Glutamate dehydrogenase isoforms in lupine roots and root nodules. Immunological studies

In roots of the lupine ( Lupinus luteus L. cv. Ventus) glutamate dehydrogenase (EC 1.4.1.2–4) was present in a single, electrophoretically homogeneous form (GDH_R), while in root nodules eight forms of the enzyme (GDH_N) were detected. Highly purified fractions of glutamate dehydrogenase from roots and nodules were used to prepare antisera in rabbits. The two antisera recognized extracts of glutamate dehydrogenase from both roots and nodules. Low concentrations of the antiserum raised against nodule glutamate dehydrogenase precipitated GDH from the nodule extract (the homologous antigen) effectively but decreased the activity of GDH from the root extract only slightly. At high concentrations, however, the antiserum totally precipitated both enzyme extracts. Ouchterlony's double diffusion test showed that seven forms of root nodule glutamate dehydrogenase arose as the result of random association of two subunits in a hexameric complex, while the eighth form probably consisted of totally distinct subunits. The question of whether the new glutamate dehydrogenase forms, present exclusively in root nodules, can be classified as nodulins, is discussed.     

Proteomic analysis of rice leaf, stem and root tissues during growth course

Rice proteins were isolated from leaf, stem and root tissues, harvesting at 1, 2, 4, 8 and 10 weeks after budding. Each tissue of each age was separately pulverized in liquid nitrogen, and the resulted tissue powders were suspended in 10% TCA-acetone and followed by acetone suspension to precipitate at low temperature, which resulted in the tissue-specific and age-specific protein mixture. The protein mixtures were separated by 2-DE using polyacrylamide gels (26 x 20 cm). The protein spots were identified by N-terminal sequence analysis and by MALDI and LC-MS/MS analyses after in-gel tryptic digestion. From a total of 4532 spots, 676 unique proteins were identified, of which 80 proteins (12%) were observed in all three tissues: leaf, stem and root. In addition, 45 (7%) were common in leaf and stem, 57 (8%) in stem and root, and 10 (2%) proteins in root and leaf. Also 141 unique proteins (21%) were observed only for leaf, 96 (14%) for stem, and 247 (36%) for root tissue. Proteins playing a role for photosynthesis and energy production were most abundant in leaf and stem, and those for cell defense were rich in roots.     

Glutamate synthase in rice root extracts and the relationship among electron donors, nitrogen donors and its activity

The presence of glutamate synthase (GOGAT) in rice root extractsand the relationship among electron donors, nitrogen donorsand its activity were studied using ¹⁵N-amido-labelled glutamine,asparagine, ¹⁴C-2-oxoglutarate and inhibitors The high molecular fraction of rice root extracts prepared bySephadex G-50 column showed ferredoxin-dependent GOGAT activity,but pyridine nucleotide dependent activity could not be detectedin it. Asparagine did not act as a nitrogen donor for rice rootGOGAT. Methyl viologen could be a substitute for ferredoxin,but GOGAT activity with it was about 1/4 of that with ferredoxin.Accordingly, rice root GOGAT was considered to be the same typeas that observed in leaves of many higher plants, but differentfrom that discovered in pea roots and cultured carrot tissues. The extracts showed high GDH activity, which was reduced bythe addition of glutamine. The GDH did not act with glutamineand asparaginc in the presence of aminooxy-acetate and did notshow GOGAT-like activity. (Received December 19, 1977; )     

Subcellular distribution of enzymes of the oxidative pentose phosphate pathway in root and leaf tissues

The subcellular distribution of enzymes of the oxidative pentose phosphate pathway was studied in plants. Root and leaf tissues from several species were separated by differential centrifugation into plastidic and cytosolic fractions. In all tissues studied, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in both plastidic and cytosolic compartments. In maize and pea root, and spinach and pea leaf, the non-oxidative enzymes of the pentose phosphate pathway (transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase) appear to be restricted to the plastid. In tobacco leaf and root, however, the non-oxidative enzymes were found in the cytosolic as well as the plastidic compartments. In the absence of ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase in the cytosol, the product of the oxidative limb of the pathway (ribulose 5-phosphate) must be transported into a compartment capable of utilizing it. Ribulose 5-phosphate was supplied to isolated intact pea root plastids and was shown to be capable of supporting nitrite reduction. The kinetics of ribulose 5-phosphate-driven nitrite reduction in isolated pea root plastids suggested that the metabolite was translocated across the plastid envelope in a carrier-mediated transport process, indicating the presence of a translocator capable of transporting pentose phosphates.Keywords: Pentose phosphate, subcellular, plastid, ribulose 5-phosphate, compartmentation      

Glutamate synthase in developing pea cotyledons: Occurrence of NADH-dependent and ferredoxin-dependent enzymes

