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Leishmaniasis, a category 1 neglected protozoan disease caused by a kinetoplastid pathogen called Leishmania, is transmitted through dipteran insect vectors (phlebotomine, sand flies) in three main clinical forms: fatal visceral leishmaniasis, self-healing cutaneous leishmaniasis, and mucocutaneous leishmaniasis. Generic pentavalent antimonials have long been the drug of choice against leishmaniasis; however, their success is plagued with limitations such as drug resistance and severe side effects, which makes them redundant as frontline therapy for endemic visceral leishmaniasis. Alternative therapeutic regimens based on amphotericin B, miltefosine, and paromomycin have also been approved. Due to the unavailability of human vaccines, first-line chemotherapies such as pentavalent antimonials, pentamidine, and amphotericin B are the only options to treat infected individuals. The higher toxicity, adverse effects, and perceived cost of these pharmaceutics, coupled with the emergence of parasite resistance and disease relapse, makes it urgent to identify new, rationalized drug targets for the improvement in disease management and palliative care for patients. This has become an emergent need and more relevant due to the lack of information on validated molecular resistance markers for the monitoring and surveillance of changes in drug sensitivity and resistance. The present study reviewed the recent advances in chemotherapeutic regimens by targeting novel drugs using several strategies including bioinformatics to gain new insight into leishmaniasis. Leishmania has unique enzymes and biochemical pathways that are distinct from those of its mammalian hosts. In light of the limited number of available antileishmanial drugs, the identification of novel drug targets and studying the molecular and cellular aspects of these drugs in the parasite and its host is critical to design specific inhibitors targeting and controlling the parasite. The biochemical characterization of unique Leishmania-specific enzymes can be used as tools to read through possible drug targets. In this review, we discuss relevant metabolic pathways and novel drugs that are unique, essential, and linked to the survival of the parasite based on bioinformatics and cellular and biochemical analyses. [ABSTRACT FROM AUTHOR]

As compared to C3, C4 plants have higher photosynthetic rates and better tolerance to high temperature and drought. These traits are highly beneficial in the current scenario of global warming. Interestingly, all the genes of the C4 photosynthetic pathway are present in C3 plants, although they are involved in diverse non-photosynthetic functions. Non-photosynthetic isoforms of carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), the decarboxylating enzymes NAD/NADP-malic enzyme (NAD/NADP-ME), and phosphoenolpyruvate carboxykinase (PEPCK), and finally pyruvate orthophosphate dikinase (PPDK) catalyze reactions that are essential for major plant metabolism pathways, such as the tricarboxylic acid (TCA) cycle, maintenance of cellular pH, uptake of nutrients and their assimilation. Consistent with this view differential expression pattern of these non-photosynthetic C3 isoforms has been observed in different tissues across the plant developmental stages, such as germination, grain filling, and leaf senescence. Also abundance of these C3 isoforms is increased considerably in response to environmental fluctuations particularly during abiotic stress. Here we review the vital roles played by C3 isoforms of C4 enzymes and the probable mechanisms by which they help plants in acclimation to adverse growth conditions. Further, their potential applications to increase the agronomic trait value of C3 crops is discussed. [ABSTRACT FROM AUTHOR]

Proteins are modular functionalities regulating multiple cellular activities in prokaryotes and eukaryotes. As a consequence of higher plants adapting to arid and thermal conditions, C4 photosynthesis is the carbon fixation process involving multi-enzymes working in a coordinated fashion. However, how these enzymes interact with each other and whether they co-evolve in parallel to maintain interactions in different plants remain elusive to date. Here, we report our findings on the global protein co-evolution relationship and local dynamics of co-varying site shifts in key C4 photosynthetic enzymes. We found that in most of the selected key C4 photosynthetic enzymes, global pairwise co-evolution events exist to form functional couplings. Besides, protein–protein interactions between these enzymes may suggest their unknown functionalities in the carbon delivery process. For PEPC and PPCK regulation pairs, pocket formation at the interactive interface are not necessary for their function. This feature is distinct from another well-known regulation pair in C4 photosynthesis, namely, PPDK and PPDK-RP, where the pockets are necessary. Our findings facilitate the discovery of novel protein regulation types and contribute to expanding our knowledge about C4 photosynthesis. [ABSTRACT FROM AUTHOR]

