Structural genes for Mg-chelatase subunits in barley:Xantha-f, -g and-h

Abstract

Barley mutants in the lociXantha-f, Xantha-g andXantha-h, when fed with 5-aminolevulinate in the dark, accumulate protoporphyrin IX. Mutant alleles at these loci that are completely blocked in protochlorophyllide synthesis are also blocked in development of prolamellar bodies in etioplasts. In contrast to wild type, thexan-f, -g and-h mutants had no detectable Mg-chelatase activity, whereas they all had methyltransferase activity for synthesis of Mg-protoporphyrin monomethyl ester. Antibodies recognising the CH42 protein ofArabidopsis thaliana and the OLIVE (OLI) protein ofAntirrhinum majus immunoreacted in wild-type barley with 42 and 150 kDa proteins, respectively. Thexan-h mutants lacked the protein reacting with antibodies raised against the CH42 protein. Twoxan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to theA. majus olive and theA. thaliana Ch-42 genes were cloned using PCR and screening of cDNA and genomic libraries. Probes for these genes were applied to Northern blots of RNA from thexantha mutants and confirmed the results of the Western analysis. The mutantsxan-f ²⁷, -f⁴⁰, -h⁵⁶ and-h ⁵⁷ are defective in transcript accumulation while-h ³⁸ is defective in translation. Southern blot analysis established thath ³⁸ has a deletion of part of the gene. Mutantsxan-f ¹⁰ and-f ⁴¹ produce both transcript and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded thatXan-f and-h genes encode two subunits of the barley Mg-chelatase and thatXan-g is likely to encode a third subunit. The XAN-F protein displays 82% amino acid sequence identity to the OLI protein ofAntirrhinum, 66% to theSynechocystis homologue and 34% identity to theRhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identity to theArabidopsis CH42 protein, 69% identity to theEuglena CCS protein, 70% identity to theCryptomonas BchA andOlisthodiscus CssA proteins, as well as 49% identity to theRhodobacter BchI subunit of Mg-chelatase. Identification of the barleyXan-f andXan-h encoded proteins as subunits required for Mg-chelatase activity supports the notion that theAntirrhinum OLI protein and theArabidopsis CH42 protein are subunits of Mg-chelatase in these plants. The expression of both theXan-f and-h genes in wild-type barley is light induced in leaves of greening seedlings, and in green tissue the genes are under the control of a circadian clock.