NADP-malic enzyme (NADP-ME) catalyzes the oxidative decarboxylation of L-malate, producing pyruvate, CO2 and NADPH. The photosynthetic role of this enzyme in C4 and Crassulacean acid metabolism (CAM) ...plants has been well established; however, the biological role of several non-photosynthetic isoforms described in C3, C4 and CAM plants is still speculative. In this study, the characterization of the NADP-ME isoforms from Nicotiana tabacum was performed. Three different nadp-me transcripts were identified in this C3 plant, two of which encode for putative cytosolic isoforms (DQ923118 and EH663836), while the third encodes for a plastidic counterpart (DQ923119). Although the three transcripts are expressed in vegetative as well as in reproductive tissues, they display different levels of expression. With regards to enzyme activity, root is the tissue that displays the highest NADP-ME activity. Recombinant NADP-MEs encoded by DQ923118 and DQ923119 were expressed in Escherichia coli and their kinetic parameters and response to different metabolic effectors were analyzed. Studies carried out with crude extracts and with the recombinant proteins indicate that the cytosolic and plastidic isoforms aggregate as tetramers of subunits of 65 and 63 kDa, respectively. Real-time reverse transcription-PCR studies show that the three nadp-me tobacco transcripts respond differently to several biotic and abiotic stress stimuli. Finally, the physiological role of each isoform is discussed in terms of the occurrence, kinetic properties and response to stress. The structure of the NADP-ME family in tobacco is compared with those of other C3 species.
Boron-aluminum interactions affect organic acid metabolism more in leaves than in roots of Citrus grandis seedlings Tang, N., Fujian Agriculture and Forestry Univ., Fuzhou (China). Inst. of Horticultural Plant Physiology, Biochemistry and Molecular Biology; Jiang, H.X., Fujian Agriculture and Forestry Univ., Fuzhou (China). Inst. of Horticultural Plant Physiology, Biochemistry and Molecular Biology; Yang, L.T., Fujian Agriculture and Forestry Univ., Fuzhou (China). Inst. of Horticultural Plant Physiology, Biochemistry and Molecular Biology ...
Biologia plantarum,
12/2011, Letnik:
55, Številka:
4
Journal Article
Recenzirano
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Sour pummelo (Citrus grandis) seedlings were irrigated with nutrient solution containing four boron concentrations (i.e., 2.5, 10, 25 and 50 microM H3BO3) and two aluminum concentrations i.e., 0 ...(-Al) and 1.2 mM AlCl3 * 6 H2O (+Al). B did not affect, but Al increased the Al content in the roots. The Al and citrate contents in the -Al leaves either did not change or slightly increased with increasing B concentration. On the other hand, the Al and citrate contents in the +Al leaves rapidly decreased as B concentration increased from 2.5 to 50 microM, then decreased at the highest B concentration. The Al and citrate contents were higher in the +Al than in the -Al leaves, except for at 25 microM B when they were similar. The leaf malate content did not change in response to B or Al, except for an increase in the +Al leaves and a decrease in the -Al leaves at 2.5 microM B. Similarly, the root malate and citrate contents did not change in response to B with or without Al, except for a decrease in the malate and citrate contents in the +Al roots at 50 microM B and an increase in the citrate content in the -Al roots at 50 microM B. The activities of acid-metabolizing enzymes were less affected by B-Al interactions in the roots than in the leaves.
Malic enzyme prepared and purified from Brevundimonas diminuta IFO13182 catalyzed the decarboxylation reaction of malate to pyruvate and CO2 using NADsup(+) as the coenzyme, and the reverse reaction ...was used in the present study for L-malic acid production with fixation of HCO3sup(-) as a model compound for carbon source. The L-malic acid production was based on electrochemical regeneration of NADH on a carbon plate electrode modified by layer-by-layer adsorption of polymer-bound mediator (Alginic acid bound viologen derivative. Alg-V), polymer-bound coenzyme (Alginic acid bound NADsup(+), Alg-NADsup(+)), and lipoamide dehydrogenase (LipDH). Electrochemical reduction of immobilized NADsup(+) catalyzed by LipDH in a multilayer film was achieved, and the L-malic acid production with HCO3sup(-) fixation system with layer-by-layer immobilization of Alg-V/LipDH/Alg-NADsup(+)/malic enzyme multilayer film on the electrode gave an L-malic acid production of nearly 11.9 mmol and an HCO3sup(-) fixation rate of nearly 47.4% in a buffer containing only KHCO3 and pyruvic acid potassium salt, using a cation exchange membrane. The total turnover number of NADH within 48 h was about 19,000, which suggests that efficient NADH regeneration and fast electron transfer were achieved within the multilayer film, and that the modified electrode is a potential method for the fixation of HCO3sup(-) without addition of free coenzyme.
