Purification and biochemical characterization of proteins which bind to the H‐box cis‐elementimplicated in transcriptional activation of plant defense genes

Abstract

The H‐box (CCTACC(N)₇CT(N)₄A), which occurs three times within the ‐154 to ‐42 region of the bean chal‐cone synthase chs15 promoter, is important for developmental regulation of chs15, and induction of chs15 and coordinately regulated defense genes by elicitors and other stress stimuli. Two protein factors, KAP‐1 and KAP‐2, which recognize conserved features in the H‐box motif, were purified from bean cell suspension cultures by a combination of ion exchange chromatography and DNA affinity chromatography. KAP‐1 is a 97 kDa polypeptide, whereas KAP‐2 comprises two polypeptides of 76 and 56 kDa. KAP‐1 and KAP‐2 also differ in the sensitivity of their DNA‐bound forms to trypsin. Dephosphorylation of KAP‐1 or KAP‐2 affects the mobility of the protein/H‐box binding complex in gel shift assays but does not inhibit DNA binding. Elicitation of bean cell suspensions with glutathione does not affect the total cellular activities of KAP‐1 or KAP‐2, but causes a rapid increase in the specific activities of both factors in the nuclear fraction, consistent with a role for these factors in the signal pathway for elicitor induction of chs15 and related defense genes.