Plant Cell Advance Online Publication
Published on March 16, 2007; 10.1105/tpc.107.050633
Received January 24, 2007
Returned for revision January 24, 2007
Accepted February 27, 2007
Chlorophyllase Is a Rate-Limiting Enzyme in Chlorophyll Catabolism and Is Posttranslationally Regulated
Smadar Harpaz-Saad 1, Tamar Azoulay 1, Tzahi Arazi 2, Eran Ben-Yaakov 1, Anahit Mett 2, Yoel M. Shiboleth 3, Stefan Hörtensteiner 4, David Gidoni 2, Amit Gal-On 3, Eliezer E. Goldschmidt 5, and Yoram Eyal 2*
1 Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, Bet-Dagan 50250, Israel; R.H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, Food, and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel
2 Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, Bet-Dagan 50250, Israel
3 Institute of Plant Protection, The Volcani Center, Agricultural Research Organization, Bet-Dagan 50250, Israel
4 Institute of Plant Sciences, University of Bern, CH-3013 Bern, Switzerland
5 R.H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, Food, and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel
* To whom correspondence should be addressed. E-mail: eyalab{at}volcani.agri.gov.il.
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 (Chlase N), which corresponds to the mature protein, led to extensive chlorophyll breakdown in both tobacco protoplasts and squash leaves. ChlaseN-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. Full-length 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.
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