Genomewide identification of genes involved in the potato response to drought indicates functional evolutionary conservation with Arabidopsis plants.

Potato is one of the four most important food crop plants worldwide and is strongly affected by drought. The following two pairs of potato cultivars, which are related in ancestry but show different drought tolerances, were chosen for comparative gene expression studies: Gwiazda/Oberon and Tajfun/Owacja. Comparative RNA-seq analyses of gene expression differences in the transcriptomes obtained from drought-tolerant versus drought-sensitive plants during water shortage conditions were performed. The 23 top-ranking genes were selected, 22 of which are described here as novel potato drought-responsive genes. Moreover, all but one of the potato genes selected have homologues in the Arabidopsis genome. Of the seven tested A. thaliana mutants with altered expression of the selected homologous genes, compared to the wild-type Arabidopsis plants, six showed an improved tolerance to drought. These genes encode carbohydrate transporter, mitogen-activated protein kinase kinase kinase 15 (MAPKKK15), serine carboxypeptidase-like 19 protein (SCPL19), armadillo/beta-catenin-like repeat-containing protein, high-affinity nitrate transporter 2.7 and nonspecific lipid transfer protein type 2 (nsLPT). The evolutionary conservation of the functions of the selected genes in the plant response to drought confirms the importance of these identified potato genes in the ability of plants to cope with water shortage conditions. Knowledge regarding these gene functions can be used to generate potato cultivars that are resistant to unfavourable conditions. The approach used in this work and the obtained results allowed for the identification of new players in the plant response to drought.

Proteomic analysis of S-nitrosylated and S-glutathionylated proteins in wheat seedlings with different dehydration tolerances.

A loss of dehydration tolerance in wheat seedlings on the fifth day following imbibition is associated with a disturbance in cellular redox homeostasis, as documented by a shift of the reduced/oxidized glutathione ratio to a more oxidized state and a significant increase in the ratio of protein thiols to the total thiol group content. Therefore, the identification and characterization of redox-sensitive proteins are important steps toward understanding the molecular mechanisms of the loss of dehydration tolerance. In the present study, proteins that were differentially expressed between fully turgid (control), dehydrated tolerant (four-day-old) and dehydrated sensitive (six-day-old) wheat seedlings were analysed. Protein spots having at least a significant (p < 0.05) two-fold change in protein abundance were selected by Delta2D as differentially expressed, identified by MALDI-TOF and LC-MS/MS, and classified according to their function. The observed changes in the proteomic patterns of the differentially S-nitrosylated and S-glutathionylated proteins were highly specific in dehydration-tolerant and -sensitive wheat seedlings. The metabolic function of these proteins indicates that dehydration tolerance is mainly related to nucleic acids, protein metabolism, and energy metabolism. It has been proven that leaf-specific thionins BTH6 and DB4, chloroplastic 50S ribosomal protein L16, phospholipase A1-II delta, and chloroplastic thioredoxin M2 are both S-nitrosylated and S-glutathionylated upon water deficiency. Our results revealed the existence of interplay between S-nitrosylation and S-glutathionylation, two redox-regulated protein posttranslational modifications that could enhance plant defence mechanisms and/or facilitate the acclimation of plants to unfavourable environmental conditions.

Maize proteomic responses to separate or overlapping soil drought and two-spotted spider mite stresses.

MAIN CONCLUSION: In maize, leaf proteome responses evoked by soil drought applied separately differ from those evoked by mite feeding or both types of stresses occurring simultaneously. This study focuses on the involvement of proteomic changes in defence responses of a conventional maize cultivar (Bosman) to the two-spotted spider mite infestation, soil drought and both stresses coexisting for 6 days. Under watering cessation or mite feeding applied separately, the protein carbonylation was not directly linked to the antioxidant enzymes' activities. Protein carbonylation increased at higher and lower SOD, APX, GR, POX, PPO activities following soil drought and mite feeding, respectively. Combination of these stresses resulted in protein carbonylation decrease despite the increased activity of all antioxidant enzymes (except the CAT). However, maize protein network modification remains unknown upon biotic/abiotic stresses overlapping. Here, using multivariate chemometric methods, 94 leaf protein spots (out of 358 considered; 2-DE) were identified (LC-MS/MS) as differentiating the studied treatments. Only 43 of them had individual discrimination power. The soil drought increased abundance of leaf proteins related mainly to photosynthesis, carbohydrate metabolism, defence (molecular chaperons) and protection. On the contrary, mite feeding decreased the abundance of photosynthesis related proteins and enhanced the abundance of proteins protecting the mite-infested leaf against photoinhibition. The drought and mites occurring simultaneously increased abundance of proteins that may improve the efficiency of carbon fixation, as well as carbohydrate and amino acid metabolism. Furthermore, increased abundance of the Rubisco large subunit-binding protein (subunit ß), fructose-bisphosphate aldolase and mitochondrial precursor of Mn-SOD and decreased abundance of the glycolysis-related enzymes in the mite-free leaf (in the vicinity of mite-infested leaf) illustrate the involvement of these proteins in systemic maize response to mite feeding.

