. Then, (exo-) Klenow DNA polymerase and one of either ?-thio-dATP, dTTP, dGTP, or dCTP were supplemented and incubated for 1 min. Pyrophosphate freed by DNA polymerase is altered to ATP by pyruvate phosphate dikinase (PPDK), and the amount of ATP is measured using the firefly luciferase reaction. This technique, which does not demand expensive equipment, can be applied to rapidly monitor one-point mutation in the gene that causes drug resistant in mycobacterium tuberculosis. The results showed a high variation in values of ATP formation through matching and mismatch bases added. So, this assay (which required only five minutes), enable to find the gene SNP causes resistant for the specific drug

Development of attenuated live vaccine candidates against swine brucellosis in a non-zoonotic B. suis biovar 2 background

Brucella is a genus of gram-negative bacteria that cause brucellosis. B. abortus and B. melitensis infect domestic ruminants while B. suis (biovars 1–3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are effective vaccines in domestic ruminants, though both can infect humans. However, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2ΔwbkF) or only producing internal O-chain precursors (Bs2Δwzm) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2ΔwadB and Bs2ΔwadD). We also investigated mutants in the pyruvate phosphate dikinase (Bs2ΔppdK) and phosphoenolpyruvate carboxykinase (Bs2ΔpckA) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (108 and 105 CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2ΔwbkF, Bs2Δwzm, Bs2ΔwadB and the Rev1 control (105 CFU). As described before for B. abortus, B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2ΔppdKΔpckA had the same metabolic phenotype as Bs2ΔppdK and ppdK mutation was enough to generate attenuation. At 105 CFU, Bs2ΔppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2ΔwbkF, the latter seems a better R vaccine candidate than Bs2Δwzm. However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2ΔwbkF is advantageous over Bs2ΔwadB or Bs2ΔppdK requires experiments in the natural host.

Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.).

C4 photosynthesis has evolved in over 60 different plant taxa and is an excellent example of convergent evolution. Plants using the C4 photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C3 and C4 species have elucidated the genetic diversity underpinning the C4 photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C4 crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C4 gene families in an important C4 crop, Sorghum bicolor (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C4 gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10−3) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10−3). Genetic diversity of C4 genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C4 photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C4 PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C4 PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C4 genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.

Responses of CO2-concentrating mechanisms and photosynthetic characteristics in aquatic plant Ottelia alismoides following cadmium stress under low CO2

The effects of cadmium (Cd) have been investigated in an aquatic plant Ottelia alismoides grown under low CO2. Under low CO2, no Cd treated O. alismoides operated three carbon dioxide-concentrating mechanisms (CCMs) efficiently, including HCO3− acquisition, C4 and CAM photosynthesis. After 4 days of treatment with 200 μM and 2000 μM Cd, O. alismoides exhibited an elevated Cd accumulation along with the increasing Cd concentration. Both Cd treatments induced appreciable phytotoxicities in O. alismoides. The leaves showed chlorosis symptoms and the anatomy as well as chloroplast ultrastructure were obviously damaged. Significant decreases in the content of pigments, chlorophyll fluorescence (Fv/Fm and Yield of PS II) and carbon isotope ratio (δ13C) were measured in leaf extracts of O. alismoides grown with both concentrations of Cd. In addition, the pH-drift technique showed that both Cd-treated O. alismoides plants could not uptake HCO3−. The maximum and minimum acidity in Cd-exposed O. alismoides were greatly decreased and the diurnal change of acidity was absent in both Cd treated plants. Furthermore, significant decreases in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), pyruvate phosphate dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPC) activities were also found at Cd treated O. alismoides plants, indicating the disturbance within C4 cycle. The alterations in the functionality of CCMs in O. alismoides induced by Cd might be related with the inhibition of the enzymes such as carbonic anhydrase (CA) and PEPC involved in inorganic carbon fixation, and the destruction of chloroplasts, as well as the re-allocation of energy and nutrients involved in CCMs and Cd detoxification.

