"plants contain both cytosolic and chloroplastic NADP- MEs (Edwards and Andreo, 1992; Drincovich et al., 2001). The cytosolic NADP-ME may provide NADPH to the oxidative pentose phosphate pathway and lignin biosynthesis (Walter et al., 1990; Schaaf et al., 1995), and also may control cytosolic pH via malate concentration (Martinoia and Rentsch , 1994; Lai et al., 2002). The chloroplastic isoform of NADP- ME is proposed to play a role in lipid metabolism (Smith et al., 1992; Eastmond et al., 1997; Gerrard-Wheeler et al., 2005). "
[Show abstract][Hide abstract] ABSTRACT: Cells associated with veins of petioles of C3 tobacco possess high activities of the decarboxylase enzymes required in C4 photosynthesis. It is not clear whether this is the case in other C3 species, nor whether these enzymes provide precursors for specific biosynthetic pathways. Here, we investigate the activity of C4 acid decarboxylases in the mid-vein of Arabidopsis, identify regulatory regions sufficient for this activity, and determine the impact of removing individual isoforms of each protein on mid-vein metabolite profiles. This showed that radiolabelled malate and bicarbonate fed to the xylem stream were incorporated into soluble and insoluble material in the mid-vein of Arabidopsis leaves. Compared with the leaf lamina, mid-veins possessed high activities of NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). Transcripts derived from both NAD-ME, one PCK and two of the four NADP-ME genes were detectable in these veinal cells. The promoters of each decarboxylase gene were sufficient for expression in mid-veins. Analysis of insertional mutants revealed that cytosolic NADP-ME2 is responsible for 80% of NADP-ME activity in mid-veins. Removing individual decarboxylases affected the abundance of amino acids derived from pyruvate and phosphoenolpyruvate. Reducing cytosolic NADP-ME activity preferentially affected the sugar content, whereas abolishing NAD-ME affected both the amino acid and the glucosamine content of mid-veins.
"The topology obtained by this method is shown. The following sequences, in addition to the four Arabidopsis NADP-MEs, were analyzed: C 4(1) -NADP-ME from maize (Rothermel and Nelson, 1989), Flaveria trinervia (Borsch and Westhoff, 1990), and Flaveria bidentis (AY863144); C 4(2) -NADP-ME from maize (Saigo et al., 2004); C 4(3) -NADP-ME from maize (AY864063); CAM 1 -NADP-ME from Mesembryanthemum crystallinum (Cushman, 1992) and Aloe arborescens (Honda et al. 2000); CAM 2 -NADP-ME from A. arborescens (Honda et al., 1997); C 3(1) -NADP-ME from bean (Phaseolus vulgaris; Walter et al., 1990), poplar (Populus spp.; van Doorsseleare et al., 1991), grape berries (Franke and Adams, 1995), tomato (Lycopersicon esculentum; AF001270), Apium graveolens (AJ132257), F. pringlei (Lai et al., 2002a), and rice (C 3(1) rice1, rice2, and rice3; Chi et al., 2004); C 3(2) -NADP-ME from rice (Fushimi et al., 1994), F. pringlei (Lipka et al., 1994), tomato (AF001269), grape berries (U67426), and Ricinus communis (AF262997). The photosynthetic isoforms were named as C 4(1) -NADP-ME and CAM 1 -NADP-ME; the plastidic nonphotosynthetic NADP-ME isoforms as C 4(2) -NADP-ME and C 3(2) -NADP-ME, while the nonphotosynthetic cytosolic isoforms as C 4(3) -NADP-ME, CAM 2 -NADP-ME, and C 3(1) -NADP-ME (Drincovich et al., 2001). "
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis (Arabidopsis thaliana) genome contains four genes encoding putative NADP-malic enzymes (MEs; AtNADP-ME1-ME4). NADP-ME4 is localized to plastids, whereas the other three isoforms do not possess any predicted organellar targeting sequence and are therefore expected to be cytosolic. The plant NADP-MEs can be classified into four groups: groups I and II comprising cytosolic and plastidic isoforms from dicots, respectively; group III containing isoforms from monocots; and group IV composed of both monocots and dicots, including AtNADP-ME1. AtNADP-MEs contained all conserved motifs common to plant NADP-MEs and the recombinant isozymes showed different kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and NADP-ME4 present the highest catalytic efficiency for NADP and malate, respectively. NADP-ME4 exists in equilibrium of active dimers and tetramers, while the cytosolic counterparts are present as hexamers or octamers. Characterization of T-DNA insertion mutant and promoter activity studies indicates that NADP-ME2 is responsible for the major part of NADP-ME activity in mature tissues of Arabidopsis. Whereas NADP-ME2 and -ME4 are constitutively expressed, the expression of NADP-ME1 and NADP-ME3 is restricted by both developmental and cell-specific signals. These isoforms may play specific roles at particular developmental stages of the plant rather than being involved in primary metabolism.
"Non-photosynthetic isoforms of NADP-ME were described to be localized in plastids as well as in the cytosol in different species and tissues . The isolation of cDNA clones of NADP-ME from C 3 plants has indicated both cytosolic and plastidic isoforms on the basis of the presence of transit peptides; for example, cytosolic forms were described in bean (Walter et al., 1990), poplar (van Doorsselaere et al., 1991) and grape berries (Franke and Adams, 1995), while plastidic forms were described in rice (Fushimi et al., 1994) and Flaveria pringlei (Lipka et al., 1994). On the other hand, NADP-ME activity was detected in chloroplasts isolated from cotyledons of Cucurbita pepo and from suspensions cultures of Glycine max (El-Shora and ap Rees, 1991), while in situ immunolocalization studies indicated the occurrence of NADP-ME in chloroplasts of wheat (Maurino et al., 1997) as well as in chloroplasts of C 3 Flaveria species (Drincovich et al., 1998). "
[Show abstract][Hide abstract] ABSTRACT: The characterization of a non-photosynthetic isoform of NADP-malic enzyme (NADP-ME) from maize roots, which represents nearly 7% of the total soluble protein of this tissue, was performed. The molecular properties of the purified protein, as well as the kinetic parameters determined, indicate that the NADP-ME isoform present in maize roots differs from the photosynthetic enzyme implicated in the C4 cycle, but is similar, or identical, to the enzyme previously characterized from etiolated maize leaves (Maurino, Drincovich and Andreo, Biochem. Mol. Biol. Int. 38 (1996) 239-250). A full-length ORF encoding a plastidic NADP-ME (almost identical to the maize root NADP-ME, GenBank accession number U39958) was cloned from a root cDNA library as well as isolated by reverse transcription (RT)-PCR using green leaves mRNA as template. These results indicate that root NADP-ME does not constitute a root-specific isoform, but represents a protein with a constitutive pattern of expression in plastids of the C4 plant maize. The amount of NADP-ME measured by activity, western and northern blot was modified when different stress conditions (including treatments with cellulase, fungal elicitors, jasmonate and hypoxic treatment) were applied to maize roots, indicating that the enzyme from maize roots is under transcriptional or post-transcriptional regulation by effectors related to plant defence responses. It is deduced that the induction of housekeeping genes, like non-photosynthetic NADP-ME, whose constitutive role may be the provision of reductive power in non-photosynthetic plastids, is likely to accompany the defence response.
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