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

Abstract

Barley mutants in the loci Xantha-f, Xantha-g and Xantha-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, the xan-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 of Arabidopsis thaliana and the 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 protein. Two xan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to the A. majus olive and the A. 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 the xantha mutants and confirmed the results of the Western analysis. The mutants xan-f27, -f40, -h56 and -h57 are defective in transcript accumulation while -h38 is defective in translation. Southern blot analysis established that h38 has a deletion of part of the gene. Mutants xan-f10 and -f41 produce both transcript and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded that X an-f -h genes encode two subunits of the barley Mg-chelatase and that X an-g is likely to encode a third subunit. The XAN-F protein displays 82% amino acid sequence identity to the OLI protein of Antirrhinum, 66% to the Synechocystis homologue and 34% identity to the Rhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identity to the Arabidopsis CH42 protein, 69% identity to the Euglena CCS protein, 70% identity to the Cryptomonas BchA and Olisthodiscus CssA proteins, as well as 49% identity to the Rhodobacter BchI subunit of Mg-chelatase. Identification of the barley X an-f and X an-h encoded proteins as subunits required for Mg-chelatase activity supports the notion that the Antirrhinum OLI protein and the Arabidopsis Ch42 protein are subunits of Mg-chelatase in these plants. The expression of both thet X an-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.

Magnesium-Chelatase from Developing Pea Leaves1

“…If porphyrin substrate is limiting, the difference in K m values is more than sufficient to account for the higher flux into the Mg 2ϩ branch of the pathway. Regulation of the branch point by relative affinity for the substrate could work in addition to a mechanism of day versus night regulation, based on the demonstrated circadian rhythmicity of subunit H expression (Gibson et al, 1996;Jensen et al, 1996b).…”

“…The same is true for the two plant genes that have been identified (Hudson et al, 1993;Nakayama et al, 1995;Gibson et al, 1996;Jensen et al, 1996b). Although most of our work on the in vitro characterization of the Mg-chelatase reaction has used membrane-containing chloroplast fractions, we have observed that Mg-chelatase activity can be solubilized by chloroplast lysis in buffers that contain low concentrations of MgCl 2 (Walker and Weinstein, 1995).…”

“…The requirement for three sub-units catalyzing an ATP-dependent metal ion insertion is analogous to the situation for Co insertion in vitamin B12 biosynthesis in Pseudomonas denitrificans (Debussche et al, 1992). Homologs for two of the bacterial Mg-chelatase genes have been identified in eukaryotic plants, olive and ch42 for bchH and bchI, respectively (Koncz et al, 1990;Hudson et al, 1993;Nakayama et al, 1995;Gibson et al, 1996;Jensen et al, 1996b). The sequence homologies between the two bacterial and plant genes and the requirement for ATP suggest that higher plants also require three subunits for Mg-chelatase activity.…”

“…The sequence homologies between the two bacterial and plant genes and the requirement for ATP suggest that higher plants also require three subunits for Mg-chelatase activity. Evidence for this supposition comes from the finding that there are three nonallelic mutations that affect Mg-chelatase activity in isolated barley (Hordeum vulgare L.) chloroplasts (Jensen et al, 1996b), and that activity can be reconstituted by mixing chloroplast extracts from any two nonallelic mutants (Kannangara et al, 1997). Although two of the plant genes have been cloned and expressed, there is currently no system available to test whether the expressed subunits are active (Nakayama et al, 1995;Gibson et al, 1996).…”

Gene silencing from plant DNA carried by a Geminivirus

“…To do this, we targeted both a nuclear transgene encoding luciferase (luc) and an endogenous gene coding for an enzyme required in chlorophyll biosynthesis. Magnesium chelatase is a multi-subunit protein that catalyzes the insertion of magnesium into protoporphyrin IX (Jensen et al, 1996). In tobacco, a mutated allele (Su) of one subunit causes the phenotype known as 'sulfur' (Nguyen, 1995).…”