Water deficit during meiosis in pollen mother cells of wheat (Triticum aestivum L.) induces male sterility, which can reduce grain set by 40 to 50%. In plants stressed during meiosis and then rewatered, division of pollen mother cells proceeds normally but subsequent pollen development is arrested 3 or 4 d later. An inhibition of starch accumulation within the pollen grain suggested that an alteration in carbohydrate metabolism or assimilate supply may be involved in pollen abortion. We measured levels of various carbohydrates and activities of key enzymes of Suc metabolism and starch synthesis at different stages of pollen development in anthers collected from well-watered and water-stressed plants. Compared to controls, soluble sugars increased in anthers stressed during meiosis, then decreased at later poststress stages. Sucrose and myoinositol accounted for part of the sugar accumulation. The activity of soluble acid invertase declined 4-fold during the stress period and never recovered thereafter. Sucrose synthase activity during starch accumulation in pollen was also lower in the anthers of plants stressed at meiosis. Stress had little negative effect on the activities of ADP-glucose pyrophosphorylase or soluble and granule-bound starch synthase during starch accumulation in pollen, although at the earlier stages, ADP-glucose pyrophosphorylase activity in stressed anthers was slightly lower compared to controls. The results suggest that carbohydrate starvation per se and inhibition of the enzymes of starch synthesis probably were not responsible for the stress-induced pollen abortion. Instead, an inability to metabolize incoming sucrose to hexoses may be involved in this developmental lesion.

Despite the importance of the stress-activated protein kinase pathways in cell death and survival, it is unclear how stressful stimuli lead to their activation. In the case of heat shock, the existence of a specific mechanism of activation has been evidenced, but the molecular nature of this pathway is undefined. Here, we found that Ask1 (apoptosis signal-regulating kinase 1), an upstream activator of the stress-activated protein kinase p38 during exposure to oxidative stress and other stressful stimuli, was also activated by heat shock. Ask1 activity was required for p38 activation since overexpression of a kinase dead mutant of Ask1, Ask1(K709M), inhibited heat shock-induced p38 activation. The activation of Ask1 by oxidative stress involves the oxidation of thioredoxin, an endogenous inhibitor of Ask1. A different activation mechanism takes place during heat shock. In contrast to p38 induction by H 2 O 2 , induction by heat shock was not antagonized by pretreatment with the antioxidant Nacetyl-L-cysteine or by overexpressing thioredoxin and was not accompanied by the dissociation of thioredoxin from Ask1. Instead, heat shock caused the dissociation of glutathione S-transferase Mu1-1 (GSTM1-1) from Ask1 and overexpression of GSTM1-1-inhibited induction of p38 by heat shock. We concluded that because of an alternative regulation by the two distinct repressors thioredoxin and GSTM1-1, Ask1 constitutes the converging point of the heat shock and oxidative stress-sensing pathways that lead to p38 activation.Heat shock affects all proteins and structures but nevertheless produces a highly specific stress response aimed at protecting the cells and re-establishing homeostasis. In addition to the well characterized transcriptional activation of the genes coding for heat shock proteins (1-3), within minutes heat shock activates a major signal transduction pathway involving the stress-activated protein kinase p38 and leading to the phosphorylation of heat shock protein 27 (HSP27) (4, 5). Phosphorylation of HSP27 activates a protective function, which may result from the known phosphorylation-modulated function of the protein at the level of the actin microfilaments (6 -8) or from other described protective activities, either as a chaperone (9 -11) or as an inhibitor of apoptotic processes (12)(13)(14). Activation of the p38 pathway and phosphorylation of HSP27 occurs within minutes at elevated temperature and constitutes a very tightly regulated response (15). After a mild heat shock, cells becomes refractory to reinduction of p38 activity by a second heat shock but remained fully responsive to reinduction by other stresses, cytokines, or growth factors (15). The specificity of this desensitization reinforces the existence of a highly specific heat shock-sensing pathway upstream of p38. Despite its importance for cell survival, the signaling components and the molecular mechanism leading to heat shock-induced p38 activation are unknown.Little is known about the mechanisms of activation of the stress-sensitive pathways. I...

In addition to inducing new transcriptional activities that lead within a few hours to the accumulation of heat shock proteins (Hsps), heat shock activates within minutes the major signaling transduction pathways involving mitogen-activated protein kinases, extracellular signal-regulated kinase, stress-activated protein kinase 1 (SAPK1)-c-Jun N-terminal kinase, and SAPK2-p38. These kinases are involved in both survival and death pathways in response to other stresses and may, therefore, contribute significantly to the heat shock response. In the case of p38, the activation leads to the phosphorylation and activation of one of the Hsps, Hsp27. Phosphorylation occurs very early during stress, is tightly regulated, and results from the triggering of a highly specific heat shock-sensing pathway.