NADH-dependent and ferredoxin-dependent glutamate synthasesfrom developing pea cotyledons were separated by gel filtrationon a Sephadex G-200 column. The substrate requirements, molecularweights and effect of some inhibitors on both glutamate synthaseenzymes were investigated. ¹ Department of Agricultural Chemistry, Kyoto University, Kyoto606, Japan. ² To whom inquiries should be addressed. (Received August 9, 1979; )     

Characterization of two sucrose synthase isoforms in sugarbeet root

Two sucrose synthase isoforms (EC 2.4.1.13) have been identified in developing sugarbeet (Beta vulgaris L.) roots. To aid in understanding the physiological significance of these multiple sucrose synthase isoforms, the two isoforms were partially purified and some of their physical and kinetic properties determined. Both isoforms were tetrameric proteins with native molecular masses of 320 kDa. The isoforms exhibited similar kinetic properties as well as similar changes in activity in response to changes in temperature. The isoforms differed, however, in their subunit composition. Sucrose synthase isoform I (SuSyI) was composed of two 84 kDa subunits and two 86 kDa subunits. Sucrose synthase isoform II (SuSyII) was a homotetramer with a subunit size of 86 kDa. The amino acid composition of the two subunits was similar, although differences in alanine, glycine, isoleucine and lysine content were noted. The activity of the two isoforms differed in response to varying pH conditions. The optimum pH for sucrose cleaving activity was observed at pH 6.0 and 6.5 for SuSyI and SuSyII, respectively. The optimum pH for sucrose synthesizing activity occurred at pH 7.5 and 7.0 for SuSyI and SuSyII, respectively. The observed differences in subunit composition and reactivity at different pH values suggest that multiple isoforms of sucrose synthase may provide a mechanism to regulate sucrose metabolism in sugarbeet root by differential regulation of expression of the two isoforms and modulation of their activity by changes in cellular pH.     

Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF(1alpha) and TXB(2) aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.     

The interaction of ferredoxin and glutamate synthase: cross-linking and immunological studies

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, glutamate synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to glutamate synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:glutamate synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on glutamate synthase, does not recognize the cross-linked ferredoxin-glutamate synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on glutamate synthase renders this epitope inaccessible to the antibody.     

Comparative proteomics of leaf,stem, and root tissues of synthetic Brassica napus

Comparative proteomics was applied to three vegetative organs of Brassica napus, the leaf, stem, and root using 2‐DE. Among the >1600 analyzed spots, 43% were found to be common to all three organs, suggesting the existence of a “basal” or ubiquitous proteome composed of housekeeping proteins. The green organs, leaf, and stem, were closely related (～80% common spots) while the root displayed more organ‐specific polypeptides (～10%). Reference maps were established using MS, allowing the identification of 93, 385, and 266 proteins in leaf, stem, and root proteomes, respectively. Bioinformatic analyses were also performed; in silico functional categorization and cellular localization allow obtaining a precise picture of the cell molecular network within vegetative organs. These proteome maps can be explored using the PROTICdb software at the following address: http://bioinformatique.moulon.inra.fr/proticdb/web_view/.     

Absolute quantification of Medicago truncatula sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry

Mass spectrometry (MS) has become increasingly important for tissue specific protein quantification at the isoform level, as well as for the analysis of protein post-translational regulation mechanisms and turnover rates. Thanks to the development of high accuracy mass spectrometers, peptide sequencing without prior knowledge of the amino acid sequence--de novo sequencing--can be performed. In this work, absolute quantification of a set of key enzymes involved in carbon and nitrogen metabolism in Medicago truncatula 'Jemalong A17' root nodules is presented. Among them, sucrose synthase (SuSy; EC 2.4.1.13), one of the central enzymes in sucrose cleavage in root nodules, has been further characterized and the relative phosphorylation state of the three most abundant isoforms has been quantified. De novo sequencing provided sequence information of a so far unidentified peptide, most probably belonging to SuSy2, the second most abundant isoform in M. truncatula root nodules. TiO(2)-phosphopeptide enrichment led to the identification of not only a phosphorylation site at Ser11 in SuSy1, but also of several novel phosphorylation sites present in other root nodule proteins such as alkaline invertase (AI; EC 3.2.1.26) and an RNA-binding protein.     

Glutamate synthase from Chlamydomonas reinhardtii: Interaction studies with its substrate ferredoxin and molecular cloning

Glutamate synthase (GOGAT) from Chlamydomonas reinhardtii is able to form functional covalent complexes with its substrate ferredoxin (Fd), either wild-type (^WTFd) or recombinant form (^rFd). However, when Fd carboxyl groups were chemically modified (^mdFd), no complexes were detected and its ability to serve as electron donor for glutamate synthase activity was also decreased. By site-directed mutagenesis, we have demonstrated that Fd glu91 and a negative core in the helix α1 are critical for Fd interaction with this enzyme and its functionality as electron carrier for glutamate synthase. As a previous step to elucidate the specific positive charged residues involved in glutamate synthase interaction with Fd, we have isolated a cDNA, CrFG-3, encoding Fd-GOGAT from C. reinhardtii. The cDNA comprised about 60% of the protein and sequence comparison showed that CrFG-3 was structurally more similar to higher plant enzymes than to the corresponding prokaryotic GOGAT. Two conserved domains were present in this protein fragment, the FMN-binding domain and the cysteines involved in the iron–sulfur cluster binding. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characteristics of ammonium absorption by excised root and leaf tissues of maize