Molecular Functional Characterisation of MechlPPDK Promoter in Transgenic Tobacco

Chloroplastic pyruvate phosphate dikinase (PPDK) (chlPPDK) is a key enzyme in the photosynthesis of C4 plants. PPDK is expressed in high abundance in C4 plants but only in trace amounts in C3 plants. The existing research reveals a higher expression of MechlPPDK in cultivated cassava varieties than that in wild cassava W14. However, knowledge about the transcriptional regulation of the MechlPPDK gene in cassava (Manihot esculenta Crantz) is insufficient. Therefore, we aim to identify the transcription profile of MechlPPDK and the core promoter region of MechlPPDK. We cloned the MechlPPDK coding sequence and its 5′-flanking sequence from the cassava variety Ku50. A series of deletion constructions fused to a uidA reporter gene were performed in the 5′-flanking sequence and stably transformed into tobacco. We found that P1 (from −590 bp to +114 bp) had the highest promoter activity among P1 to P3. The 5′-flanking sequence of MechlPPDK responded differently to varied abiotic stresses. Our results will further the understanding of the regulation of MechlPPDK expression.

Genomic dissection and expression analysis of stress-responsive genes in C4 panicoid models, Setaria italica and Setaria viridis

The study reports the identification and expression profiling of five major classes of C4 pathway-specific genes, namely, carbonic anhydrase (CaH), phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), NADP-dependent malate dehydrogenase (MDH) and NADP-dependent malic enzyme (NADP-ME), in the model species, Setaria italica and Setaria viridis. A total of 42 and 41 genes were identified in S. italica and S. viridis, respectively. Further analysis revealed that segmental and tandem duplications have contributed to the expansion of these gene families. RNA-Seq derived expression profiles of the gene family members showed their differential expression pattern in tissues and dehydration stress. Comparative genome mapping and Ks dating provided insights into their duplication and divergence in the course of evolution. Expression profiling of candidate genes in contrasting S. italica cultivars subjected to abiotic stresses and hormone treatments showed distinct stress-specific upregulation of SiαCaH1, SiβCaH5, SiPEPC2, SiPPDK2, SiMDH8, and SiNADP-ME5 in the tolerant cultivar. Overexpression of SiNADP-ME5 in heterologous yeast system enabled the transgenic cells to survive and grow in dehydration stress conditions, which highlights the putative role of SiNADP-ME5 in conferring tolerance to dehydration stress. Altogether, the study highlights key genes that could be potential candidates for elucidating their functional roles in abiotic stress response.

A New SNP in Rice Gene Encoding Pyruvate Phosphate Dikinase (PPDK) Associated with Floury Endosperm.

Rice varieties with suitable flour-making qualities are required to promote the rice processed-food industry and to boost rice consumption. A rice mutation, Namil(SA)-flo1, produces grains with floury endosperm. Overall, grains with low grain hardness, low starch damage, and fine particle size are more suitable for use in flour processing grains with waxy, dull endosperm with normal grain hardness and a high amylose content. In this study, fine mapping found a C to T single nucleotide polymorphism (SNP) in exon 2 of the gene encoding cytosolic pyruvate phosphate dikinase (cyOsPPDK). The SNP resulted in a change of serine to phenylalanine acid at amino acid position 101. The gene was named FLOURY ENDOSPERM 4-5 (FLO4-5). Co-segregation analysis with the developed cleaved amplified polymorphic sequence (CAPS) markers revealed co-segregation between the floury phenotype and the flo4-5. This CAPS marker could be applied directly for marker-assisted selection. Real-time RT-PCR experiments revealed that PPDK was expressed at considerably higher levels in the flo4-5 mutant than in the wild type during the grain filling stage. Plastid ADP-glucose pyrophosphorylase small subunit (AGPS2a and AGPS2b) and soluble starch synthase (SSIIb and SSIIc) also exhibited enhanced expression in the flo4-5 mutant.

Cold acclimation of mesophyll conductance, bundle-sheath conductance and leakiness in Miscanthus×giganteus.