Leishmaniasis, a category 1 neglected protozoan disease caused by a kinetoplastid pathogen called Leishmania, is transmitted through dipteran insect vectors (phlebotomine, sand flies) in three main clinical forms: fatal visceral leishmaniasis, self-healing cutaneous leishmaniasis, and mucocutaneous leishmaniasis. Generic pentavalent antimonials have long been the drug of choice against leishmaniasis; however, their success is plagued with limitations such as drug resistance and severe side effects, which makes them redundant as frontline therapy for endemic visceral leishmaniasis. Alternative therapeutic regimens based on amphotericin B, miltefosine, and paromomycin have also been approved. Due to the unavailability of human vaccines, first-line chemotherapies such as pentavalent antimonials, pentamidine, and amphotericin B are the only options to treat infected individuals. The higher toxicity, adverse effects, and perceived cost of these pharmaceutics, coupled with the emergence of parasite resistance and disease relapse, makes it urgent to identify new, rationalized drug targets for the improvement in disease management and palliative care for patients. This has become an emergent need and more relevant due to the lack of information on validated molecular resistance markers for the monitoring and surveillance of changes in drug sensitivity and resistance. The present study reviewed the recent advances in chemotherapeutic regimens by targeting novel drugs using several strategies including bioinformatics to gain new insight into leishmaniasis. Leishmania has unique enzymes and biochemical pathways that are distinct from those of its mammalian hosts. In light of the limited number of available antileishmanial drugs, the identification of novel drug targets and studying the molecular and cellular aspects of these drugs in the parasite and its host is critical to design specific inhibitors targeting and controlling the parasite. The biochemical characterization of unique Leishmania-specific enzymes can be used as tools to read through possible drug targets. In this review, we discuss relevant metabolic pathways and novel drugs that are unique, essential, and linked to the survival of the parasite based on bioinformatics and cellular and biochemical analyses.

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H1- pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30±0.01 h21) and the biomass yield by 38% (0.18±0.00 gbiomass gsubstrate 21), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. [ABSTRACT FROM AUTHOR]

Abstract Background NADP-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. Results We 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. Conclusion This 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.

Drought is one of the major stress which affects agricultural crop productivity around the world. In this study the role of Phos phoenol pyruvate carboxylase (PEPC, EC 4.1.1.31), NADP-malic enzyme (NADP-ME, EC 1.1.1.40), and pyruvate, phosphate dikinase (PPDK, EC 2.7.9.1) in different onion cultivars (Allium cepa L.) under drought stress was investigated. An elevated specific activity of these three enzymes was monitored in onion cultivars after 8 h of drought stress. With the increasing concentration of drought there was an increase in C4 enzyme activities. PEPC showed 3.5-fold increase after 24 h, NADP-ME 3.9-fold, and PPDK 3.2-fold increase was observed in Arkalalima (AL)onion after 48 h when compared to control. Statistical analysis including regression analysis suggested significant results to onion drought resistant cultivars under drought stress condition. The results revealed that the variety AL exhibited best response followed by Arkakirthaman (AK) and Bellary(BL). Therefore PEPC, PPDK and NADPME participate in the drought response of onion, a C3 Plant. Future research will allow further understanding of the drought stress mechanism of adaptation of the onion germplasm to drought. [ABSTRACT FROM AUTHOR]

Summary: 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 Δ13C 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: NADP-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. Results: We 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. Conclusion: This 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. [ABSTRACT FROM AUTHOR]

• PPDK and PEPC, in lieu of Rubisco, increased almost two-fold for ammonium tolerant species • Overproduction of PEPC and PPDK led to improved photosynthesis, better ammonium assimilation and overall ammonium detoxification • These findings propose likelihood for the existence of a complementary ammonium detoxification pathway that targets carbon metabolism Possible ammonium detoxification mechanisms have been proposed recently, on submerged macrophytes, evidently illustrating that glutamate dehydrogenase (GDH) plays a greater role in ammonium detoxification compared to the primary glutamine synthetase/glutamate oxaloacetate transaminase (GS/GOGAT) pathway. In the current investigation, we cultured three submerged macrophytes to extreme concentrations of [NH 4 +-N] of up to 50 mg/L with the aim of clarifying the interaction between carbon and nitrogen metabolisms. The activities of carboxylation enzymes pyruvate orthophosphate dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPC), in lieu of Rubisco, increased almost two-fold for ammonium tolerant species P. maackianus and M. spicatum , compared with the sensitive species P. lucens. While these enzymes are well known for their central role in CO 2 fixation, their inference in conferring resistance to ammonium stress has not been well elucidated before. In this study, we demonstrate that the overproduction of PEPC and PPDK led to improved photosynthesis, better ammonium assimilation and overall ammonium detoxification in M. spicatum and P. maackianus. These findings propose likelihood for the existence of a complementary ammonium detoxification pathway that targets carbon metabolism, thus, presenting a relatively efficient linkage between nitrogen and carbon metabolisms and identify candidate species for practical restoration of fresh water resources. [ABSTRACT FROM AUTHOR]

