Minisymposium 3: Hormones I

Abs # 13002: Molecular characterization of His-Asp phosphorelay system implicated in cytokinin signaling in maize

Presenter:	Sakakibara, Hitoshi , sakaki@postman.riken.go.jp
Authors	Sakakibara, Hitoshi  (A)   Yonekura-Sakakibara, Keiko  (A)
Affiliations:	(A): Plant Science Center, RIKEN
Web Site:	http://www.riken.go.jp/engn/r-world/research/lab/plant/mfr/com/index.html

Cytokinin is involved in various processes of growth and development, such as cell division, chloroplast differentiation, inhibition of leaf senescence, and nutrient signaling. The signal is mediated via histidyl-aspartyl (His-Asp) phosphorelay system composed of sensory histidine-kinase (HK), histidine phosphotransmitter (HP) and response regulator (RR). The modules transmit cytokinin signal by transferring phosphoryl group between His and Asp residues. In maize (Zea mays L.), genes for HP (ZmHP1 - ZmHP3) and RR (ZmRR1 - ZmRR10) were identified, and their expression patterns and biochemical properties were characterized [1] whereas the sensory HK has been remained. We isolated three genes for HK, ZmHK1, ZmHK2 and ZmHK3, whose deduced amino acid sequences were similar to those of AHK4/CRE1, AHK3 and AHK2, respectively, encoding cytokinin-responsive HKs of Arabidopsis thaliana. Analyses of ligand preference of ZmHK1 and ZmHK2 in a heterologous system with Escherichia coli demonstrated that ZmHK1 was more responsive to free-base type cytokinins such as trans-zeatin and isopentenyladenine than to their ribosides. Isopentenyladenine was more effective for ZmHK1 and trans-zeatin was for ZmHK2. Interestingly, both HKs could also respond to cis-zeatin with a comparable sensitivity to trans-zeatin whereas such reactivity was not observed in AHK4. This suggests a physiological significance of cis-type cytokinin in maize, whose role has not been understood well. In terms of the downstream signaling path, some possible combinations between ZmHPs and ZmRRs were suggested by the yeast two-hybrid assay. Single substitution of aspartic acid residue of receiver domain of ZmHK1 to glutamic acid, which is the putative phosphorylation site, enabled it to interact with ZmHPs. This implies that His-Asp phosphotransfer with physical interaction between the ZmHK and the ZmHPs occurs in the signaling pathway. Transient expression of fusion products with green fluorescent protein suggested endoplasmic reticulum localization of ZmHK1, cytosolic and nuclear localizations of ZmHPs and ZmRRs. Putting together these results, we will discuss His-Asp phosphorelay networks with special reference to cytokinin signal transduction in maize leaves. [1] Asakura et al. Plant Mol Biol. (2003) in press.