Candidate Genes for Freezing and Drought Tolerance Selected on the Basis of Proteome Analysis in Doubled Haploid Lines of Barley.

Plant tolerance to environmental stress is determined by a very complicated network composed of many intra- and extracellular factors. The aim of this study was to select candidate genes involved in responses to freezing and drought in barley on the basis of previous proteomic studies and to analyze changes in their expression caused by application of both stress factors. Six candidate genes for freezing tolerance (namely the genes encoding elongation factor 1 alpha (EF1A), ferredoxin-NADP reductase, a 14-3-3a protein, ß-fructofuranosidase, CBF2A and CBF4B) and six for drought tolerance (encoding transketolase, periplasmic serine protease, triosephosphate isomerase, a protein with a co-chaperon region (GroEs), pfam14200 and actin) were chosen arbitrarily on the basis of in silico bioinformatic analyses. The expression levels of these genes were measured under control and stress conditions in six DH (doubled haploid) lines with differing freezing and drought tolerance. The results of gene expression analysis confirmed the roles of the candidate genes preselected in this study on the basis of previous proteome analysis in contributing to the differences in freezing and drought tolerance observed in the studied population of DH lines of winter barley.

Changes in protein abundance and activity involved in freezing tolerance acquisition in winter barley (Hordeum vulgare L.).

The changes in protein abundance induced by cold hardening were analysed by 2 DE in ten doubled haploid (DH) lines of winter barley, highly differentiated with respect to freezing tolerance level. Among 45 differential proteins identified by MALDI-TOF/TOF, the majority was classified as related to photosynthesis, carbohydrate metabolism, oxidation-reduction reactions and stress response. Among the detected proteins, higher abundance of RuBisCO large and small subunits, RuBisCO activase, two Oxygen-evolving enhancer proteins, Ferredoxin-NADP reductase, Cytochrome P450-dependent fatty acid hydroxylase and 14-3-3 protein was associated with higher freezing tolerance level. Lower relative level of hypothetical ATP synthase beta subunit, uncharacterized mitochondrial protein AtMg00810 and ribosomal RNA small subunit methyltransferase G also seems to be important. The results of proteomic studies were complemented by the evaluation of photosynthetic apparatus acclimation, showing distinctive differences between the studied genotypes in the number of active PSII reaction centres (RC/CSm). Additionally, the analysis of antioxidative enzyme activities suggests the importance of H2O2 as a signalling molecule possibly involved in the initiation of cold-induced plant acclimation. However, in DH lines with high freezing tolerance, H2O2 generation during cold hardening treatment was accompanied by more stable activity of catalase, H2O2-decomposing enzyme. SIGNIFICANCE: In the study, the changes in protein abundance induced by cold hardening treatment were analysed by two-dimensional gel electrophoresis in ten doubled haploid (DH) lines of winter barley. Harnessing DH technology resulted in distinctive widening of genetic variation with respect to freezing tolerance level. Both the cold-hardening effect on the protein pattern in an individual winter barley DH line as well as the variation among the selected DH lines were investigated, which resulted in the identification of 45 differentiated proteins classified as involved in 14 metabolic pathways and cellular processes. Among them, eight proteins: (1) the precursor of RuBisCO large subunit, (2) RuBisCO small subunit (partial), (3) RuBisCO activase small isoform, (4) the precursor of Oxygen-evolving enhancer protein 1-like (predicted protein), (5) Oxygen-evolving enhancer protein 2, (6) the leaf isozyme of Ferredoxin-NADP reductase, (7) hypothetical protein M569_12509 Cytochrome P450-dependent fatty acid hydroxylase-like and (8) hypothetical protein BRADI_1g11290 (14-3-3 protein A-like) were accumulated to a higher level in leaves of cold-hardened seedlings of freezing tolerant winter barley DH lines in comparison with susceptible ones. Three others: (9) hypothetical protein BRADI_5g05668 F1 ATP synthase beta subunit-like, (10) predicted protein uncharacterized mitochondrial protein AtMg00810-like and (11) BnaA02g08010D Ribosomal RNA small subunit methyltransferase G-like were detected at lower level in freezing tolerant seedlings in comparison with susceptible genotypes. The last two were for the first time linked to cold acclimation. The results of complementary analyses indicate that PSII activity and stability of antioxidative enzymes under low temperature are also very important for freezing tolerance acquisition.

Changes in protein abundance and activity induced by drought during generative development of winter barley (Hordeum vulgare L.).

The present study investigated drought-induced changes in proteome profiles of ten DH lines of winter barley, relatively varied in water deficit tolerance level. Additionally, the parameters describing the functioning of the photosynthetic apparatus and the activity of the antioxidative system were analysed. Water deficit (3-week growth in soil with water content reduced to ca. 35%) induced significant changes in leaf water relations and reduced photosynthetic activity, probably due to decreased stomatal conductance. It was associated with changes in protein abundance and altered activity of antioxidative enzymes. From 47 MS-identified proteins discriminating more tolerant from drought-sensitive genotypes, only two revealed distinctly higher while seven revealed lower abundance in drought-treated plants of tolerant DH lines in comparison to sensitive ones. The majority were involved in the dark phase of photosynthesis. Another factor of great importance seems to be the ability to sustain, during drought stress, relatively high activity of enzymes (SOD and CAT) decomposing reactive oxygen species and protecting plant cell from oxidative damages. Low molecular weight antioxidants seem to play less important roles. Our findings also suggest that high tolerance to drought stress in barley is a constitutively controlled trait regulated by the rate of protein synthesis and their activity level. BIOLOGICAL SIGNIFICANCE: According to our knowledge, this is the first comparative proteomic analysis of drought tolerance performed for the model set of several winter barley doubled haploid (DH) lines. We analysed both the drought impact on the protein pattern of individual winter barley DH lines as well as comparisons between them according to their level of drought tolerance. We have identified 47 proteins discriminating drought-tolerant from drought-sensitive genotypes. The majority was involved in the dark phase of photosynthesis. Another factor of great importance in our opinion seems to be the ability to sustain, during drought stress, relatively high activity of antioxidative enzymes (SOD and CAT) decomposing reactive oxygen species and protecting plant cell from oxidative damages. Our findings also suggest that high tolerance to drought stress in barley is a constitutively-controlled trait regulated by the rate of protein synthesis and their activity level.