Background: Esters of quinoxaline-7-carboxylate 1,4-di-N-oxide (7-carboxylate QdNOs) derivatives are compounds that inhibit the growth of Entamoeba histolytica, the causative agent of amebiasis. Although these compounds cause changes in the redistribution of glycogen deposits within the parasite, it is unknown whether these compounds interact with enzymes of the glycolytic pathway., Objective: The aim of this study was to test the binding affinity of these compounds to pyrophosphate- dependent phosphofructokinase (PPi-PFK), triosephosphate isomerase (TIM), and pyruvate phosphate dikinase (PPDK) from E. histolytica as a possible mechanism of action., Methods: The molecular docking study of the 7-carboxylate QdNOs derivatives and the proteins was performed using AutoDock/Vina software. Molecular dynamics simulation was performed for 100 ns., Results: Among all the selected compounds, T-072 exhibited the best binding affinity to EhPPi- PFK and EhTIM proteins, while T-006 interacted best with EhPPDK. ADMET analysis revealed that T-072 was non-toxic, while T-006 could become harmful to the host. In addition, molecular dynamics showed that T-072 has stable interaction with EhPPi-PFK and EhTIM., Conclusion: Including all aspects, these data indicated that these compounds might inhibit the activity of key enzymes in energy metabolism leading to parasite death. Furthermore, these compounds may be a good starting point for the future development of new potent antiamebic agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The occurrence of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate orthophosphate dikinase (PPDK) was determined in the ripe pericarps of several grape genotypes. Moreover, the abundance pattern of PEPCK during the development of grape berries was studied in a range of cultivars. In addition, the presence of PEPCK in grape leaves and the location/abundance of PEPCK in developing grape seeds were investigated. PEPCK was present in the pericarp of all the cultivars of Vitis vinifera studied and in that of the hybrid 420A (Vitis berlandieri × Vitis riparia). By contrast, PPDK was either not present or present at very low abundance. The results strongly indicated that the PEPCK and not the PPDK pathway is the predominant one utilised in gluconeogenesis in the berry pericarp of grape genotypes. Further, the results showed that the abundance of PEPCK increased greatly at the start of ripening in all the cultivars, showing a similar pattern in all of them. PEPCK was not present (or at very low abundance) in grape leaves throughout their development. In developing grape seeds, the results indicated a similar abundance of PEPCK in both the seed coat and the tissues enclosed by it. [ABSTRACT FROM AUTHOR]

Remobilization of leaf nitrogen (N) contributes greatly to grain N in maize, but leads to low photosynthetic rate (P n ). P n is determined by various N components involving in light harvest and CO 2 reduction. However, it is less clear which N component is the major contributor for the reduction of photosynthesis in modern stay-green maize hybrids. In this study, we analyzed the relationship between remobilization of different N components and P n during grain filling stage under low N (no N application) and high N (180 kg N ha −1 ) in a field experiment. The remobilization efficiency of photosynthetic enzymes (PEPc, PPDK and Rubisco) in the leaf was much higher than that of thylakoid N and other N components. Low N supply increased the remobilization efficiency of all the leaf N components. During grain filling stage, the amount of all the N components decreased together with P n . The ratio of P n to the N in the PEPc, PPDK and Rubisco kept increase in the whole grain filling stage, while the ratio of P n to chlorophyll and thylakoid-N decreased. Correlation analysis indicated that P n was more related to the content of photosynthetic enzymes than to chlorophyll and thylakoid N. It is concluded that photosynthetic enzymes serve as an N storage reservoir at early grain filling stage and their degradation is critical in the reduction of P n during later grain filling stage. Future breeding targets may be focused on enhancing the efficiency of photosynthetic enzymes during late grain filling stage. [ABSTRACT FROM AUTHOR]

