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, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed 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 employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.