Jean Rivoal

Biosynthesis of 3-Dimethylsulfoniopropionate in Wollastonia biflora (L.) DC. (Evidence That S-Methylmethionine Is an Intermediate)

The compatible solute 3-dimethylsulfoniopropionate (DMSP) is accumulated by certain salt-tolerant flowering plants and marine algae. It is the major biogenic precursor of dimethylsulfide, an important sulfur-containing trace gas in the atmosphere. DMSP biosynthesis was investigated in Wollastonia biflora (L.) DC. [=Wedelia biflora (L.) DC., Melanthera biflora (L.) Wild, Asteraceae]. After characterizing DMSP and glycine betaine accumulation in three diverse genotypes, a glycine betaine-free genotype was chosen for radiotracer and stable isotope-labeling studies. In discs from young leaves, label from [U-14C]methionine was readily incorporated into the dimethylsulfide and acrylate moieties of DMSP. This establishes that DMSP is derived from methionine by deamination, decarboxylation, oxidation, and methylation steps, without indicating their order. Five lines of evidence indicated that methylation is the first step in the sequence, not the last. (a) In pulse-chase experiments with [14C]methionine, S-methylmethionine (SMM) had the labeling pattern expected of a pathway intermediate, whereas 3-methylthiopropionate (MTP) did not. (b) [14C]SMM was efficiently converted to DMSP but [14C]MTP was not. (c) The addition of unlabeled SMM, but not of MTP, reduced the synthesis of [14C]DMSP from [14C]methionine. (d) The dimethylsulfide group of [13CH3,C2H3]SMM was incorporated as a unit into DMSP. (e) When [C2H3,C2H3]SMM was given together with [13CH3]methionine, the main product was [C2H3,C2H3]DMSP, not [13CH3,C2H3]DMSP or [13CH3,13CH3]DMSP. The stable isotope labeling results also show that the SMM cycle does not operate at a high level in W. biflora leaves.

Metabolic Control of Anaerobic Glycolysis (Overexpression of Lactate Dehydrogenase in Transgenic Tomato Roots Supports the Davies-Roberts Hypothesis and Points to a Critical Role for Lactate Secretion

Roots of all plants examined so far have the potential for both ethanol and lactate fermentation. A short burst of lactate fermentation usually occurs when plant tissues are transferred from normoxic to anoxic conditions. According to the Davies-Roberts hypothesis, the consequent pH drop both initiates ethanol fermentation and blocks further production of lactate by inhibiting lactate dehydrogenase (LDH). However, the role of LDH in this pH control mechanism is still a matter of debate. To perturb the control system in a defined way, a barley LDH cDNA under the control of the cauliflower mosaic virus 35S promoter was introduced into tomato (Lycopersicon esculentum Mill. cv VFMT) using Agrobacterium rhizogenes. The transgenic root clones expressed up to 50 times the LDH activity of controls. The fermentative metabolism of these clones was compared using roots grown previously in normoxic conditions or roots given a 3-d hypoxic pretreatment. During the transition from normoxia to anoxia, lactate accumulation was no faster and no more extensive in transgenic roots than in controls. Similarly, during prolonged anoxia the flux of 14C from [U-14C] glucose to lactate and ethanol was not modified by the expression of the transgene. However, in both transgenic and control roots, hypoxic pretreatment increased the flux to lactate and promoted lactate export to the medium. These results show that LDH has a very low flux control coefficient for lactate fermentation, consistent with the Davies-Roberts hypothesis. Moreover, they suggest that lactate secretion exerts major control over long-term lactate glycolysis in vivo.

