園田 雅俊

The melon 1-aminocyclopropane-1-carboxylic acid oxidase gene (CM-ACO1) is an ethylene responsive gene and plays a role in ethylene synthesis in ripening fruits. To elucidate the regulatory mechanism of the CM-ACO1 gene during the ripening stage, we isolated two cDNAs encoding melon Ethylene Insensitive 3 (EIN3)-like genes (CmEIL1 and CmEIL2) from ripening melon (Cucumis melo L cv. Andes) fruits, and studied their properties. The predicted amino acid sequences of CmEIL1 and CmEIL2 were highly homologous to that of EIN3 (60 and 52% similarity for CmEIL1 and CmEIL2, respectively). CmEIL1 and CmEIL2 proteins interacted with a CM-ACO1 promoter in the yeast one-hybrid system, and activated transcription of CM-ACO1 promoter in melon leaf discs by a transient experiment with the luciferase reporter gene. The mRNA levels of CM-ACO1. CmEIL1 and CmEIL2 genes were very low in Immature melon fruits, but increased markedly in fruits during the ripening stage Ethylene did not affect the expression levels of CmEIL1 and CmEIL2 genes in melon leaf tissues These results suggest that CmEIL1 and CmEIL2 were ripening-related genes, and that their products acted as transcriptional activators of the CM-ACO1 gene in ripening melon fruits. (C) 2010 Elsevier Ireland Ltd All rights reserved

WRKY genes encode proteins which belong to a large family of transcription factors that are involved in various developmental and physiological processes, response to pathogen infections and wound stress in plants. The molecular characteristics of WRKY genes involved in these are becoming clear in model plants, like Arabidopsis. However, knowledge of WRKY genes in other plants, for instance vegetable, is still not enough. In the present studies, using a yeast one-hybrid system we isolated a cDNA from a spinach cDNA library and characterized its function. The cDNA, designated SoWRKY1, encodes a putative polypeptide of 362 amino acids which is highly homologous to Arabidopsis WRKY40 (AtWRKY40). The putative primary structure of SoWRKY1 contains a single WRKY domain of Cys2His2 zinc finger motif and a potential nuclear localization signal (NLS), whose structure is characteristic of group IIa type WRKY protein. SoWRKY1-sGFP fusion protein was localized to the nucleus when the protein was expressed in onion epidermal cells. SoWRKY1 showed high binding affinity to DNA molecules containing TTGAC(C/T) W-box sequences and transcriptional activation activity in yeast. SoWRKY1 transcripts in spinach leaves were transiently induced by wounding treatment and salicylic acid (SA). The transcripts accumulated following treatment with cycloheximide (CHX), a protein biosynthesis inhibitor. Functional analysis of SoWRKY1 in vivo was performed by overexpression in Arabidopsis, and pathogenesis-related PR1 and PR2 gene expression level increased in the transgenic plants. These results suggest that SoWRKY1 might be involved as a transcription factor in defense-related signaling transduction pathways of spinach.