Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism

Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, por phyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was clone d (pHvRCCR). The gene was expressed at all stages of leaf development and i n roots. By comparison with different databases, genomic sequences and expr essed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was em ployed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC red uction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accu mulated. The reaction required reduced ferredoxin and was sensitive to oxyg en. AtRCCR encoded a 35 kDa protein which was used for chloroplast import e xperiments. Upon transport, it was processed to a mature form of 31 kDa. Th e significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.