Differential Induction of Pyruvate Decarboxylase Subunits and Transcripts in Anoxic Rice Seedlings

In 2-d-old rice (Oryza sativa L.) seedlings subjected to anoxic stress, pyruvate decarboxylase (PDC) activity increased 9-fold during a 168-h period. A polyclonal PDC antiserum that recognized [alpha]- and [beta]-subunits was used to quantify PDC protein by an enzyme-linked immunosorbant assay and showed a 5.6-fold increase, suggesting that the anoxically induced enzyme has a higher specific activity than the PDC isoform present under normoxia. Immunoblot analysis showed that levels of both PDC subunits were induced by anoxia. Immunoprecipitation of proteins labeled in vivo during anoxic treatment demonstrated that the [alpha]-subunit was preferentially synthesized at the onset of anoxia Two partial cDNAs, including a novel sequence, were cloned from a cDNA library made from seedlings subjected to anoxia for 6 h. Gene-specific probes used to quantify northern blots showed that two or three PDC mRNAs are differentially induced by anoxia in rice seedlings. Immunoprecipitation of in vitro translation products of mRNAs isolated at different times of anoxic treatment confirmed this finding. Our results suggest that anoxic induction of rice PDC involves transcriptional and posttranscriptional regulation of gene expression as well as differences in enzyme characteristics.

Evidence for a Large and Sustained Glycolytic Flux to Lactate in Anoxic Roots of Some Members of the Halophytic Genus Limonium

Soil salinity and anaerobiosis often occur together. This led us to investigate the fermentative metabolism in roots of species from the halophytic genus Limonium (Plumbaginaceae). Root segments from hypoxically induced plants were incubated for 8 h under strict anoxia in the presence of [U-14C]glucose. In three species (Limonium latifolium, L. nashii, and L. humile), the pattern of 14C-labeled end products was typical of higher plants, with a 14C flux to ethanol higher than that to lactate. However, in four species (L. ramosissimum, L. gougetianum, L perezii, and L. sinuatum), the rate of lactate fermentation was exceptionally high, and in the latter two species the 14C flux to lactate exceeded that to ethanol. These two species secreted most of the lactate produced into the medium. Calculations indicated that the cytoplasm would have been lethally acidified had this secretion not occurred. The effects of factors that might control lactate fermentation or secretion (O2 partial pressure, pH, salt concentration) were studied in two contrasting species: L. sinuatum and L. latifolium. In both species, the lactate:ethanol ratio was higher under hypoxia (0.1–)3 kPa O2 partial pressure) than under strict anoxia. In L. sinuatum, this ratio was slightly increased by increasing the pH of the medium from 5.5 to 7.5, but salinity treatment had no effect. The potential contribution of lactate fermentation to the overall carbon and energy metabolism of halophytes is discussed.

Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway

A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed.

Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato

A cDNA encoding Solanum chacoense cytosolic NDPK (NDPK1, EC 2.7.4.6) was isolated. The open reading frame encoded a 148 amino acid protein that shares homology with other cytosolic NDPKs including a conserved N-terminal domain. S. chacoense NDPK1 was expressed in Escherichia coli as a 6×His-tagged protein and purified by affinity chromatography. The recombinant protein exhibited a pattern of abortive complex formation suggesting that the enzyme is strongly regulated by the NTP/NDP ratio. A polyclonal antibody generated against recombinant NDPK1 was specific for the cytosolic isoform in Solanum tuberosum as shown from immunoprecipitation experiments and immunoblot analysis of chloroplasts and mitochondria preparations. NDPK activity and NDPK1 protein were found at different levels in various vegetative and reproductive tissues. DEAE fractogel analyses of NDPK activity in root tips, leaves, tubers and cell cultures suggest that NDPK1 constitutes the bulk of extractable NDPK activity in all these organs. NDPK activity and NDPK1 protein levels raised during the exponential growth phase of potato cell cultures whereas no rise in activity or NDPK1 protein was observed when sucrose concentration in the culture was manipulated to limit growth. Activity measurements, immunoblot analysis as well as immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that NDPK1 was predominantly localized in the meristematic zones and provascular tissues of the apical regions. These data suggest that NDPK1 plays a specific role in the supply of UTP during early growth of plant meristematic and provascular tissues.