In C4 plants, the pyruvate phosphate dikinase regulatory protein (PDRP) regulates the C4 pathway enzyme pyruvate phosphate dikinase (PPDK) in response to changes in incident light intensity. In maize (Zea mays) leaves, two distinct isoforms of PDRP are expressed, ZmPDRP1 and ZmPDRP2. The properties and C4 function of the ZmPDRP1 isoform are well understood. However, the PDRP2 isoform has only recently been identified and its properties and function(s) in maize leaves are unknown. We therefore initiated an investigation into the maize PDRP2 isoform by performing a side by side comparison of its enzyme properties and cell-specific distribution with PDRP1. In terms of enzyme functionality, PDRP2 was found to possess the same protein kinase-specific activity as PDRP1. However, the PDRP2 isoform was found to lack the phosphotransferase activity of the bifunctional PDRP1 isoform except when PDRP2 in the assays is elevated 5- to 10-fold. A primarily immuno-based approach was used to show that PDRP1 is strictly expressed in mesophyll cells and PDRP2 is strictly expressed in bundle sheath strand cells (BSCs). Additionally, using in situ immunolocalization, we establish a regulatory target for PDRP2 by showing a significant presence of C4 PPDK in BSC chloroplasts. However, a metabolic role for PPDK in this compartment is obscure, assuming PPDK accumulating in this compartment would be irreversibly inactivated each dark cycle by a monofunctional PDRP2. [ABSTRACT FROM AUTHOR]

* Background and Aims The high productivity of Miscanthus x giganteus has been at least partly ascribed to its high chilling tolerance compared with related C4 crops, allowing for a longer productive growing season in temperate climates. However, the chilling tolerance of M. x giganteus has been predominantly studied under controlled environmental conditions. The understanding of the underlying mechanisms contributing to chilling tolerance in the field and their variation in different miscanthus genotypes is largely unexplored. * Methods Five miscanthus genotypes with different sensitivities to chilling were grown in the field and scored for a comprehensive set of physiological traits throughout the spring season. Chlorophyll fluorescence was measured as an indication of photosynthesis, and leaf samples were analysed for biochemical traits related to photosynthetic activity (chlorophyll content and pyruvate, Pi dikinase activity), redox homeostasis (malondialdehyde, glutathione and ascorbate contents, and catalase activity) and water-soluble carbohydrate content. * Key Results Chilling-tolerant genotypes were characterized by higher levels of malondialdehyde, raffinose and sucrose, and higher catalase activity, while the chilling-sensitive genotypes were characterized by higher concentrations of glucose and fructose, and higher pyruvate, Pi dikinase activity later in the growing season. On the early sampling dates, the biochemical responses of M. x giganteus were similar to those of the chilling-tolerant genotypes, but later in the season they became more similar to those of the chilling-sensitive genotypes. * Conclusions The overall physiological response of chilling-tolerant genotypes was distinguishable from that of chilling-sensitive genotypes, while M. x giganteus was intermediate between the two. There appears to be a trade-off between high and efficient photosynthesis and chilling stress tolerance. Miscanthus x giganteus is able to overcome this trade-off and, while it is more similar to the chilling-sensitive genotypes in early spring, its photosynthetic capacity is similar to that of the chilling-tolerant genotypes later on. [ABSTRACT FROM AUTHOR]

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling- lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression. [ABSTRACT FROM AUTHOR]

Aluminum is one of the most important heavy metals inducing stress during plant growth and development. In this study, transgenic rice (Oryza sativa L., cv. Kitaake) plants expressing the maize C4 PEPC and PPDK genes were evaluated for aluminum tolerance. A 4.3 and 19.1 folds increase of PPDK and PEPC activities in transgenic rice produced increases in root exudation of oxalate, malate, and citrate (1.20, 1.41, and 1.65 times, respectively) compared to untransformed (WT) plants. Transgenic rice had enhanced aluminum tolerance compared to WT based on chlorophyll fluorescence and chlorophyll levels. Transgenic plants under aluminum stress also had decreased lipid membrane oxidative damage and higher levels of ROS-scavenging enzyme activity. The PEPC and PPDK genes play an important role in aluminum stress tolerance by increasing the effluxes of organic acids. [ABSTRACT FROM AUTHOR]

