Molecular biological and enzymatic studies confirm that <i>xantha -f</i> , <i>-g</i> and <i>-h</i> mutants of barley are defective in magnesium chelatase.
Molecular biological and enzymatic studies confirm that xantha -f , -g and -h mutants of barley are defective in magnesium chelatase.

¹Robert D. Willows, ¹Ute C. Vothknecht, ²Bent L. Petersen and ²Poul Erik Jensen
¹Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10,
DK-2500 Copenhagen-Valby, Denmark
²Dept. of Ecology and Molecular Biology, Royal Vet. and Agr. Univ.,
Bülowsvej 13, DK-1870 Frederiksberg C, Denmark



In barley a number of genetically characterized xantha mutants are available (barley mutant stock list). When grown in light they accumulate no chlorophyll or reduced levels of this pigment compared to wild type due to defects in chloroplast biogenesis (von Wettstein et al., 1971). Intermediates of chlorophyll synthesis accumulate in the leaves of several xantha mutants when they are treated with 5-aminolevulinate indicating blocks at specific steps of the chlorophyll biosynthetic pathway. Protoporphyrin IX accumulated in the leaves of xan-f, -g and -h mutants in the dark when they were treated with 5-aminolevulinate and it was established that these mutants are blocked at the magnesium chelatase step (Henningsen et al., 1993). Recently it has been shown that in higher plants the insertion of magnesium into protoporphyrin IX requires at least two separate components (Walker and Weinstein, 1994) while in Rhodobacter sphaeroides three separate proteins are required (Gibson et al., 1995, in press). The 140 kDa protein encoded by the oli gene of Anthirhinum majus (Hudson et al., 1993) and the 42 kDa protein encoded by the chlorata-42 gene (ch42) of Arabidopsis (Koncz et al., 1990) have been proposed as subunits of the magnesium chelatase. We have cloned and sequenced from wildtype barley the genes homologues to oli and ch42 and analysed transcription and translation of these genes in wild type and xan-f, -g and -h mutants. These studies show that xan-f corresponds to the oli gene and xan-h to the ch-42 gene.

Comparision of enzyme activities and Northern and Western blot analysis of several xantha mutants relative to wild type are given in table 1. Significant levels of magnesium chelatase activity was observed with chloroplasts isolated from wild type barley leaves. However the chloroplasts isolated from the leaves of xan-f, -g and -h mutants were unable to insert magnesium into protoporphyrin IX. Western blot analysis showed that wild type barley contained a 140 kDa protein that was recognized by an antibody raised against the oli gene product of Antirhinum majus. This protein was absent in xan-f^27^ and -f^40^ mutants. Xan-f^10^ and -f^41^ mutants produced excess 140 kDa protein compared to wild type. However there was no difference in the levels of transcripts in these xantha-f mutants. These results show that xantha-f of barley corresponds to the oli gene of Antirhinum majus. The excess protein observed in xan-f^10^ and xan-f^41^ mutants probably results from defects in transport of the protein into the chloroplast. When the proteins from wild type and the xan-h mutants (-h^30^, -h^38^, -h^56^ and -h^57^) were probed with an antibody raised against the 42 kDa chlorata (ch42) gene product only wild type showed an immunoreactive band and all xan-h mutants lacked this band. Northern analysis with the barley ch42 probe showed that xan-h^56^ lacked this transcript. It is concluded that xan-h corresponds to the chlorata-42 gene of Arabidopsis

Table 1. Comparison of enzyme activity data, Northern and Western blot analysis of wild type and xantha mutants representing the three gene loci xan-f, -g and -h. Northern blots of total RNA were hybridized with probes from the cloned barley genes homologous to the ch42 and olive genes. Total protein extracts were analysed by Western blot using antibodies directed against the CH42 protein of Arabidopsis thaliana and the OLI protein of Antirhinum majus.

______________________________________________________________________________
          Mg-chelatase         mRNA for the             Protein reacting with:
          activity                                                           
                       42 kDa protein  140 kDa protein  Anti CH42    Anti OLI
______________________________________________________________________________
wild type       +            +               +            +            +    
xan-f^10^         -            +               +            +            +    
xan-f^26^         -            +               +            +            +    
xan-f^27^         -            +               -            +            -     
xan-f^41^         -            +               +            +            +    
xan-g^28^         -            +               +            +            +    
xan-g^37^         -            +               +            +            +    
xan-g^45^         -            +               +            +            +    
xan-h^56^         -            -               +            -            +    
______________________________________________________________________________

thaliana. Three mutants at the xan-g locus (-g^28^, -g^37^, -g^45^) were studied by Western and Northern blot analysis. Both these mutants had chlorata-42 and oli transcripts and the proteins in levels comparable to those of wild type.The presence of three different loci xan-f, xan-g and xan-h indicate that the barley magnesium chelatase is composed of three subunits as the enzyme from Rhodobacter sphaeroides. BchH gene encoding a 140 kDa subunit of the bacterial magnesium chelatase is homologous to the oli gene, while the BchI which encodes a 40 kDa polypeptide is homologous to the chlorata-42. BchD gene of Rhodobacter encodes a 60 kDa polypeptide and a homologue for this gene has not yet been described in higher plants. We suggest that xan-g represents the BchD homologue and that three polypeptides are required for the insertion of magnesium into protoporphyrin IX in barley.

Acknowledgements

The authors like to thank Profs. Diter von Wettstein and K.W. Henningsen and Dr. C.G. Kannangara for invaluable help. We also acknowledge financial support from the Danish Agricultural Research Council (no. 13-4827-1) for Dr. P. E. Jensen and from the Danish Research Council (no. 11-0544-1) for Dr. U.C. Vothknecht and from the Danish Biotechnology Programme to D. von Wettstein.

References

Gibson, L.C.D., Willows, R.D., Kannangara, C.G., von Wettstein, D. and Hunter, C.N. (1995) Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, I and D genes expressed in E.coli. Proc. Natl. Acad. Sci., USA (in press).

Henningsen, K.W., Boynton J.E. and von Wettstein, D. (1993) Mutants at xantha and albina loci in relation to chloroplast biogenesis in barley (Hordeum vulgare L.) Biologiske Skrifter 42, 1-349: The Royal Danish Academy of Sciences and Letters. Available from Munksgaard Export and Subsciption Service. Nørre Søgade 35, Dk-1370 Copenhagen.

Hudson, A., Carpenter, R., Doyle, S. and Coen, E.S. (1993) Olive: a key gene required for chlorophyll biosynthesis in Antirrhinum majus. EMBO J. 12, 3711-3719.

Koncz, C., Mayerhofer, R., Konch-Kalman, Z., Nawrath, C., Reiss, B., Redei, G.P. and Schell, J. (1990). Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana. EMBO J. 9, 1337-1346.

Simpson, D.J. and von Wettstein, D. (1991) Coordinators report: Nuclear genes affecting the chloroplast. Stock list of mutants kept at the Carlsberg Laboratory. Barley Genetics Newsletter 21, 102-108.

Walker C.J. and Weinstein, J.D. (1994) The magnesium-insertion step of chlorophyll biosynthesis is a two-stage reaction. Biochem. J. 299, 277-284.

Wettstein, D. von, Henningsen, K.W., Boynton, J.E, Kannangara, G.C. and Nielsen, O.F. (1971) The genic control of chloroplast development in barley. In: Autonomy and Biogenesis of Mitochondria and Chloroplasts, 205-223. (Eds. Boardman, N.K., Linnane, A.W. and Smillie, R.M.) North-Holland, Amsterdam.