The effectiveness of microspore embryogenesis (ME) is determined by a complex network of internal and environmental factors. In the present study on triticale and barley, strong positive correlation (r = 0.85) between the generation of hydrogen peroxide (H2O2) and ME effectiveness confirmed the important role of reactive oxygen species in microspore reprogramming. However, for high effectiveness of ME induction, intensive H2O2 generation had to be associated with high activity of antioxidative enzymes, superoxide dismutase and catalase. The strong seasonal effect on the physiological status of microspores revealed in the study suggests a kind of ‘biological clock’ controlling plant reproduction, crucial for microspore viability and embryogenic potential. Although the effect of various modifications of ME-inducing stress tiller pre-treatment was determined mainly by the physiological condition of microspores, at higher stress intensity positive effects induced by antioxidant molecules—reduced glutathione and its precursor, l-2-oxothiazolidine-4-carboxylic acid—were observed. High level of variation in the response to ME-inducing stress tiller pre-treatment was also revealed between the two DH lines of triticale and two cultivars of barley and among microspores isolated from subsequently developed spikes.
Ten doubled haploid (DH) lines of winter barley with an increased range of freezing/drought tolerance were used to identify phytohormones involved in plant stress acclimation. Cold hardening and drought stress were applied at the most critical stages of plant development on young seedlings and heading plants, respectively. The level of the phytohormones was significantly higher at heading, more than 5-fold in respect of salicylic acid (SA) and total brassinosteroids (BRs) and 1.7-fold in the case of abscisic acid (ABA). Moreover, the spectrum of detectable BRs increased from one-homocastasterone (HCS)-found in seedlings to four BRs identified in heading plants [HCS, castasterone (CS), teasterone and dolicholide], with the last one detected for the first time in cereal species. To some extent freezing tolerance seems to be determined by native hormonal status as control seedlings of tolerant DH lines contained 1.4-and 2.3-fold lower amount of ABA and HCS and 2.3-fold higher amount of SA in comparison to freezing-sensitive ones. Such dependency was not observed in heading plants as significant variation in CS content was the only detected difference. Under stress treatments, tolerant DH lines accumulated significantly lower (75-81%) amount of ABA, which probably reflected lower stress intensity resulting from another defence strategy. In contrast, stress-induced significant almost 2-fold increase in HCS/CS and 2-3-fold decrease in SA content specific for tolerant DH lines of barley suggest the involvement of these molecules in freezing/drought defence. Detected correlations suggest their interaction with nonspecific peroxidase and low molecular weight antioxidants.
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.
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