[Redox modifications of cysteine residues in plant proteins]. / Modyfikacje oksydoredukcyjne reszt cysteinowych bialek roslinnych.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS), considered as toxic by-products of aerobic metabolism, play a key role in intracellular signaling pathways. The putative mechanism of cell responses to ROS and RNS is thiol modifications of cysteine residues, which can be oxidized to varying degrees to cause changes in protein conformation and activity. These post-translational modifications include generation of disulphide bridges, formation of sulphenic, sulphinic and sulphonic acids as well as S-glutathionylation and S-nitrosylation. The main feature of these modifications is their potential reversibility (with the exception of sulphonic acid). There is increasing evidence for the involvement of thioredoxins, peroxiredoxins and glutaredoxins in regulation of thiol modifications. This review provides an overview of recent findings and discusses the modifications of protein cysteine residues and their contribution to the maintenance and control of cellular homeostasis.

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.).

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65kDa and both of them are monomers in native state. Nonsignificant differences between K(m) using alanine as substrate and catalytic efficiency (k(cat)/K(m)) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50kDa. The estimated K(m) for GGAT1 (3.22M) and GGAT2 (1.27M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.

Non-parametric multivariate analysis of variance in the proteomic response of potato to drought stress.

Spot detection is a mandatory step in all available software packages dedicated to the analysis of 2D gel images. As the majority of spots do not represent individual proteins, spot detection can obscure the results of data analysis significantly. This problem can be overcome by a pixel-level analysis of 2D images. Differences between the spot and the pixel-level approaches are demonstrated by variance analysis for real data sets (part of a larger research project initiated to investigate the molecular mechanism of the response of the potato to drought stress). As the method of choice for the analysis of data variation, the non-parametric MANOVA was chosen. NP-MANOVA is recommended as a flexible and very fast tool for the evaluation of the statistical significance of the factor(s) studied.

Differential regulation of alanine aminotransferase homologues by abiotic stresses in wheat (Triticum aestivum L.) seedlings.

Wheat (Triticum aestivum L.) seedlings contain four alanine aminotransferase (AlaAT) homologues. Two of them encode AlaAT enzymes, whereas two homologues act as glumate:glyoxylate aminotransferase (GGAT). To address the function of the distinct AlaAT homologues a comparative examination of the changes in transcript level together with the enzyme activity and alanine and glutamate content in wheat seedlings subjected to low oxygen availability, nitrogen and light deficiency has been studied. Shoots of wheat seedlings were more tolerant to hypoxia than the roots as judging on the basis of enzyme activity and transcript level. Hypoxia induced AlaAT1 earlier in roots than in shoots, while AlaAT2 and GGAT were unaffected. The increase in AlaAT activity lagged behind the increase in alanine content. Nitrogen deficiency has little effect on the activity of GGAT. In contrast, lower activity of AlaAT and the level of mRNA for AlaAT1 and AlaAT2 in wheat seedlings growing on a nitrogen-free medium seems to indicate that AlaAT is regulated by the availability of nitrogen. Both AlaAT and GGAT activities were present in etiolated wheat seedlings but their activity was half of that observed in light-grown seedlings. Exposure of etiolated seedlings to light caused an increase in enzyme activities and up-regulated GGAT1. It is proposed that hypoxia-induced AlaAT1 and light-induced peroxisomal GGAT1 appears to be crucial for the regulation of energy availability in plants grown under unfavourable environmental conditions. Key message In young wheat seedlings, both AlaAT and GGAT are down-regulated by nitrogen deficiency, whereas AlaAT1 is upregulated by hypoxia and GGAT1 by light.

Acclimation to frost alters proteolytic response of wheat seedlings to drought.

A comparative examination of cysteine proteinases in winter wheat (Triticum aestivum L.) seedlings differing in sensitivity to frost and drought revealed many similarities and differences in response to water deprivation. Azocaseinolytic activity was enhanced under water deficiency, but the enhancement was significantly lower in the tolerant genotype (Kobra cultivar). On the contrary, acclimation of wheat seedlings at low temperature had no effect on the proteolytic activity of the tolerant cultivar and depressed the azocaseinolytic activity of the sensitive cultivar (Tortija). However, the observed depression of enzyme activity was fully reversible under dehydration. The content of soluble proteins was reduced in dehydrated non-acclimated and in acclimated seedlings of the frost-sensitive cultivar, but increased in acclimated seedlings of the tolerant cultivar. The cysteine proteinases were preferentially induced under water deficiency when assessment was based on the inhibitory effect of iodoacetate on azocasein hydrolysis. Separation of cysteine proteinases by SDS-PAGE containing gelatin as a substrate showed two bands with apparent molecular masses of 36 and 38 kDa in the sensitive cultivar, and a third band was detected (42 kDa) in the resistant cultivar. Water deficit and low temperature induced the new cysteine proteinases of molecular masses about 29, 33 and 42 kDa in sensitive non-acclimated seedlings. Polyclonal antibodies raised against Arabidopsis proteinase responsive to drought (RD21) cross-reacted with the protein in the 33 kDa region, and a slight signal was obtained in the 42 kDa region, but only in dehydrated seedlings acclimated to frost. Several polypeptides of molecular masses of 30, 22, 20 and 18 kDa were recognized by the Arabidopsis aleurain-like proteinase (AtALEU) antibodies. The results presented indicate that cysteine proteinases are potentially responsible for both low temperature and drought tolerance.

Multifunctional role of plant cysteine proteinases.

Cysteine proteinases also referred to as thiol proteases play an essential role in plant growth and development but also in senescence and programmed cell death, in accumulation of storage proteins such as in seeds, but also in storage protein mobilization. Thus, they participate in both anabolic and catabolic processes. In addition, they are involved in signalling pathways and in the response to biotic and abiotic stresses. In this review an attempt was undertaken to illustrate these multiple roles of cysteine proteinases and the mechanisms underlying their action.