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.

Evidence for the critical role of sucrose synthase for anoxic tolerance of maize roots using a double mutant

The induction of the sucrose synthase (SuSy) gene (SuSy) by low O2, low temperature, and limiting carbohydrate supply suggested a role in carbohydrate metabolism under stress conditions. The isolation of a maize (Zea mays L.) line mutant for the two known SuSy genes but functionally normal showed that SuSy activity might not be required for aerobic growth and allowed the possibility of investigating its importance during anaerobic stress. As assessed by root elongation after return to air, hypoxic pretreatment improved anoxic tolerance, in correlation with the number of SuSy genes and the level of SuSy expression. Furthermore, root death in double-mutant seedlings during anoxic incubation could be attributed to the impaired utilization of sucrose (Suc). Collectively, these data provide unequivocal evidence that Suc is the principal C source and that SuSy is the main enzyme active in Suc breakdown in roots of maize seedlings deprived of O2. In this situation, SuSy plays a critical role in anoxic tolerance.

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 a different times of anoxic treatment confirmed this findings Our results suggest that anoxic induction of rice PDC involves transcriptional and posttranscriptional regulation of gene expression as well as differences in enzyme characteristics.

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 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.

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.

Mammalian beta-D-mannosidase and beta-mannosidosis.

Lysosomal beta-D-mannosidase is the last exoglycosidase involved in the sequential degradation of the N-glycosylproteins glycans. Research on this enzyme was restricted before the discovery of its hereditary deficiency, first in goat (1981) and later in man (1986). We describe the biochemical aspects of these beta-mannosidosis and the properties of the beta-mannosidases of mammalian origin. Our own results concerning human enzyme (from kidney and urine, seminal plasma and blood cells) suggest that, apart from the case of the inherited disease, beta-mannosidase may become a useful tool in other pathologies.

Anaerobic stress induces the transcription and translation of sucrose synthase in rice.

Sucrose synthase activity increased in 2-day-old rice (Oryza sativa) seedlings submitted to anaerobic stress. Likewise, both denaturing and native Western blot analysis detected a rise in the cellular concentration of sucrose synthase protein. Significantly higher steady-state levels of sucrose synthase mRNA, as determined by Northern blots and by the ability of total RNA to direct in vitro synthesis of sucrose synthase, were also induced by anaerobic treatment. Analysis of run-on transcripts showed increased transcription of sucrose synthase genes as early as 60 minutes after initiation of anaerobic stress. Together, these results indicate that sucrose synthase is a typical anaerobic protein in rice.

Lactate Dehydrogenase in Oryza sativa L. Seedlings and Roots: Identification and Partial Characterization.

A lactate dehydrogenase activity is present in rice (Oryza sativa L.) seedlings and roots. Under aerobic conditions, lactate dehydrogenase activity is barely detectable in rice seedlings and is very low in rice roots. In 30 day old roots, the activity is increased two to three times by an anoxic or hypoxic treatment and can be detected on immunoblots by an antiserum raised against barley lactate dehydrogenase. The activity present in aerobic seedlings was partially purified. The native enzyme has a molecular mass of 160 kilodaltons, and is a tetramer of 2 subunit (38 and 39 kilodaltons) randomly associated. Studies of substrate specificity, native gel electrophoresis, and immunoblot analysis indicate that the partially purified enzyme is a typical lactate dehydrogenase. However, no increase of lactate dehydrogenase activity or protein was observed in seedlings transferred to anoxia.

Purification and partial characterization of pyruvate decarboxylase from Oryza sativa L.

Pyruvate decarboxylase(PyrDC) was purified from rice bran to a specific activity of 1 mu kat/mg and partially characterized. The holoenzyme is a tetramer of two types of subunits with molecular masses 64 kDa and 62 kDa. Purified rice PyrDC exhibits positive cooperative kinetics with respect to pyruvate and functions with a significant lag phase. When compared to other plant PyrDC, the lag phase was shorter at low pyruvate concentrations and the S0.5 was smaller. The optimum pH (6.25) was also less acidic and the enzyme retained 30% of its maximal activity at neutral pH. In contrast to other plant PyrDC, rice PyrDC could be active at the onset of anoxia and would be activated by small changes in pyruvate concentration.

Human urinary and renal alpha-L-fucosidases. A comparative study.

1. Enzymatic forms of alpha-L-fucosidase from human renal tissue and urine were investigated. 2. In renal tissue two different isoenzymatic patterns were obtained by chromatofocusing of either directly soluble or detergent solubilized extracts. 3. On the other hand the urinary isoenzymatic pattern is similar to that obtained for the renal soluble extract.

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.

Photoaffinity labeling of ANF receptor in cultured brain neurones.