Ottelia alismoides is a constitutive C4 plant and bicarbonate user, and has facultative crassulacean acid metabolism (CAM) at low CO2. Acclimation to a factorial combination of light and CO2 showed that the ratio of phosphoenolpyruvate carboxylase (PEPC) to ribulose-bisphosphate carboxylase/oxygenase (Rubisco) (>5) is in the range of that of C4 plants. This and short-term response experiments showed that the activity of PEPC and pyruvate phosphate dikinase (PPDK) was high even at the end of the night, consistent with night-time acid accumulation and daytime carbon fixation. The diel acidity change was maximal at high light and low CO2 at 17-25 μequiv g-1 FW. Decarboxylation proceeded at ~2-3 μequiv g-1 FW h-1, starting at the beginning of the photoperiod, but did not occur at high CO2; the rate was greater at high, compared with low light. There was an inverse relationship between starch formation and acidity loss. Acidity changes account for up to 21% of starch production and stimulate early morning photosynthesis, but night-time accumulation of acid traps <6% of respiratory carbon release. Ottelia alismoides is the only known species to operate CAM and C4 in the same tissue, and one of only two known aquatic species to operate CAM and bicarbonate use. [ABSTRACT FROM AUTHOR]

The presence of CO2-concentrating mechanisms (CCMs) is believed to be one of the characteristics that allows diatoms to thrive in many environments and to be major contributors to global productivity. Here, the type of CCM and the responses to variable CO2 concentration were studied in marine and freshwater diatoms. At 400 ppm, there was a large diversity in physiological and biochemical mechanisms among the species. While Phaeodactylum tricornutum mainly used HCO3-, Thalassiosira pseudonana mainly used CO2. Carbonic anhydrase was an important component of the CCM in all species and C4 metabolism was absent, even with T. weissflogii. For all species, at 20 000 ppm, the affinity for dissolved inorganic carbon was lower than at 400 ppm CO2 and the reliance on CO2 was higher. Despite the difference in availability of inorganic carbon in marine and fresh waters, there were only small differences in CCMs between species from the two environments, and Navicula pelliculosa behaved similarly when grown in the two environments. The results suggest that species-specific differences are great, and more important than environmental differences in determining the nature and effectiveness of the CCM in diatoms. [ABSTRACT FROM AUTHOR]

In this study, we used ELISA for quantification of PPDK in photosynthesizing leaves of miscanthus. We cloned a fragment of the gene encoding PPDK, purified the resulting protein by affinity chromatography, identified it using MALDI mass spectrometry, and obtained monoclonal antibodies by immunizing BALB/c mice. Selectivity of monoclonal antibodies was assessed by Western blot using the protein extracts of Soranovskii. The presence of PPDK was again verified by MALDI mass spectrometry. Therefore, we developed and tested the method for determining PPDK quantity in miscanthus using ELISA. [ABSTRACT FROM AUTHOR]

More efficient photosynthesis has allowed C plants to adapt to more diverse ecosystems (such as hot and arid conditions) than C plants. To better understand C photosynthesis, we investigated the expression patterns of C genes ( C4PPDK and PCK1) and their non-C homologous genes ( CyPPDK1, CyPPDK2, and PCK2) in the different organs of maize ( Zea mays). Both C genes and non-C genes showed organ-dependent expression patterns. The mRNA levels of C genes were more abundant in leaf organ than in seeds at 25 days after pollination (DAP), while non-C genes were mainly expressed in developing seeds. Further, acetylation of histone H3 lysine 9 (H3K9ac) positively correlates with mRNA levels of C genes ( C4PPDK and PCK1) in roots, stems, leaves, and seeds at 25 DAP, acetylation of histone H4 lysine 5 (H4K5ac) in the promoter regions of both C ( C4PPDK and PCK1) and non-C genes ( CyPPDK1, CyPPDK2, and PCK2) correlated well with their transcripts abundance in stems. In photosynthetic organs (stems and leaves), dimethylation of histone H3 lysine 9 (H3K9me2) negatively correlated with mRNA levels of both C and non-C genes. Taken together, our data suggest that histone modification was involved in the transcription regulation of both C genes and non-C genes, which might provide a clue of the functional evolution of C genes. [ABSTRACT FROM AUTHOR]