Glutathione is an essential metabolite for plant life best known for its role in the control of reactive oxygen species (ROS). Glutathione is also involved in the detoxification of methylglyoxal (MG) which, much like ROS, is produced at low levels by aerobic metabolism under normal conditions. While several physiological processes depend on ROS and MG, a variety of stresses can dramatically increase their concentration leading to potentially deleterious effects. In this review, we examine the structure and the stress regulation of the pathways involved in glutathione synthesis and degradation. We provide a synthesis of the current knowledge on the glutathione-dependent glyoxalase pathway responsible for MG detoxification. We present recent developments on the organization of the glyoxalase pathway in which alternative splicing generate a number of isoforms targeted to various subcellular compartments. Stress regulation of enzymes involved in MG detoxification occurs at multiple levels. A growing number of studies show that oxidative stress promotes the covalent modification of proteins by glutathione. This post-translational modification is called S-glutathionylation. It affects the function of several target proteins and is relevant to stress adaptation. We address this regulatory function in an analysis of the enzymes and pathways targeted by S-glutathionylation.

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In plant cells, an increase in cellular oxidants can have multiple effects, including the promotion of mixed disulfide bonds between glutathione and some proteins (S-glutathionylation). The present study focuses on the cytosolic isoform of the glycolytic enzyme triosephosphate isomerase (cTPI) from Arabidopsis thaliana and its reversible modification by glutathione. We used purified recombinant cTPI to demonstrate the enzyme sensitivity to inhibition by N-ethylmaleimide, hydrogen peroxide and diamide. Treatment of cTPI with diamide in the presence of reduced glutathione (GSH) led to a virtually complete inhibition of its enzymatic activity by S-glutathionylation. Recombinant cTPI was also sensitive to the oxidized form of glutathione (GSSG) in the micromolar range. Activity of cTPI was restored after reversion of S-glutathionylation by two purified recombinant A. thaliana cytosolic glutaredoxins (GRXs). GRXs-mediated deglutathionylation of cTPI was dependent on a GSH-regenerating system. Analysis of cTPI by mass spectrometry after S-glutathionylation by GSSG revealed that two Cys residues (Cys127 and Cys218) were modified by glutathione. The role of these two residues was assessed using site-directed mutagenesis. Mutation of Cys127 and Cys218 to Ser separately or together caused different levels of decrease in enzyme activity, loss of stability, as well as alteration of intrinsic fluorescence, underlining the importance of these Cys residues in protein conformation. Comparison of wild-type and mutant proteins modified with biotinyl glutathione ethyl ester (BioGEE) showed partial binding with single mutants and total loss of binding with the double mutant, demonstrating that both Cys residues were significantly S-glutathionylated. cTPI modification with BioGEE was reversed using DTT. Our study provides the first identification of the amino acid residues involved in cTPI S-glutathionylation and supports the hypothesis that this reversible modification could be part of an oxidative stress response pathway.

Triosephosphate isomerase (TPI, EC 5.3.1.1) catalyzes the interconversion of dihydroxyacetone-P and glyceraldehyde 3-P in the glycolytic pathway. A constitutively expressed antisense construct for cytosolic TPI was introduced into potato (Solanum tuberosum) using Agrobacterium rhizogenes to examine the metabolic effects of a reduction in cytosolic TPI in roots. We obtained a population of transgenic root clones displaying ~36 to 100 % of the TPI activity found in control clones carrying an empty binary vector. Ion exchange chromatography and immunoblot analysis showed that the antisense strategy significantly decreased the cytosolic TPI isoform, while levels of plastidial TPI activity remained apparently unaffected. Transgenic roots were characterized with respect to the activity of glycolytic enzymes, their metabolite contents and carbon fluxes. Metabolite profiling of sugars, organic acids, amino acids and lipids showed elevated levels of sucrose, glucose, fructose, fumarate, isocitrate, 4-aminobutyrate, alanine, glycine, aromatic amino acids and saturated long chain fatty acids in roots containing the lowest TPI activity. Labelings with (14)C-glucose, (14)C-sucrose and (14)C-acetate indicated that a reduction of cytosolic TPI activity in roots increased carbon metabolism through the pentose phosphate pathway, O(2) uptake and catabolism of sucrose to CO(2), and capacity for lipid synthesis. These results demonstrate that a large reduction of cytosolic TPI alters the distribution of carbon in plant primary metabolism.

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 6xHis-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.