Biological Determination Of Phosphorylation Of ABI5 At Ser-42, A Key Transcription Factor Involved In Plant ABA Responses

Abscisic acid(ABA) is an important plant hormone that regulates plant growth, development and in response to environmental stresses. In the model plant Arabidopsis thaliana, the ABA reaction and its mechanism were studied. The phosphorylation of key regulators has been shown to play a fundamental role in the initialation of ABA responses. In terms of the molecular basis of core network of early ABA signaling, phosphorylation of ABI5-like bZIP transcription factors(ABI5 and ABFs) by SnRK1-or SnRK2-type proein kinase is an essential step to establishe plant ABA response. Previous study revealed that SnRK2-type proein kinase PKS5 take part in the modulation of plant ABA response. PKS5 could phosphorylate ABI5 in vitro. However, the molecular basis remains elusive. Here, we determined the main phosphorylation site of PKS5 upon ABI5. Moreover, also examine the biological significance of this phosphorylation.We obtained following results:(1) Studies show that PKS5 can interact with ABI5 by phosphorylating ABI5, and the main phosphorylation sites of PKS5 are on ABI5-N1 region. In order to further determine the major phosphorylation site of ABI5, we obtained different phosphorylated sites clones of ABI5.Then we utilizeed the floral dip method to infect the model plant Arabidopsis. After a series of identification and screening test for the genetic stability of transgenic plants, finally we extracted protein of transgenic plants, and by protein immunoblotting experiment-s on these mutants of phosphorylation assay to determine that Ser-42 of ABI5 is the princi-pal amino acid phosphorylated by PKS5.(2)In order to detect whether the ABI5 phosphorylation of specific antibody can specifically recognize ABI5 Ser-42 residues or not, we prepared the ABI5 phosphorylation specific antibodies and through the phosphorylation assays of ABI5 Ser-42 residues in vivo and vitro, we detected the specificity and titer of the antibody.The results show that phosphorylation of ABI5 at Ser-42 is induced by ABA treatment via ABI5 Ser-42 phosphorylation site-specific antibodies based western blot.(3) In order to understand the biological significance of phosphorylation modification of Ser-42 on ABI5, we investigated whether the phosphorylation of Ser42 residues in ABI5 is necessary to inhibit the germination of ABA seeds.Through the construction and identification of the mutants, the transgenic plants of abi5-8,and their phenotype and function were analyzed. The above results showed phosphorylation of Ser residue at position 42 in ABI5 is important for ABI5 function in Arabidopsis.(4) The biological significance of Ser42 ABI5 residues phosphorylation site on ABI5 transcription activity was studied by detecting the activity of firefly luciferase LUC and yeast single impurity in Arabidopsis thaliana. The results showed that phosphorylation of Ser residue at position 42 in ABI5 is important for ABI5 transactivation.Taken together, these data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for plant ABA response.