The green-tide-forming macroalga Ulva linza was profiled by transcriptome sequencing to ascertain whether the alga carries both C and C photosynthesis genes. The key enzymes involved in C metabolism including pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK) were found. When measured under normal and different stress conditions, expression of rbcL was higher under normal conditions and lower under the adverse conditions, whereas that of PPDK was higher under some adverse conditions, namely desiccation, high salinity, and low salinity. Both ribulose-1, 5-biphosphate carboxylase (RuBPCase) and PPDK were found to play a role in carbon fixation, with significantly higher PPDK activity across the stress conditions. These results suggest that elevated PPDK activity alters carbon metabolism in U. linza leading to partial operation of the C carbon metabolism, a pathway that, under stress conditions, probably contributes to the hardy character of U. linza and thus to its wide distribution. [ABSTRACT FROM AUTHOR]

To improve the efficiency of CO fixation in C photosynthesis, C-cycle genes were overexpressed in Arabidopsis plants. In this study, a full-length cDNA for pyruvate orthophosphate dikinase ( PPDK) with 971 amino acids was isolated from Zea mays using LA-PCR. The PPDK gene (GenBank accession number GU363532) and the phosphoenolpyruvate carboxylase ( PEPC) gene (GenBank accession number: FJ415327) derived from a previous study were introduced into wild-type Arabidopsis both individually and in combination. The vector pCAMBIA3301 (p3301) was introduced into Arabidopsis as a control using an Agrobacterium-mediated system. The integration, transcription, and translation of the PPDK and the PEPC genes were confirmed by Southern blot, quantitative real-time PCR, and Western blot analysis. The average enzyme activity of C- PPDK and C- PEPC peaked at 4.63- and 5.81-fold compared to that of controls, respectively. The insertion of PPDK was found to increase the activity of PEPCase and vice versa. The photosynthetic rates of C- PPDK, C- PEPC, and C- PKC( PPDK+ PEPC) transgenic lines increased to 123.3, 136.4, and 146.6 % compared to that of controls, respectively. [ABSTRACT FROM AUTHOR]

Pyruvate, orthophosphate dikinase (PPDK) is a key enzyme in the C4 photosynthetic pathway of maize. To improve the cold tolerance of the enzyme in maize, we designed two genomic sequence-based constructs in which the carboxy-terminal region of the enzyme was modified to mimic the amino acid sequence of the cold-tolerant PPDK of Flaveria brownii (Asteraceae). A large amount of PPDK was found to have accumulated in the leaves of many of the maize plants transformed with one of these constructs – that which introduced 17 amino acid substitutions without any alteration of the exon-intron structure – although there was a wide range of variation in the amount of PPDK among the separate plants. In contrast, the production was much less in maize transformed with the second construct in which a cDNA fragment for the same carboxy-terminal region was inserted. The specific activity of PPDK in the plants transformed with the gene with the amino acid substitutions was inversely correlated with the amount of enzyme in the leaves. In addition, the activity of the cold-tolerant recombinant enzyme was judged to be regulated by the PPDK regulatory protein, similar to that of the native PPDK. The cold tolerance of PPDK in crude leaf extracts was greatly improved in plants that produced a large amount of the engineered PPDK. The photosynthetic rate at 8°C increased significantly (by 23%, p<0.05), but there was no obvious effect at higher temperatures. These results support the hypothesis that PPDK is one of the limiting factors in the C4 photosynthesis of maize under cold conditions. [ABSTRACT FROM AUTHOR]

Abstract: Pyruvate, orthophosphate dikinase (PPDK; E.C. 2.7.9.1) catalyzes the synthesis of the primary inorganic carbon acceptor, phosphoenolpyruvate in the C4 photosynthetic pathway and is reversibly regulated by light. PPDK regulatory protein (RP), a bifunctional serine/threonine kinase-phosphatase, catalyzes both the ADP-dependent inactivation and the Pi-dependent activation of PPDK. Attempts to clone the RP have to date proven unsuccessful. A bioinformatics approach was taken to identify the nucleotide and amino acid sequence of the protein. Based on previously established characteristics including molecular mass, known inter- and intracellular location, functionality, and low level of expression, available databases were interrogated to ultimately identify a single candidate gene. In this paper, we describe the nucleotide and deduced amino acid sequence of this gene and establish its identity as maize PPDK RP by in vitro analysis of its catalytic properties via the cloning and expression of the recombinant protein. [Copyright &y& Elsevier]

