AtCRK1 positively regulates salt stress tolerance in Arabidopsis through the modulation of stress-responsive genes

Plant molecular improvement group at University of Shizuoka, Japan, previously identified 18 potential salt tolerance mutants using an activation-tagging approach at the cellular level. In one of these mutants, stc8, two genes AtbHLH106 and AtCRK1 were activated adjacent to the borders of T-DNA. AtbHLH106 is known to regulate salt stress by interacting with the G-box (CACGTG) present in the promoters of abiotic stress responsive genes. Here we report that AtCRK1 contributes to salt-stress regulation in Arabidopsis. In this study, TAIL-PCR revealed that AtCRK1 was present 5567 bp from the RB of the T-DNA and activated on both MS without salt and MS supplemented with 150 mM NaCl. Real-time PCR showed that AtCRK1 was enhanced 1.7 times on MS without salt and 2.7 times on MS with 150 mM NaCl. Quantitative expression in different tissues of plant and calli showed that AtCRK1 was highly expressed in leaves and flowers and least expressed in silique. In addition, expression analysis at different levels of NaCl showed that AtCRK1 was salt-inducible. Expression of AtCRK1 was significantly reduced in KO lines and the KO lines showed sensitive phenotype under salt stress, compared with Col-0 plants. We demonstrated that overexpressed calli of AtCRK1 were tolerant to NaCl and ABA, compared with control calli, and the survival percentage of overexpression lines was higher than in KO and wild-type plants. On vertical plates, shoots of overexpression lines did not show significantly stress-tolerant phenotypes, compared with wildtype and KO plants, but roots of overexpression plants were still longer than KO and wildtype plants. Furthermore, microarray analysis of stc8 mutant and KO lines showed that activation of AtCRK1 leads to upregulation of several heat shock proteins: myoinositol phosphate synthase gene, cold binding factor, ABC transporters, and CAT genes. Together, these findings indicate that AtCRK1 plays an important role during abiotic stress in plants through regulation of several abiotic responsive genes.