Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

Abstract

Two distinct cDNA clones encoding NAD(H)-dependent glutamate dehydrogenase (NAD[H]-GDH) in Arabidopsis thaliana were identified and sequenced. The genes corresponding to these cDNA clones were designated GDH1 and GDH2. Analysis of the deduced amino acid sequences suggest that both gene products contain putative mitochondrial transit polypeptides and NAD(H)- and α--ketoglutarate-binding domains. Subcellular fractionation confirmed the mitochondrial location of the NAD(H)-GDH isoenzymes. In addition, a putative EF-hand loop, shown to be associated with Ca2+ binding, was identified in the GDH2 gene product but not in the GDH1 gene product. GDH1 encodes a 43.0-kD polypeptide, designated α-, and GDH2 encodes a 42.5-kD polypeptide, designated [beta]. The two subunits combine in different ratios to form seven NAD(H)-GDH isoenzymes. The slowest-migrating isoenzyme in a native gel, GDH1, is a homohexamer composed of α- subunits, and the fastest-migrating isoenzyme, GDH7, is a homohexamer composed of [beta] subunits. GDH isoenzymes 2 through 6 are heterohexamers composed of different ratios of α- and [beta] subunits. NAD(H)-GDH isoenzyme patterns varied among different plant organs and in leaves of plants irrigated with different nitrogen sources or subjected to darkness for 4 d. Conversely, there were little or no measurable changes in isoenzyme patterns in roots of plants treated with different nitrogen sources. In most instances, changes in isoenzyme patterns were correlated with relative differences in the level of α- and [beta] subunits. Likewise, the relative difference in the level of α- or [beta] subunits was correlated with changes in the level of GDH1 or GDH2 transcript detected in each sample, suggesting that NAD(H)-GDH activity is controlled at least in part at the transcriptional level.

Tobacco Isoenzyme 1 of NAD(H)-Dependent Glutamate Dehydrogenase Catabolizes Glutamate in Vivo

“…in C sinks, during darkness, and during C starvation of explants) and repressed by a high cellular C to N ratio (e.g. in C sources, during light, and during supply of Suc to explants; Melo-Oliveira et al, 1996;Turano et al, 1997;Ficarelli et al, 1999;Masclaux et al, 2000;Masclaux-Daubresse et al, 2002;Tercé-Laforgue et al, 2004b), agreeing with GDH activity data (Restivo et al, 1989;Robinson et al, 1992;Masclaux et al, 2000;Morkunas et al, 2000;Tercé-Laforgue et al, 2004b). In source leaves, both subunits are localized to mitochondria of phloem companion cells.…”

Glutamate Dehydrogenase of Tobacco Is Mainly Induced in the Cytosol of Phloem Companion Cells When Ammonia Is Provided Either Externally or Released during Photorespiration

“…This lytic activity of the vacuole already reported in the senescing flowers of grapevine (Paczek et al, 2002) and possibly in senescing leaves (Dubois et al, 2003) is the result of carbon starvation (Matile and Winkenbach, 1971;Robinson et al, 1992;Aubert et al, 1996). These results suggest that GDH is sensitive to the C status of the plant via the vascular tissue if we consider the well-established fact that the expression of GDH is repressed when the cellular level of sugars is high (Turano et al, 1997, Masclaux-Daubresse et al, 2002.…”

Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis

“…There were previous reports of the isolation of mitochondria from green Arabidopsis tissues (e.g. Turano et al, 1997;Berkemeyer et al, 1998;Yasuno and Wada, 1998;Fan et al, 1999). However, some of the generated mitochondrial fractions were not tested for chloroplast contamination, some were tested and found to contain chloroplast proteins, and some others were tested and found to be pure, but the results are based on monitoring single enzymes of single chloroplast subcompartments in the mitochondrial fractions.…”