Uloga transkripcionog faktora DREB2A i malih RNK miR398a/b i miR408 u odgovoru graška (Pisum sativum L.) na dehidrataciju / The role of DREB2A transcription factor and small RNAs miR398a/b and miR408 in dehidration response of pea (Pisum sativum L.)

Adverse environmental stresses, such as drought, low temperature and soil salinity have a strong influence on agricultural production and sustainability. A major limitation to yield and quality in many crop species is water availability throughout or at critical times during the growing season. We investigated the effect of dehydration on expression of DREB2A transcription factor in pea, as well as on the expression of conserved miRNAs-miR398a/b and miR408. Partial cDNA (PsDREB2A), which encodes a DREB2A transcription factor in pea, was obtained by PCR with primers designed according to DREB2A gene from the model legume Medicago truncatula. The resulting sequence (495 bp) has been deposited in Gene Bank (HM229349). Comparison of nucleotide sequences showed that the obtained sequence is most similar to the sequence of DREB2A from M. truncatula, and with EREBP (ethylene responsive element binding protein) from the same species. Analysis of deduced amino acid sequence showed that PsDREB2A has a structure characteristic of the DREB2A protein - presence of AP2 domains (17-55 a.a.), and has the highest homology with DREB2A and ERBP protein from M. truncatula. Computer prediction of post-translational modification of protein revealed the presence of a potential site for protein sumoylation, at the position of Lys157 (MKQE), and the presence of several potential sites for phosphorylation. To study the expression of PsDREB2A we performed the analysis of mRNA level in pea root and shoot in a normal physiological state, as well as during dehydration for 7 and 10 days, and rehydration. Real-time PCR using a probe specific for PsDREB2A has been applied. It has been shown that the expression profile was different in roots and aerial parts of the plants. In the roots the maximum level of expression PsDREB2A was determined after 10 days of treatment, while in the above-ground parts of plants the maximum was rached after 7 days of dehydration. We also studied the expression of conserved micro RNAs- miR398a/b and miR408 during dehydration and rehydration of plants. Northern blot analysis, using the appropriate LNA probes, showed that RNAs were most abundant in the control group of plants. During dehydration stress, there was a progressive reduction of the level of investigated miRNAs, both in roots and in the above-ground parts of plants.. In addition, using Real-time PCR changes in the expression level of potential target genes of selected miRNAs - CSD, COX5b and P1B-ATPase have been investigated. The level of COX5b gene has not been changed dehydration and rehydration of plants. It is noted that the CSD expression level correlated inversely with the expression of the corresponding miRNA, so that the lowest level of expression was noted in the control group of plants, while under treatment the level of expression of CSD has been increased. Expression of P1B-ATP-ase was changed during dehydration only in above-ground parts of plants, and the biggest change was observed in rehydrated plants. In the roots the expression of this gene was unaffected.