Absorption of ammonium from solutions of ammonium chloride by maize ( Zea mays L. cv. GS-2) tissue was studied. In contrast to an initial rapid phase of absorption in root tissue, a one hour lag period was recorded in leaf tissue. The maximum rate of uptake was observed at 5–10 m M NH₄Cl in both tissues. Roots had a K_m value of 1.0 m M and V_max of 24.3 μmol ammonium (g fresh weight)⁻¹ h⁻¹, whereas the leaf tissue had a higher K_m (4.1 m M ) and a lower V_max (8.7 μmol). There was a concentration dependent increase in ethanol soluble and insoluble fractions of organic nitrogen during ammonium supply. The optimum pH for ammonium absorption for both tissues was 7.4. The optimal concentration of CaCl₂ for ammonium absorption was 5 m M whereas that of KCl was only 1 m M . In both tissues, the absorption was inhibited substantially by DCMU, DNP, cycloheximide, lincomycin, sodium tungstate, sodium arsenate and to some extent also by the anions nitrate and sulfate. It is suggested that a carrier is involved in an active uptake of ammonium in the leaf tissues.     

Dissecting substrate specificity of two rice BADH isoforms: Enzyme kinetics, docking and molecular dynamics simulation studies

Fragrance rice (Oryza sativa) contains two isoforms of BADH, named OsBADH1 and OsBADH2. OsBADH1 is implicated in acetaldehyde oxidation in rice plant peroxisomes, while the non-functional OsBADH2 is believed to be involved in the accumulation of 2-acetyl-1-pyrroline, the major compound of aroma in fragrance rice. In the present study, site-directed mutagenesis, molecular docking and molecular dynamics simulation studies were used to investigate the substrate specificity towards Bet-ald and GAB-ald. Consistent with our previous study, kinetics data indicated that the enzymes catalyze the oxidation of GAB-ald more efficiently than Bet-ald and the OsBADH1 W172F and OsBADH2 W170F mutants displayed a higher catalytic efficiency towards GAB-ald. Molecular docking analysis and molecular dynamics simulations for the first time provided models for aldehyde substrate-bound complexes of OsBADHs. The amino acid residues, E262, L263, C296 and W461 of OsBADH1 and E260, L261, C294 and W459 of OsBADH2 located within 5 Å of the OsBADH active site mainly interacted with GAB-ald forming strong hydrogen bonds in both OsBADH isoforms. Residues W163, N164, Q294, C296 and F397 of OsBADH1–Bet-ald and Y163, M167, W170, E260, S295 and C453 of OsBADH2–Bet-ald formed the main interaction sites while E260 showed an interaction energy of −14.21 kcal/mol. Unconserved A290 in OsBADH1 and W288 in OsBADH2 appeared to be important for substrate recognition similar to that observed in PsAMADHs. Overall, the results here help to explain how two homologous rice BADHs recognize the aldehyde substrate differently, a key property to their biological role.     

Characterization of protoheme levels in etiolated and greening plant tissues

The protoheme content of etiolated, greening, and fully greened bean (Phaseolus vulgaris L. var. Light Red Kidney) leaves has been studied. The protoheme level in etiolated and fully greened leaf tissue stays relatively constant from age 7 to 14 days. In agreement with the studies reported for barley (Castelfranco and Jones 1975 Plant Physiol 55: 485-490), the protoheme content of greening bean and barley (Hordeum vulgare var. Larker) leaves does not change appreciably during the first 9 hours of illumination, but the level rises significantly by the 24th hour of illumination (cf. Hendry and Stobart 1977 Phytochemistry 16: 1545-1548). This increase also occurs in seedlings returned to the dark for 24 to 48 hours following a 10-minute pulse of light. These results demonstrate a limited correlation with previous studies on the development of b-type cytochromes during greening of these tissues (Gregory and Bradbeer 1973; Planta 109: 317-326).     

Multiple, distinct isoforms of sucrose synthase in pea

Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum). The genes have distinct patterns of expression in different organs of the plant, and during organ development. Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties. Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms. Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1. One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95%. The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected. The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell.     

Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature.     

Qualitative and quantitative studies of some enzymes in the nodule,root and shoot ofPisum sativum

Summary The specific activity of enzymes involved in glutamate metabolism in the nodule were found to be approximately 2–4 times higher than those of the root and 5–10 higher than those of the shoot. The occurence of multiple forms of these enzymes in nodule, shoot and root is investigated by using starch gel electrophoresis.