Bérénice Ricard

The Role of Sugars, Hexokinase, and Sucrose Synthase in the Determination of Hypoxically Induced Tolerance to Anoxia in Tomato Roots

Hypoxic pretreatment of tomato (Lycopersicon esculentum M.) roots induced an acclimation to anoxia. Survival in the absence of oxygen was improved from 10 h to more than 36 h if external sucrose was present. The energy charge value of anoxic tissues increased during the course of hypoxic acclimation, indicating an improvement of energy metabolism. In acclimated roots ethanol was produced immediately after transfer to anoxia and little lactic acid accumulated in the tissues. In nonacclimated roots significant ethanol synthesis occurred after a 1-h lag period, during which time large amounts of lactic acid accumulated in the tissues. Several enzyme activities, including that of alcohol dehydrogenase, lactate dehydrogenase, pyruvate decarboxylase, and sucrose synthase, increased during the hypoxic pretreatment. In contrast to maize, hexokinase activities did not increase and phosphorylation of hexoses was strongly inhibited during anoxia in both kinds of tomato roots. Sucrose, but not glucose or fructose, was able to sustain glycolytic flux via the sucrose synthase pathway and allowed anoxic tolerance of acclimated roots. These results are discussed in relation to cytosolic acidosis and the ability of tomato roots to survive anoxia.

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.

Expression of alcohol dehydrogenase in rice embryos under anoxia.

SummaryAlcohol dehydrogenase (ADH) activity was present in roots and shoots of 48-h rice embryos and rose in response to anoxia. The increase was accompanied by changes in the ADH isozyme pattern. Translatable levels of mRNA for two ADH peptides increases as early as 1 h after the beginning of anoxic treatment. Adh mRNA was detected in aerobically grown rice embryos by hybridization to maize Adh1 cDNA: its level increased significantly after 3 h of anoxia.

The tomato hexokinase LeHXK1 cloning, mapping, expression pattern and phylogenetic relationships

Abstract A tomato ( Lycopersicon esculentum L. Mill.) hexokinase, LeHXK1 , was cloned from a tomato fruit cDNA library. Functional complementation of triple-mutant yeast cells ( hxk1 hxk2 glk1 ) that lack the ability to phosphorylate glucose and fructose confirmed the identity of LeHXK1 as a hexokinase. LeHXK1 was mapped to tomato chromosome 3-distinct from LeHXK2 which mapped to chromosome 6. Interestingly, expression of LeHXK1 was very low in leaves and very high in young tomato fruits. Two hexokinase isozymes, HXKl and HXK2, were chromatographically separated from young fruit extracts, Antisense StHXK1 the homologue of, LeHXK1 , knocked out the HXKl isozyme, identifying HXKl as the gene product of LeHXK1 . Phylogenetic analysis of eukaryotic and prokaryotic hexose phosphorylating enzymes indicates that tomato hexokinase genes, LeHXK1 and LeHXK2 , and the known eukaryotic hexokinases and ‘glucokinases’ belong to the same hexokinase family whose members are unrelated to the glucose-specific glucokinases of bacteria. Hence, the data suggest that thus far no true eukaryotic glucokinases have been identified, neither from plants, nor from fungi or mammalian cells.

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.

Cloning and characterization of a cDNA encoding hexokinase from tomato

Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato (Lycopersicon esculentum) by RT-PCR using degenerate primers. Southern blot analysis suggested the existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK2 contained an open reading frame encoding a 496 amino acid protein that has 69% identity with the two Arabidopsis HXKs, 83 and 85% identity with potato StHXK1 and tobacco NtHXK, respectively. However, this clone had 97% amino acid identity with potato StHXK2 and, therefore, was named LeHXK2. LeHXK2 cDNA was expressed in a triple mutant yeast (Saccharomyces cerevisiae) strain which lacked the ability to phosphorylate glucose and fructose and, therefore, was unable to grow on these sugars as carbon sources. Mutant cells expressing LeHXK2 grew on both glucose and fructose with shorter doubling time on glucose. The kinetic properties of LeHXK2 expressed in yeast were determined after the purification of LeHXK2 by HPLC-ion exchange chromatography, confirming the identity of LeHXK2 as hexokinase with higher affinity to glucose. LeHXK2 mRNA was detected by RT-PCR expression analysis in all organs and tissues and at all stages of fruit development. However, semi-quantitative RT-PCR analysis showed that LeHXK2 was most highly expressed in flowers.