Pyruvate phosphate dikinase (PPDK) was recently reported in trypanosomatids, but its metabolic function is not yet known. The present work deals with the cellular localization and the function of the Trypanosoma cruzi enzyme. First, we show by digitonin titration and cell fractionation that the enzyme was essentially present in the glycosome matrix of the epimastigote form. Second, we address the issue of the direction of the reaction inside the glycosome for one part, our bibliographic survey evidenced a quite exergonic ΔG°′ (at least -5.2 kcal/mol at neutral pH and physiologic ionic strength); for another part, no pyrophosphatase (PPase) could be detected in fractions corresponding to the glycosomes; therefore, glycosomal PPDK likely works in the direction of pyruvate production. Third, we address the issue of the origin of the glycosomal pyrophosphate (PPi): several synthetic pathways known to produce PPi are already considered to be glycosomal. This work also indicates the presence of an NADP+-dependent β-oxidation of palmitoyl-CoA in the glycosome. Several pyruvate-consuming activities, in particular alanine dehydrogenase (ADH) and pyruvate carboxylase (PC), were detected in the glycosomal fraction. PPDK appears therefore as a central enzyme in the metabolism of the glycosome of T. cruzi by providing a link between glycolysis, fatty acid oxidation and biosynthetic PPi-producing pathways. Indeed, PPDK seems to replace pyrophosphatase in its classical thermodynamic role of displacing the equilibrium of PPi-producing reactions, as well as in its role of eliminating the toxic PPi. [Copyright &y& Elsevier]

Pyruvate, orthophosphate dikinase (PPDK, E.C. 2.7.9.1) is a cardinal carbon-assimilating, stromal enzyme of the C4 photosynthetic pathway. Like several other photosynthetic pathway enzymes, its activity is strictly and reversibly regulated by light. This regulation is conferred by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase that catalyzes the ADP-/Pi-dependent, reversible phosphorylation of an active-site threonine residue. In this minireview, we highlight how plastidic PPDK in leaves and developing seeds of C3 plants is regulated in an identical manner as C4 PPDK via a putative C3-RP isoform. Additionally, we also detail the progress in research concerning C4 RP, since this highly unusual regulatory enzyme was last reviewed nearly two decades ago. [Copyright &y& Elsevier]

The gene for C-pyruvate,orthophosphate dikinase (PPDK) from maize ( Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein. [ABSTRACT FROM AUTHOR]

A simple, single-tube radiolsotopic method has been developed to assay the relative phosphorylation (inaetivation) activity of the bifunctional regulatory protein (RP) of C-leaf pyruvate,orthophosphate dikinase (PPDK) in desalted leaf homogenates and partially purified preparations. RP catalyzes the inactivation of maize PPDK by phosphorylation of Thr-456, utilizing [β-P]ADP as the specific phosphoryl donor. Existing spectrophotometric and radioisotopic assays for the detection of RP activity are either relatively insensitive or labor-intensive and timeconsuming. We describe a modified radioisotopic assay that couples the synthesis of [β-P]ADP by exogenous adenylate kinase with the subsequent RP-catalyzed [β-P]ADP-dependent phosphorylation of exogenous maize PPDK. The incorporation of [β-P] is dependent on the initial concentrations of ATP and PPDK, as well as the presence of active RP. Desalted leaf homogenates of C species fail to catalyze P incorporation into exogenous maize PPDK. Conversely, heterologous systems containing the maize target enzyme and leaf homogenats of other C species result in PPDK-specific P-incorporation. This simple radioisotopic assay is at least 40-times more sensitive than the routine spectrophotometric assay, and qualitatively exhibits comparable sensitivity and requires significantly less time than the currently available radioisotopic RP assay. The present assay reliably generates [β-P]ADP and as such may be useful for studies of other systems requiring β-labeled ADP, which is not commercially available. [ABSTRACT FROM AUTHOR]

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 CO 2 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 (O 2 · - , H 2 O 2 ) 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 CO 2 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 C 3 crops.

Immunolocalization analysis is a principal tool to study protein expression and subcellular distribution in plant cells or tissues. In this chapter, we present the method of the preparation of lightly fixed fresh rice leaf tissue for immunolocalization analysis and detection of the protein of interest using fluorescent probes by fluorescent microscopy. This method especially does not need the process of embedding plant materials that saves time and prevents alterations of cellular compounds and structure during sample preparation. Using this method, the C 4 rice project compared the expressions of the proteins of interest among C 4 model plants, wild-type rice, and transgenic or mutant plants and successfully selected the transgenic plants with the correct location of each protein to create a C 4 rice prototype.