'Tonewase' Japanese persimmon fruit (Diospyros kaki Thunb.) grown in a heated plastic-house softens rapidly within several days of harvest, which is a major problem in marketing of this cultivar. In this study, we elucidated the involvement of water stress-induced ethylene in fruit softening and investigated the induction mechanism of this ethylene biosynthesis occurring in specific tissues at the molecular level. Two instances of increase in ethylene production were observed in fruit held in ambient low humidity conditions (40-60% RH), an initial increase on the 1st and 2nd days and a second increase on the 6th and 8th days after harvest. Increase in ethylene production was accompanied by rapid softening in these fruit. Fruit held in high humidity conditions (&gt
95%) neither produced detectable levels of ethylene nor softened rapidly. Moreover, treatment of the fruit held in low humidity with 1-methylcyclopropene (1-MCP), a strong inhibitor of ethylene action, inhibited fruit softening remarkably. These results suggest the involvement of water stress-induced ethylene in fruit softening. 1-MCP also suppressed the second increase in ethylene production but not the initial increase, indicating that the initial increase is induced directly in response to the primary water stress signal while the second is induced autocatalytically by the ethylene produced during the initial phase. During the initial increase in ethylene, the calyx produced more than 5 nl g-1 h-1 of ethylene accompanied by increased accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) and expression of DK-ACS2. In pulp, 0.5 nl g-1 h-1 of ethylene was detected but no increase in ACC content or expression of any ethylene biosynthetic genes was observed. During the second increase in ethylene, ethylene production in the calyx was not detected whereas the pulp produced 0.2-0.4 nl g-1 h-1 of ethylene with a marked increase in ACC content and expression of the two ACC synthase (DK-ACS1, DK-ACS2) and one ACC oxidase (DK-ACO1) genes. These results suggest that in plastic-house 'Tonewase' persimmon fruit, ethylene production is initiated in the calyx in response to water stress through activated expression of DK-ACS2, and this ethylene in turn induces autocatalytic ethylene production in the pulp. As the flesh firmness decreased markedly just after the initial ethylene production, the results also indicate that the fruit softening is due to the action of ethylene produced in the calyx. © 2002 Elsevier Science B.V. All rights reserved.