Differential expression of two tomato lactate dehydrogenase genes in response to oxygen deficit

Two different cDNAs encoding lactate dehydrogenase (LDH) were isolated from a library of hypoxically treated tomato roots and sequenced. The use of gene-specific probes on northern blots showed that Ldh2 mRNA was predominant in well-oxygenated roots and levels remained stable upon oxygen deficit; in contrast, Ldh1 mRNA accumulated to high levels within 2 h of hypoxia or anoxia. Immunoblot analyses of native gels using a polyclonal antiserum raised against an LDH1 fusion protein indicated that LDH2 homotetramer was the major isoform present in aerobic roots. Levels of both LDH1 and LDH2 subunits increased during an 18 h hypoxic treatment, together with a 5-fold rise in activity. These results suggest that the regulation of ldh1 expression is primarily at the transcriptional level while that of ldh2 is post-transcriptional. Increases in Ldh1 mRNA and LDH activity were not correlated with lactic acid production, which was maximal at the onset of anoxia in unacclimated roots and then declined. Taken together, our results indicate that LDH2 present in aerobic roots is principally responsible for lactic acid production occurring transiently upon imposition of anoxia. Possible physiological roles for LDH1 are discussed.

Rice embryos can express heat-shock genes under anoxia.

Heat-shock proteins (hsps) are induced by a number of oxidative stresses. The proposal that the reduction products of oxygen initiate hsp induction was tested in rice embryos, capable of coleoptile growth under oxygen-free conditions. In such embryos, hsps could be detected by both in vivo labeling and in vitro translation of RNA using the reticulocyte lysate system. It is therefore improbable that the mechanism for hsp induction involves oxygen.

Messenger properties of TYMV-RNA.

Abstract Turnip yellow mosaic virus (TYMV) RNA extracted from virions (“virion” TYMV-RNA) contains two types of molecules of molecular weight 2 × 10 6 : one which resists heat denaturation (“intact” TYMV-RNA) and one which yields smaller fragments upon heat denaturation (TYMV-RNA containing hidden breaks). A small RNA molecule of 0.2 × 10 6 MW is also present in virions. “Virion” TYMV-RNA can therefore be separated into “intact” RNA and smaller molecules by heat denaturation followed by sucrose gradient centrifugation. The RNA from the sucrose gradient fractions were analyzed by formamide PAGE: The small RNA with a molecular weight of 0.2 × 10 6 was separated from “intact” TYMV-RNA. The amount of coat protein synthesized by the RNA fractions when added to the wheat germ cell-free system was quite proportional to the amount of small RNA present. We conclude that coat protein synthesis is not directed by “intact” TYMV-RNA but by the small RNA. In addition, infectivity studies show that “intact” TYMV-RNA is fully infectious and thus it also contains the coat protein cistron.

Two ldh genes from tomato and their expression in different organs, during fruit ripening and in response to stress

Two different ldh genes have been isolated from a tomato genomic library and sequenced. Both contain a single intron and correspond to cDNA clones LeLdh1 and LeLdh2 isolated from a library constructed from hypoxically induced tomato roots. Southern blots indicate that the two genes comprise the entire ldh gene family in tomato. Both genes are expressed at low levels in leaves, fruit and roots. Their transcript levels do not change during fruit ripening. Ldh1 but not ldh2 is inducible by oxygen deficit in both roots and fruit.