Chlorophyll biosynthesis: characterization of the CHL I subunit of the magnesium chelatase in Arabidopsis reveals two gene products with different properties

Abstract

Magnesium chelatase is a heterotrimeric enzyme complex that catalyzes a key regulatory reaction in chlorophyll biosynthesis, the insertion of Mg2+ into protoporphyrin IX. Studies of the enzyme complex reconstituted in vitro have shown that all three of its subunits, CHL I, CHL D, and CHL H, are required for enzymatic activity. In barley and tobacco CHL I is encoded by a single gene and consequently the xantha-h mutant of barley, which has a mutation in the Chl I gene, is unable to synthesize chlorophyll. However, a new T-DNA knockout mutant of the chlorina locus, ch42-3 (Chl I1), in Arabidopsis is still able to accumulate some chlorophyll despite the absence of Chl I message and protein. We identified an open reading frame that apparently encodes a second Chl I gene, Chl I2, as it has 82% similarity at the nucleotide level to the Chl I1 sequence. Chl I1 and Chl I2 mRNA accumulate to similar levels in wild-type, yet CHL I2 protein is not detectable in wild-type or ch42-3 although the protein is translated and stromally processed. Suprisingly, CHL D accumulates to wild-type levels in ch42-3, which is in contrast to findings that CHL D is not stable in CHL I deficient backgrounds of barley. Our results show that CHL D accumulation and some magnesium chelatase activity can occur in the absence of a stable CHL I subunit. We discuss implications of a second Chl I gene that is post-translationally regulated in Arabidopsis and its significance to our understanding of the mechanism for magnesium chelatase activity.