title: Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization.
abstract: Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.
authors.affiliation: Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, Bet-Dagan 50250, Israel.
authors.forename: Tamar
authors.initials: T
authors.lastname: Azoulay Shemer
authors.forename: Smadar
authors.initials: S
authors.lastname: Harpaz-Saad
authors.forename: Eduard
authors.initials: E
authors.lastname: Belausov
authors.forename: Nicole
authors.initials: N
authors.lastname: Lovat
authors.forename: Oleg
authors.initials: O
authors.lastname: Krokhin
authors.forename: Victor
authors.initials: V
authors.lastname: Spicer
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Standing
authors.forename: Eliezer E
authors.initials: EE
authors.lastname: Goldschmidt
authors.forename: Yoram
authors.initials: Y
authors.lastname: Eyal
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002734:Chlorophyll / Q000378:metabolism*; D002957:Citrus / Q000201:enzymology*; D004792:Enzyme Precursors / Q000378:metabolism*; D005030:Ethylenes / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005638:Fruit / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D018087:Plastids / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D005030:Ethylenes; D002734:Chlorophyll; C036216:ethylene; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase