Barbara Zagdańska

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

AbstractWheat (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.

ROS as Signaling Molecules and Enzymes of Plant Response to Unfavorable Environmental Conditions

Research concern plant response to unfavourable environmental conditions is becoming increasingly important and most climate-change scenarios suggest an increase in aridity in many areas of the globe (Petit et al. 1999, Blum 2011). On a global principle, drought (taking into account soil and/or atmospheric water deficit) in combination with coincident high temperature and radiation, determines the most important environmental constraints to plant survival and to crop productivity (Boyer 1982). Agriculture is a major user of water resources and abiotic stress is the principal cause of decreasing the average yield of major crops by more than 50%, which causes losses worth hundreds of millions of dollars each year. Understanding mechanism of abiotic stress tolerance and defense is important for crop improvement. Many works concerning this problem were developed over last decades, discussing subjects from plant strategies to control water status under drought (Schulze 1986) to the physiological and biochemical processes underlying plant response to water shortage (Chaves 1991, Cornin and Massacci 1996) and oxidative stress (Smirnoff 1998). In this chapter various aspects of reactive oxygen species and enzymes in plant response to drought (abiotic stress) are discussed.

Influence of Water Stress upon Photosynthetic Carbon Metabolism in Wheat

Two kinds of effects of water stress on flag leaves of spring wheat (Triticum aestivum L. cv. Kolibri) were recognized: the direct effect, when drought affected the expanded blade of the flag leaf and the indirect one, when drought pretreatment affected the leaf blade during tissue differentiation. As a direct effect of water stress on the leaf, a decreased amount of (14)C incorporated into all analyzed photosynthates was observed. Differences in (14)C distribution among the studied photosynthates indicated that water stress decreased the carbon flow from intermediates to end products of the photosynthetic metabolism. Drought pretreatment protected (to some extent) the carbon photosynthetic metabolism against the inhibitory effect of the subsequent water stress. The modifications of photosynthetic carbon metabolism observed seem to be involved in adaptation of plants to the water stress conditions.

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.

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.

ATP-dependent proteolysis contributes to the acclimation-induced drought resistance in spring wheat

The effect of water deficit on the ATP-dependent proteolysis and total protein degradation was estimated in the leaves of spring wheat (Triticum aestivum L.) acclimated and non-acclimated to drought. The rate of ATP-dependent proteolysis, quantified as a difference between degradation of 125I-lysozyme under ATP-regenerating and ATP-depleting systems, accounted for about 55 % of total 125I-lysozyme degradation in fully turgid wheat leaves. In the non-acclimated leaves dehydration decreased sharply ATP-dependent proteolysis catalyzed by proteasome down to about 5% while in the leaves acclimated to drought water deficit raised ATP-dependent proteolysis to 87 % of total 125I-lysozyme hydrolysis.

Insect-resistant Bt-maize response to the short-term non-target mite-pest infestation and soil drought

Transgenic lepidopteran insect-resistant maize expressing the cry1Ab gene (Bt) and its non-transgenic counterpart at the 12-leaf-stage (V12) were infested by the two-spotted spider mite or dehydrated by cessation of soil watering to check Bt-maize capacity to respond to other stresses than those assured by the presence of Cry protein. Since the antioxidant enzymes are key components of plant defence against biotic and abiotic stresses, the engagement of leaf superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) in response to 6-day mite feeding and soil drought has been investigated. The reduction of leaf hydration and soluble protein content in the fully expanded 8th leaf was independent of genotype and more pronounced in response to water cessation than mite infestation. Similarly, the changes in enzyme activities depended more on the kind of stress than the presence of the transgene. Water shortage in the soil enhanced the activity of all enzymes, whereas mite feeding decreased the activity of SOD and CAT, and markedly increased POX in the 8th leaf of both cultivars. In mite-infested leaves of the non-transgenic plant, the CAT activity remained unaffected, whereas decreased in leaves of Bt maize due to the hampered activity of CAT-2. In comparison to the control, all enzyme activity in the 10th non-infested leaf of mite-infested non-transgenic maize decreased, whereas it changed in the 10th leaf of Bt maize in the same way as in the 8th mite-infested leaf. The results suggest that SOD, CAT and POX can strongly confer short-term drought-stress response in both maize cultivars, whereas POX is the only responsive enzyme in mite-infested Bt maize.

Changes in the Thiol/Disulfide Redox Potential in Wheat Leaves Upon Water Deficit

Summary Regulation of the thiol: disulfide redox potential requires about 30 % of the total respiratory energy in wheat leaves. Drought lowered the energy input on regulation of SH/SS equilibrium with a concomitant decrease in the SH/SS ratio. The decrease in SH content should be attributed to protein SH since nonprotein SH remained unchanged. Acclimation to drought required more energy for maintenance of the redox potential, despite a lower SH/SS ratio than in the control unaffected leaves. The discrepancy between energy input and the protein SH level seems to be apparent since acclimation resulted in a pronounced increase in total SH groups, which were non-accessible for DTNB titration in the native form. The level of reduced glutathione and specific activity of glutathione reductase were significantly elevated in the acclimated water deficient leaves; moreover, the Km values for GSSG decreased, indicating changes in the isoform pattern of the enzyme. Acclimated leaves under drought required the same energy input for providing SH/SS equilibrium as fully turgid control leaves.

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

Summary 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.

Protein Oxidation and Redox Regulation of Proteolysis

Reactive oxygen species (ROS), beyond the role of toxic by-products of aerobic metab‐ olism, contribute to cell redox homeostasis and are signalling molecules in pathogen defence and abiotic stress tolerance. The putative mechanism of cell responses to ROS is thiol modifications of cysteine residues, which cause changes in protein conforma‐ tion and activity. These post-translational modifications include generation of disul‐ phide bridges and formation of sulphenic, sulphinic, and sulphonic acids, as well as S-glutathionylation and S-nitrosylation. S-nitrosylation or reversible modification may change the activity of enzymes related to the metabolism of nitric oxide, ROS, and cel‐ lular metabolism, whereas S-glutathionylation regulates the activity of proteins that contain in their structure the active cysteine residue, regulates the oxidoreductive pathway of signal transduction, and participates in the regeneration of antioxidant en‐ zymes. Carbonylation, an irreversible, non-enzymatic modification of proteins is the most commonly occurring oxidative protein modification. The formation of carbonyl groups can be linked to abnormal translation, altered chaperone system and respons‐ es to stress factors. Carbonylated proteins are marked for proteolysis mediated by dif‐ ferent pathways in different cell compartments to counteract the formation of high molecular weight aggregates and accumulation of inactive proteins. However, prod‐ ucts of proteolysis of carbonylated proteins could function as secondary ROS messen‐ gers that target the cell nucleus.