Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate

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Abstract
Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase fromChenopodium album, screened theC. albumcDNA library, and cloned a cDNA (CaCLH,C. albumchlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop).CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) inArabidopsis thaliana(AtCLH1). We expressed the gene products ofAtCLH1and ofCaCLHinEscherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on anArabidopsisgenomic fragment and expressed it inE. coli, demonstrating the presence of the secondArabidopsisCLH gene (AtCLH2). No typical feature of signal sequence was identified inAtCLH1, whereasAtCLH2 had a typical signal sequence for chloroplast.AtCLH1mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h.AtCLH2, however, did not respond to MeJA.