The cold acclimations of mesophyll conductance (gm ), bundle-sheath conductance (gbs ) and the CO2 concentrating mechanism (CCM) of C4 plants have not been well studied. Here, we estimated the temperature response of gm , gbs and leakiness (ϕ), the amount of concentrated CO2 that escapes the bundle-sheath cells, for the chilling-tolerant C4 plant Miscanthus×giganteus grown at 14 and 25°C. To estimate these parameters, we combined the C4 -enzyme-limited photosynthesis model and the Δ13 C discrimination model. These combined models were parameterised using in vitro activities of carbonic anhydrase (CA), pyruvate, phosphate dikinase (PPDK), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and phosphoenolpyruvate carboxylase (PEPc). Cold-grown Miscanthus plants increased in vitro activities of RuBisCO and PPDK but decreased PEPc activity compared with warm-grown plants. Mesophyll conductance and gbs responded strongly to measurement temperatures but did not differ between plants from the two growth temperatures. Furthermore, modelling showed that ϕ increased with measurement temperatures for both cold-grown and warm-grown plants, but was only marginally larger in cold-grown compared with warm-grown plants. Our results in Miscanthus support that gm and gbs are unresponsive to growth temperature and that the CCM is able to acclimate to cold through increased activity of PPDK and RuBisCO.

Identification and evolution of C4 photosynthetic pathway genes in plants

BackgroundNADP-malic enzyme (NAPD-ME), and pyruvate orthophosphate dikinase (PPDK) are important enzymes that participate in C4 photosynthesis. However, the evolutionary history and forces driving evolution of these genes in C4 plants are not completely understood.ResultsWe identified 162 NADP-ME and 35 PPDK genes in 25 species and constructed respective phylogenetic trees. We classified NADP-ME genes into four branches, A1, A2, B1 and B2, whereas PPDK was classified into two branches in which monocots were in branch I and dicots were in branch II. Analyses of selective pressure on the NAPD-ME and PPDK gene families identified four positively selected sites, including 94H and 196H in the a5 branch of NADP-ME, and 95A and 559E in the e branch of PPDK at posterior probability thresholds of 95%. The positively selected sites were located in the helix and sheet regions. Quantitative RT-PCR (qRT-PCR) analyses revealed that expression levels of 6 NADP-ME and 2 PPDK genes from foxtail millet were up-regulated after exposure to light.ConclusionThis study revealed that positively selected sites of NADP-ME and PPDK evolution in C4 plants. It provides information on the classification and positive selection of plant NADP-ME and PPDK genes, and the results should be useful in further research on the evolutionary history of C4 plants.

Detection of Single Nucleotide Polymorphisms (SNPs) for Genes Cause Drug-Resistant in Iraqi Mycobacterium Tuberculosis isolates by new Pyrophosphate Technique.

In this search, a new pyrophosphate technique was proved. The technique was employed to single- nucleotide polymorphisms (SNPs), which diagnosis using a one-base extension reaction. Three Mycobacterium tuberculosis genes were chosen (Rpob, InhA, KatG) genes. Fifty-four specimens were used in this study fifty-three proved as drug-resistant specimens by The Iraqi Institute of Chest and Respiratory Diseases in Baghdad.; also one specimen was used as a negative control. The steps of this technique were by used a specific primer within each aliquot that has a short 3-OH end of the base of the target gene that was hybridized to the single-stranded DNA template. Then, the Taq polymerase enzyme and one of either α-thio-dATP, dTTP, dGTP, or dCTP were supplemented and incubated for 1 min. ATP is synthesis by convert Pyrophosphate freed by DNA polymerase using pyruvate phosphate dikinase (PPDK), and the amount of ATP estimates by the firefly luciferase reaction. This technique, which does not demand expensive equipment, can be applied to rapidly monitor a one-point mutation in the gene that causes drug-resistant in mycobacterium tuberculosis. The results showed a high variation in values of ATP formation through matching and mismatch bases added. So, this assay (which required only five minutes), enable to find the gene SNP causes resistant for the specific drug.