Function Analysis Of Arabidopsis WRKY25 And A Nodulin-related Protein1 (NRP1) Encoded By At2g03440 In Heat Stress

Heat stress is an important factor which induces injury to plants and limits plants growth and productivity in agriculture. In this study we investigated WRKY25 and At2g03440(NRP1)expression pattern in response to heat stress with Northern blot. WRKY25 is induced by heat stress (42℃), and AtNRP1 is suppressed by heat stress. In order to further determine the function of these two genes, we identified T-DNA insertion mutants and constructed overexpressing transgenic plants of the two genes.The Arabidopsis WRKY25 is one of the group I WRKY proteins and responses to both abiotic and biotic stress. Northern blotting analysis revealed WRKY25 was rapidly accumulated at 30min under heat stress, and reached a maximum level of transcript around 4-6 hours. Assays of hypocotyl, root elongation and electrolyte leakage measurements revealed that wrky25 mutants were thermosensetive comparing to WT at different growth stages. Moreover, WRKY25-overexpressing transgenic lines enhanced thermotolerance in seeds germination when compared to WT.We compared the expression of a number of heat-inducible genes in wrky25 and WT plants after heat stress, such as HsfA2, HsfB1, HsplOl and oxidative stress-responsive genes such as Zat10 and APX2. In consistence with the role of WRKY25 in heat stress, the expression level of HsfA2, HsfBl, Hsp101 Zat10 and APX2 were down-regulated in wrky25 comparing to WT after 42℃treatment. It is indicated that the functions of WRKY25 in thermal adaptation might regulate some of theses heat-inducible genes or be regulated by some of these heat-inducible genes.The molecular function of Nodulin-related protein 1 (NRP1) encoded by At2g03440 has so far not been reported, and neither recognized common motifs nor conserved domains were found by bioinformatics analysis. GUS staining showed that the AtNRP1 expressed mainly in meristematic tissues such as root tips, root tips, vasculature and vegetative shoot apex; AtNRPl is also expressed in developing organs such as floral buds, stamens and pistils of flowers, stigmas and abscission layers of young siliques. The expression of AtNRP1 gene was induced by low temperature (4℃) while suppressed by heat stress (42℃) and abscisic acid (ABA) treatment.The nrpl plants were similar to wild-type seedlings in thermotolerance, but AtNRP1-overexpressing transgenic plants conferred increased thermosensitivity. Further study found that the expression of some known heat-induced genes, such as HsfA2, MBF1c, Hsp101 and Hsp70 did not alter among AtNRP1-overexpressing transgenic plants, wild type (WT) and nrpl mutants under heat stress. But ABA accumulation of/ AtNRP1-overexpressing lines was significantly lower than that of WT and nrpl mutants under heat stress, moreover, the heat-suppressed expression of AtNRP1 was abolished in aba2-3 mutants. Therefore, we deduced that AtNRP1 might play a role in thermotolerance through ABA signaling pathway.