Abiotic stresses including drought and salt stress have critical effects on plant growth and reproduction. Analysis of the water stress responses is necessary to develop strategies to increase stress tolerance in crops. Abscisic acid (ABA) is a plant hormone which is accumulated during drought stress and has critical roles to induce various water stress responses. The stress-inducible accumulation of ABA in seedlings is regulated by gene induction of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3 (NCED3) encoding a rate-limiting enzyme in the ABA biosynthetic pathway. However, the molecular mechanisms of how the gene expression level of NCED3 is controlled under water stress conditions remain to elucidate.
To identify novel transcriptional regulatory factors on the NCED3 promoter, we are trying to develop a screen method, inserted chromatin precipitation (iChIP) in which repetitive motifs are fused to a specific promoter and isolate transcriptional complexes on the promoter through coimmunoprecipitation of the binding protein to the repetitive motifs. As the results, we identified several candidate factors of transcription, epigenetics and chromatin conformation which are located on the NCED3 promoter to control the expression level in response to water stress. Knockout mutations of some genes resulted in phenotypic alteration under water stress conditions and changes of the NCED3 expression. These results suggest that the iChIP method can be applied to identify transcriptional complexes on a specific promoter region.