STRUCTURAL GENES FOR MG-CHELATASE SUBUNITS IN BARLEY - XANTHA-F, XANTHA-G AND XANTHA-H

Barley mutants in the loci Xantha-f; Xantha-g and Xantha-h, when fed w ith 5-aminolevulinate in the dark, accumulate protoporphyrin IX. Mutan t alleles at these loci that are completely blocked in protochlorophyl lide synthesis are also blocked in development of prolamellar bodies i n etioplasts. In contrast to wild type, the xan-f; -g and -h mutants h ad no detectable Mg-chelatase activity, whereas they all had methyltra nsferase activity for synthesis of Mg-protoporphyrin monomethyl ester. Antibodies recognising the CH42 protein of Arabidopsis thaliana and t he OLIVE (OLI) protein of Antirrhinum majus immunoreacted in wild-type barley with 42 and 150 kDa proteins, respectively. The xan-h mutants lacked the protein reacting with antibodies raised against the CH42 pr otein. Two xan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to the A. majus olive and t he A. thaliana Ch-42 genes were cloned using PCR and screening of cDNA and genomic libraries. Probes for these genes were applied to Norther n blots of RNA from the xantha mutants and confirmed the results of th e Western analysis. The mutants xan-f(27), -f(40), -h(56) and -h(57) a re defective in transcript accumulation while -h(38) is defective in t ranslation. Southern blot analysis established that h(38) has a deleti on of part of the gene. Mutants xan-f(10) and -f(41) produce both tran script and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded that Xan-f and -h gen es encode two subunits of the barley Mg-chelatase and that Xan-g is li kely to encode a third subunit. The XAN-F protein displays 82% amino a cid sequence identity to the OLI protein of Antirrhinum, 66% to the Sy nechocystis homologue and 34% identity to the Rhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identi ty to the Arabidopsis CH42 protein, 69% identity to the Euglena CCS pr otein, 70% identity to the Cryptomonas BchA and Olisthodiscus CssA pro teins, as well as 49% identity to the Rhodobacter BchI subunit of Mg-c helatase. Identification of the barley Xan-f and Xan-h encoded protein s as subunits required for Mg-chelatase activity supports the notion t hat the Antirrhinun OLI protein and the Arabidopsis CH42 protein are s ubunits of Mg-chelatase in these plants. The expression of both the Xa n-f and -h genes in wild-type barley is light induced in leaves of gre ening seedlings, and in green tissue the genes are under the control o f a circadian clock.