Chlorophyllase Is a Rate-Limiting Enzyme in Chlorophyll Catabolism and Is Posttranslationally Regulated

Abstract

Chlorophyll is a central player in harvesting light energy for photosynthesis, yet the rate-limiting steps of chlorophyll catabolism and the regulation of the catabolic enzymes remain unresolved. To study the role and regulation of chlorophyllase (Chlase), the first enzyme of the chlorophyll catabolic pathway, we expressed precursor and mature versions of citrus (Citrus sinensis) Chlase in two heterologous plant systems: (1) squash (Cucurbita pepo) plants using a viral vector expression system; and (2) transiently transformed tobacco (Nicotiana tabacum) protoplasts. Expression of full-length citrus Chlase resulted in limited chlorophyll breakdown in protoplasts and no visible leaf phenotype in whole plants, whereas expression of a Chlase version lacking the N-terminal 21 amino acids (ChlaseDN), which corresponds to the mature protein, led to extensive chlorophyll breakdown in both tobacco protoplasts and squash leaves. ChlaseDN-expressing squash leaves displayed a dramatic chlorotic phenotype in plants grown under low-intensity light, whereas under natural light a lesion-mimic phenotype occurred, which was demonstrated to follow the accumulation of chlorophyllide, a photodynamic chlorophyll breakdown product. Fulllength and mature citrus Chlase versions were localized to the chloroplast membrane fraction in expressing tobacco protoplasts, where processing of the N-terminal 21 amino acids appears to occur. Results obtained in both plant systems suggest that Chlase functions as a rate-limiting enzyme in chlorophyll catabolism controlled via posttranslational regulation.

Different Mechanisms Are Responsible for Chlorophyll Dephytylation during Fruit Ripening and Leaf Senescence in Tomato

“…Thus, citrus CLH was detected in chloroplasts by in situ immunofluorescence labeling. Furthermore, the enzyme is proteolytically processed at the N-and C termini, posttranslational modifications that are unrelated to chloroplast targeting but were shown to be important for activity (Harpaz-Saad et al, 2007;Azoulay Shemer et al, 2008;Azoulay-Shemer et al, 2011). Finally, citrus CLH is transcriptionally up-regulated during ethylene-induced citrus ripening (Jacob-Wilk et al, 1999).…”

“…In summary, these data indicate that the pathways of chlorophyll breakdown during fruit ripening and leaf senescence are identical. Yet, the identification of PPH as the major dephytylating enzyme of leaf senescence ) challenges this view, because, contrary to the situation in leaves, CLH was shown to be involved during ethylene-induced ripening of citrus fruits (Jacob-Wilk et al, 1999;Harpaz-Saad et al, 2007;Azoulay Shemer et al, 2008).…”

Update on the biochemistry of chlorophyll breakdown

“…Furthermore, expression patterns of neither AtCLHs nor CLHs of broccoli or Chinese flowering cabbage correlate with Chl breakdown Zimmermann et al 2004;Büchert et al 2011;Zhang et al 2011). By contrast, CLH was convincingly shown to participate in Chl breakdown during fruit ripening of Citrus species (Azoulay Shemer et al 2008;Harpaz-Saad et al 2007). Further analysis is demanded to elucidate the role of CLHs in leaves and fruits, ideally in a plant species that allows simultaneous analysis of leaf senescence and fruit ripening, such as tomato.…”

“…in the cytosol or the vacuole, and subsequent localization experiments gave ambiguous results. For example, the two Arabidopsis CLHs, AtCLH1 and AtCLH2, localized to the cytosol when tagged with green fluorescent protein ), while lemon CLH was shown by in situ immunofluorescence to reside inside the chloroplast in lemon flavedo tissue and to co-purify with chloroplast membranes after heterologous expression in tobacco mesophyll protoplasts (Harpaz-Saad et al 2007;Azoulay Shemer et al 2008). Contradictory results were also obtained in experiments with de-regulated CLH expression; absence of AtCLH1 and/or AtCLH2 in respective mutants ) as well as silencing of AtCLH1 (Kariola et al 2005) or AtCLH2 ) had little effect on senescence-related Chl breakdown, while antisense-suppression of broccoli CLH delayed rates of postharvest broccoli head yellowing .…”

A Kelch domain-containing F-box coding gene negatively regulates flavonoid accumulation in Cucumis melo L.

“…VIGE is an established experimental procedure to analyze the effect of gene overexpression in cucurbits (Harpaz-Saad et al, 2007). Our chemical analysis of muskmelon leaves suggested that flavonoid accumulation in leaves is irrelevant to flavonoid accumulation in muskmelon fruit rind (Supplemental Fig.…”