A monoiodo derivative of rat atrial natriuretic factor (rANF) was shown to specifically bind to rat brain neurones in culture with low binding site capacity (10-20 fmoles per mg of protein) and high affinity (Kd = 50-100 pM). Several analogs of both rat and human ANF competed with 125I-rANF. No change in the number of binding sites was detected upon morphological differentiation of neurones in vitro. Finally a photoreactive derivative of 125I-rANF was prepared and photoaffinity labeling experiments carried out on cultured neurones. After reduction of disulfide bridges, a single band of Mr 60,000 was specifically labeled whereas without reduction, two labeled components of Mr 60,000 and 117,000 were detected.

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.

Expression of alcohol dehydrogenase in rice embryos under anoxia.

Alcohol 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.

A DNA topoisomerase type I from wheat embryo mitochondria.

In order to study DNA replication and expression in wheat mitochondria our laboratory has been seeking to develop a system that supports DNA synthesis and transcription, either in isolated mitochondria from wheat embryos or in a mitochondrial lysate from the same source deprived of endogenous DNA in vitro. We have characterized some of the enzymes involved in the DNA synthesis and transcription process. In this study we describe a DNA topoisomerase activity.Broken mitochondria from wheat embryos can actively relax negatively supercoiled DNA (pBR322, pAT153, etc...). The enzyme is intramitochondrial: the activity is detected only when intact organelles are broken by non-ionic detergent. Most of the topoisomerase activity found in the broken mitochondria is recovered in the mitochondrial lysate. It is stimulated by Mg(2+) and has an optimum salt concentration, KCl or NaCl, between 50 mM and 100 mM. ATP has no effect on this activity. Ethidium bromide, berenil, novobiocine and nalidixic acid, compounds currently used to characterize DNA topoisomerases, do not effect the relaxation of supercoiled DNA by the wheat mitochondrial activity. On the other hand N-ethylmaleimide has a strong inhibitory effect indicating that sulfhydryl groups are essential for enzyme activity. The molecular weight of the enzyme as determined by glycerol gradient sedimentation, is about 110 kd. Another important feature of the mitochondrial lysate DNA topoisomerase is the ability to relax positively supercoiled DNA, a property of eukaryotic topoisomerases I.

DNA synthesis in isolated mitochondria and mitochondrial extracts from wheat embryos.

DNA synthesis was studied using purified wheat embryo mitochondria as well as mitochondrial lysates deprived of endogenous DNA. The optimal conditions for DNA synthesis are very similar in both systems: ATP stimulates dramatically mitochondrial DNA synthesis and magnesium is a better co-factor than manganese, contrary to what has been reported in animal mitochondrial systems. Wheat mitochondrial DNA synthesis is resistant to aphidicolin and strongly inhibited by dideoxythymidine triphosphate and ethidium bromide. Thus, the DNA polymerase involved in this system seems to be the same as that previously purified and characterized from wheat embryo mitochondria (Christopheet al., Plant Science Letters 21: 181, 1981). Two different approaches: restriction endonuclease digestion followed by electrophoresis, and autoradiography and cesium chloride equilibrium centrifugation of mitochondrial DNA, where BrdUTP has been incorporated instead of TTP, show that long stretches of the mitochondrial genome have been synthesized.

Guanine-plus-cytosine content, hybridization percentages, and EcoRI restriction enzyme profiles of spiroplasmal DNA.

The guanine-plus-cytosine (G + C) content of spiroplasmal DNA was calculated from the melting temperature determined spectrophotometrically and the buoyant density determined by equilibrium density gradient centrifugation in CsCl. Only two ranges of G + C values were found: 25-27 mol% and 29-32 mol%. The DNA of the following spiroplasmas has 25-27 mol% G + C: Spiroplasma citri (serogroup I-1); the spiroplasmas pathogenic to the honeybee (KC3, BC3, and B63; serogroup I-2); the corn stunt strain (E275; serogroup I-3); the tick strain 277F (serogroup I-4); the drosophila strain (serogroup II); and one group of flower spiroplasmas (serogroup III). The DNA of a second group of flower spiroplasmas (serogroup IV) and the SMCA strain (serogroup V) has a G + C content of 29-31 mol/. The classification of flower spiroplasmas into two groups on the basis of G + C content agrees well with the groupings based on serologic and protein analysis. Spiroplasmas isolated from honeybees in Morocco (B13) or froghoppers in Corsica (L89) have 29-31 mol% G + C, a value that corroborates the relatedness of these strains and the flower spiroplasmas of serogroup IV found by serologic analysis. Reannealing experiments between the vivo-labeled DNA of S. citri and unlabeled DNA of other spiroplasmas gave the following percentages of hybridization: 64% with honeybee spiroplasma DNA, 49% with corn stunt spiroplasma DNA, and 19% with tick spiroplasma 277F DNA; no significant hybridization was observed with DNA of any other spiroplasma. The taxonomic position of the tick spiroplasma 277F within serogroup I was confirmed by hybridization experiments involving [3H]DNA of this strain. The value of polyacrylamide gel analysis of DNA fragments produced by the action of EcoRI restriction enzyme on DNAs from various spiroplasmas is also discussed.