Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L

Introduction Leaf senescence involves degradation of proteins, The activities of chlorophyllase, contents of pigments chlorophylls, nucleic acids, membranes, and subsequent including chlorophyll a and b, chlorophyllide a and b, transport of some of the degradation products to other and phaeophorbide a during leaf senescence under parts of the plant (Buchanan-Wollaston, 1997; Noodén, low oxygen (0.5% O 2 ) and control (air) were investi1988). The yellowing of the leaves due to chlorophyll gated in a non-yellowing mutant and wild-type leaves degradation is the most obvious visible symptom. In the of snap beans (Phaseolus vulgaris L.). Chlorophyllase recent years, significant progress in the understanding of from leaf tissues had maximum activity when incubchlorophyll degradation has been achieved. A four step ated at 40 °C in a mixture containing 50% acetone. In pathway of chlorophyll degradation was demonstrated both mutant and wild type, chlorophyllase activity was (Vicentini et al., 1995). the highest in freshly harvested non-senescent leaves The initial step is the removal of phytol by chloroand decreased sharply in the course of senescence, phyllase (Matile et al., 1989; Shimokawa et al., 1978). indicating that the loss of chlorophylls in senescing Chlorophyllase is considered the first enzyme in the leaves is not directly related to the activity of chloropathway of chlorophyll degradation (Matile et al., 1997; phyllase and that chlorophyllase activity is not altered Hendry et al., 1987). However, the activity of chloroin the mutant. The wild type had higher ratios of phyllase tends to decrease with senescence (Minguezchlorophyll a to chlorophyll b than the mutant and Mosquera et al., 1996; Yamauchi and Watada, 1991), chlorophyll a5b ratios increased during senescence in indicating that factors other than chlorophyllase activity both types. In the senescent mutant leaves, accumularegulate chlorophyll degradation. Very recently, Matile tions of chlorophyllide a and chlorophyllide b were et al. (1997) reported in barley and oilseed rape leaves detected, but no phaeophorbide a was found. that chlorophyllase is located in the inner envelope memChlorophyllide b had a greater accumulation than brane of chloroplast. This finding supports the hypothesis chlorophyllide a in the early stage of senescence. Low that chlorophyllase and chlorophyll are spatially separoxygen treatment not only delayed chlorophyll ated, which prevents the hydrolysis of chlorophyll before degradation but also enhanced the accumulations of senescence (Fernandez-Lopez et al., 1991; Schoch and chlorophyllide a and b and lowered the ratios of Brown, 1987). Although it was found that chlorophyllase chlorophyll a to chlorophyll b. activity was detectable in both thylakoid membrane and