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase contains two subunits differentially regulated by anaerobiosis

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is composed of two subunits of different molecular weights. Cytosolic GAPDH activity and protein both decreased immediately after transfer of 48-h rice seedlings to anaerobic conditions. Subsequent increase in activity and protein was accompanied by a change in isoenzyme profile and was preceded by an increase in steady-state messenger levels. One and two-dimensional electrophoretic analyses of in vivo and in vitro labeled GAPDH suggested that the change in isoenzyme profile under anaerobic conditions is due to preferential synthesis of one of the two GAPDH subunits caused by a specific increase in its mRNA.

Choline-O-Sulfate Biosynthesis in Plants (Identification and Partial Characterization of a Salinity-Inducible Choline Sulfotransferase from Species of Limonium (Plumbaginaceae)

Choline-O-sulfate is a compatible osmolyte accumulated under saline conditions by members of the halophytic genus Limonium and other Plumbaginaceae. A choline sulfotransferase (EC 2.8.2.6) responsible for the formation of choline-O-sulfate was characterized in Limonium species. A simple radiometric assay was developed in which [14C]choline was used as substrate, and the h [14C]choline-O-sulfate product was isolated by ion-exchange chromatography. The choline sulfotransferase activity was soluble, required 3[prime]-phosphoadenosine-5[prime]-phosphosulfate as the sulfate donor, and showed a pH optimum at 9.0. Apparent Km values were 25 [mu]M for choline and 5.5 [mu]M for 3[prime]-phosphoadenosine-5[prime]-phosphosulfate. Choline sulfotransferase activity was detected in various Limonium species but was very low or absent from species that do not accumulate choline-O-sulfate. In roots and leaves of Limonium perezii, the activity was increased at least 4-fold by salinization with 40% (v/v) artificial sea water. Choline sulfotransferase activity was also induced in cell cultures of L. perezii following salt shock with 20% (v/v) artificial sea water or osmotic shock with 19% (w/v) polyethylene glycol 6000. Labeling experiments with [14C]choline confirmed that the enzyme induced in cell cultures was active in vivo.

Characterization of a mitochondrial NADP-dependent isocitrate dehydrogenase in axes of germinating sunflower seeds

Abstract An NADP-dependent isocitrate dehydrogenase (NADP-IDH) activity was measured in a Percoll purified mitochondrial fraction from axes of germinating sunflower (Helianthus annuus, L.) seeds. Fraction enrichment in mitochondria was revealed by marker enzyme activities and by electron microscopy which showed no contamination by other organelles and indicated that no fragmentation of mitochondria had occurred during their preparation. More than 95% of the NADP-IDH activity was latent; it was found to be different from the mitochondrial NAD-IDH activity by competition assay. Mitochondrial NADP-IDH was electrophoretically and immunologically different from the NAD-specific and the cytosolic NADP-specific isoforms. The enzyme activity from mitochondrial matrix extract was eluted from Superose 12 with a molecular weight of 70 kDa. A possible physiological role of the mitochondrial NADP-IDH is discussed.

Clues to the functions of plant NDPK isoforms.

This review describes the five nucleoside diphosphate kinase (NDPK) genes found in both model plants Arabidopsis thaliana (thale cress) and Oryza sativa L. (rice). Phylogenetic and sequence analyses of these genes allow the definition of four types of NDPK isoforms with different predicted subcellular localization. These predictions are supported by experimental evidence for most NDPK types. Data mining also provides evidence for the existence of a novel NDPK type putatively localized in the endoplasmic reticulum. Phylogenic analyses indicate that plant types I, II, and III belong to the previously identified Nme group I whereas type IV belongs to Nme group II. Additional analysis of the literature offers clues supporting the idea that the various plant NDPK types have different functions. Hence, cytosolic type I NDPKs are involved in metabolism, growth, and stress responses. Type II NDPKs are localized in the chloroplast and mainly involved in photosynthetic development and oxidative stress management. Type III NDPKs have dual targeting to the mitochondria and the chloroplast and are principally involved in energy metabolism. The subcellular localization and precise function of the novel type IV NDPKs, however, will require further investigations.