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

Abstract

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, β-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.

Keywords: barley; candidate genes; doubled haploid lines; drought tolerance; freezing tolerance; gene expression.

Figure 1

The expression of selected genes during cold acclimation or drought treatment relative to untreated plants in six double haploid lines of winter barley. FS, freezing-susceptible; DS–FT, drought-susceptible–freezing-tolerant; FDT, freezing- and drought-tolerant; DS, drought-susceptible; DT, drought-tolerant. Error bars represent standard error between means of three biological replicates, each with three instrumental repetitions. Analysis of variance was made separately for each gene. Values marked with the same letter do not differ according to Tukey’s test (p ≤ 0.05).

Figure 2

(A) Relative expression of genes before (NA) and after cold acclimation (CA) in freezing-tolerant (mean for DH602 and DH534) vs. freezing-susceptible (mean for DH158 and DH575) lines. (B) Relative expression of genes in well-watered plants (C) and under drought (D) in drought-tolerant (mean for DH561 and DH534) vs. susceptible (mean for DH435 and DH602) lines. Error bars represent standard error between the means of two lines (each with three biological replicates and three instrumental repetitions).