The effects of abiotic stresses on the NADP-dependent malic enzyme in the leaves of the hexaploid wheat Fu, Z.Y.,Chinese Academy of Sciences, Shijiazhuang (China). Inst. of Genetic and Developmental Biology; Zhang, Z.B.,Chinese Academy of Sciences, Shijiazhuang (China). Inst. of Genetic and Developmental Biology; Liu, Z.H.,Qingdao University of Science and Technology (China). Inst. of Life Sciences ...
Biologia plantarum,
03/2011, Letnik:
55, Številka:
1
Journal Article
Recenzirano
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The objective of this study was to examine the effects of different abiotic stresses on the activity of an NADP-dependent malic enzyme (NADP-ME) and the corresponding gene transcription in the leaves ...of the hexaploid Triticum aestivum. The activity of the NADP-ME was increased by water stress (20 % polyethylene glycol 6000), low temperature (4 deg C), darkness, salinity (200 mM NaCl), abscisic acid and salicylic acid. The transcription of the TaNADP-ME1 gene decreased in response to all of the stresses except darkness and NaCl. In addition, the transcription of TaNADP-ME2 was down-regulated by all of the tested treatments and could not be detected under dark stress.
NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) are specifically expressed in bundle sheath cells (BSCs) in NADP-ME-type and PCK-type C4 plants, respectively. Unlike the high ...activities of these enzymes in the green leaves of C4 plants, their low activities have been detected in the leaves of C3 plants. In order to elucidate the differences in the gene expression system between C3 and C4 plants, we have produced chimeric constructs with the beta-glucuronidase (GUS) reporter gene under the control of the maize NADP-Me (ZmMe) or Zoysia japonica Pck (ZjPck) promoter and introduced these constructs into rice. In leaves of transgenic rice, the ZmMe promoter directed GUS expression not only in mesophyll cells (MCs) but also in BSCs and vascular cells, whereas the ZjPck promoter directed GUS expression only in BSCs and vascular cells. Neither the ZjPck nor ZmMe promoters induced GUS expression due to light. In rice leaves, the endogenous NADP-Me (osMe1) was expressed in MCs, BSCs and vascular cells, whereas the rice Pck (OsPck1) was expressed only in BSCs and vascular cells. Taken together, the results obtained from transgenic rice demonstrate that the expression pattern of ZmMe or ZjPck in transgenic rice was reflected by that of its counterpart gene in rice.
Malic enzyme (ME) was purified as an electrophoretically homogenous protein from Rhodopseudomonas palustris No. 7. The molecular weight of ME was estimated to be 650 kDa and that of its subunit, 86 ...kDa. ME activity was remarkably enhanced by di- and mono-valent cations, and the K
a
values for Mg
2+
and NH
4
+
were 0.26 and 0.56 mM respectively. Purified ME used both NAD
+
and NADP
+
as electron acceptors, with K
m
values of 0.11 and 1.8 mM. The K
m
value for L-malate was 1.7 mM using NAD
+
as electron acceptor. Gene cloning of the ME indicated that the ME from R. palustris strain No. 7 was composed of 774 amino acids encompassing the ME and phosphotransacetylase domains, although purified ME displayed no phosphotransacetylase activity. ME activity was inhibited by acetyl-CoA, oxaloacetate, and fructose-6-phosphate. These results suggest that ME plays an important role in the metabolic regulation of R. palustris No. 7 under photoheterotrophic conditions.
Ribulose bisphosphate carboxylase-oxygensae (Rubisco) is inhibited by O2 and, as a consequence, atmospheric CO2 does not saturate C3 photosynthesis. The O2 effect has two components: direct ...inhibition of carboxylation and an oxygenase reaction that initiates photorespiration. C4 photosynthesis concentrates CO2 for Rubisco, which minimizes both components, and increases photosynthesis up to 50%. Although atmospheric CO2 is projected to reach 550 μbar by 2050, it will not eliminate adverse O2 effects. Rice yields will increase, but the benefit may be offset by projected higher temperatures and reductions in rice Rubisco protein. Hydrilla verticillata is a monocot that operates a facultative, single-cell C4 system. Based on this single-cell premise, rice plants have been transformed with C4-cycle enzymes to improve photosynthesis and yield, but the results have been disappointing. The Hydrilla system can provide clues to the essential elements needed for an effective CO2-concentrating mechanism (CCM) because the C4 and C3 cycles operate in series in the same C3 cell, without the bundle sheath anatomy of terrestrial C4 plants. In Hydrilla, phosphoenolpyruvate carboxylase (PEPC) in the cytosol is segragated from Rubisco and the decarboxylase, NADP-dependent malic enzyme (NADP-ME), in the chloroplasts, where CO2 is concentrated. Multiple isoforms of PEPC and NADP-ME exist in Hydrilla, but hvpepc4 and hvme1 are up-regulated in C4 leaves and encode proteins with characteristics specific to C4 photosynthesis. A β-carbonic anhydrase (CA) is also up-regulated, presumably in the cytosol to aid PEPC fixation, but we hypothesize that CA is down-regulated in C4 chloroplasts. To maintain the the NADPH/NADP+ ratio in the granal chloroplasts of C4 leaves, oxaloacetate and/or aspartate may be imported and reduced to malate for decarboxylation. A major unknown is how the Hydrilla chloroplasts, which in the C3 state must maximize CO2 conductance for Rubisco, minimize this permeability to reduce leakage from the CCM when the C4 system is induced. The down-regulation of chloroplast envelope aquaporins may be involved, and the Hydrilla system provides a means to study this crucial component. If chloroplast leakage is not regulated in a single-cell C4 rice plant, even a high-capacity C4 pump will be ineffective, and low quantum yields will compromise productivity. Hydrilla studies indicate that transporter and permeability issues, and the nuances of enzyme regulation, should be incorporated in the design of a single-cell C4 rice plant to produce an effective CCM.
C3 plants lose a significant part of previously fixed CO2 in the process of photorespiration. Reduction in photorespiration is expected to increase the productivity of crop plants and reduce the ...requirements for irrigation and fertilization. For more than ten years, research at our institute has focused on the genetic engineering of dicotyledonous crops plants toward improved CO2 fixation. In this paper, we summarize results from our work vis-a-vis reports from other laboratories and define future challenges. Furthermore, we introduce an alternative approach based on the installation of bypass of photorespiration in the chloroplast.
Isoenzyme polymorphism of almond genotypes selected in the region of northern Serbia Colic, S.,Institute for Science Application in Agriculture, Belgrade (Serbia); Milatovic, D.,University of Belgrade (Serbia). Faculty of Agriculture; Nikolic, D.,University of Belgrade (Serbia). Faculty of Agriculture ...
Horticultural science (Praha),
01/2010, Letnik:
37, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Isoenzyme polymorphism was studied in 20 almond genotypes selected from seedling populations of unknown almond cultivars in the region of northern Serbia (Vojvodina). Fourteen enzyme systems were ...studied using the method of vertical polyacrylamide gel electrophoresis. Ten systems were polymorphic in twelve loci. This polymorphism allowed unique identification of all studied genotypes. The most useful enzyme for analysis of almond genetic variability was menadione reductase. Polymorphism identified for alkaline phosphatase, formate dehydrogenase, glutamate dehydrogenase, malic enzyme, and menadione reductase was reported for the first time in almond. Cluster analysis was used to construct a dendrogram on which five clusters with different number of genotypes could be identified.
A pfl ldhA double mutant Escherichia coli strain NZN111 was used to produce succinic acid by overexpressing the E. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of ...malic acid as well as succinic acid. After the analyses of the metabolic pathways, the fumB gene encoding the anaerobic fumarase of E. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducible trc promoter.
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