Rice EPSP synthase gene resides 45-kb downstream of the Waxy gene

46. Rice EPSP synthase gene resides 45-kb downstream of the Waxy gene T. Sato, L.-H. Wu, H. NAGANO, Y. KISHIMA and Y. SANO Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase that catalyzes the reversible addition of the enolpyruvyl moiety of phosphoenolpyruvate to shikimate 3-phosphate in the shikimate pathway is localized in the plastids of higher plants. This enzyme is known to be targeted by glyphosate which has been employed as nonselective herbicide (Steinrucken and Amrhein 1980). Here we present data on the chromosomal location of the rice EPSP synthase gene and its expression pattern. Previously, we obtained a 300-kb BAC contig surrounding the Waxy (Wx) gene of a rice cultivar, Shimokita (Japonica type), and subsequently constructed a fine physical map for HindIII sites (Nagano et al. 1999). At the same time, a cDNA library was prepared from the rice anther tissues (Wu eta!. 1997). To detect regions transcribed in the anthers, the overlapping subclones of the contig around the Wx gene were probed with crude cDNAs prepared from the developing anthers. The hybridized subclones were then characterized by sequencing. A dozen of the known genes were identified among the hybridized fragments with the anther cDNAs (data not shown). Of these identified genes, the EPSP synthase gene was detected to be located 45-kb downstream of the Wx gene (Fig. 1). This fragment also included the ribosoma! protein small subunit 40 (rps40) gene and an unknown transcription product. Genomic hybridization patterns showed that the EPSP synthase gene exists as a single copy in the genome of Japonica rice (data not shown). We screened the anther cDNA library, and selected a clone with sufficient length comparable to the maize EPSP synthase gene sequence deposited in the database (accession no. X63374). Although this clone appears to possess a functional sequence for EPSP synthase, the sequence for its transit peptide to the plastids do not resemble those of petunia and tomato (Gasser eta!. 1988). This might be due to the possibility that the isolated clone in this study is an incomplete sequence for the transit peptides of the EPSP synthases. Northern blot analysis revealed that the EPSP synthase gene produces several transcripts with varying sizes (data not shown), suggesting apossibility of alternative splicing events. The alternative splicing might produce such novel transit peptides for the EPSP synthase. We have attempted to use 5� RACE PCR to extend the sequence for the transit peptide, but no extension product has been produced. This fact could lead to another interpretation that the EPSP synthase in monocot plants (at least rice and maize) may utilize different transit peptides from those of dicot plants. The transcripts of EPSP synthase in rice anthers have been observed to accumulate in relatively small amounts compared to those of the other organs such as shoot, root and inflorescent. The two enzymes, Wx protein and EPSP synthase, function in plastids, and both genes reside in close vicinity to each other in the Japonica rice genome. Further investigation will be focused on identification of other plastid-targeted genes present in the vicinity of this region and the colinearity of this region in the other grass species. The nucleotide sequence data for the rice EPSP synthase cDNA was reported to the DDBJ, GenBank and EMBL Nucleotide Sequence Databases under the accession number AB016765. References Gasser, C.S., J.A. Winter, C.M. Hironaka, D.M. Shah, 1988. Structure, expression, and evolution of the 5- enolpyruvylshikimate-3-phosphate synthase genes of petunia and tomato. J. Biol. Chem. 263: 4280- 4289. Nagano, H., L-H. Wu, S. Kawasaki, Y. Kishima andY. Sano, 1999. Genomic organization of the 260 kb surrounding the waxy locus in aJaponica rice. Genome 42: 1121-1126. Steinrucken, H.C., and N. Amrhein, 1980. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phospate syntase. Biochem. Biophys.Res. Commun. 94: 1207-1212.. Wu, L., H. Nagano, S. Kawasaki, Y. Kishima andY. Sano, 1997. Identification of the DNA fragments located in the vicinity of waxy locus respoosible for galnate eliminator. RGN. 14: 123-124.

Previously, we obtained a 300-kb BAC contig surrounding the Waxy (Wx) gene of a rice cultivar, Shimokita (Japonica type), and subsequently constructed a fine physical map for HindIII sites (Nagano et al. 1999). At the same time, a cDNA library was prepared from the rice anther tissues (Wu eta!. 1997). To detect regions transcribed in the anthers, the overlapping subclones of the contig around the Wx gene were probed with crude cDNAs prepared from the developing anthers. The hybridized subclones were then characterized by sequencing. A dozen of the known genes were identified among the hybridized fragments with the anther cDNAs (data not shown). Of these identified genes, the EPSP synthase gene was detected to be located 45-kb downstream of the Wx gene (Fig. 1). This fragment also included the ribosoma! protein small subunit 40 (rps40) gene and an unknown transcription product. Genomic hybridization patterns showed that the EPSP synthase gene exists as a single copy in the genome of Japonica rice (data not shown). We screened the anther cDNA library, and selected a clone with sufficient length comparable to the maize EPSP synthase gene sequence deposited in the database (accession no. X63374). Although this clone appears to possess a functional sequence for EPSP synthase, the sequence for its transit peptide to the plastids do not resemble those of petunia and tomato (Gasser eta!. 1988). This might be due to the possibility that the isolated clone in this study is an incomplete sequence for the transit peptides of the EPSP synthases. Northern blot analysis revealed that the EPSP synthase gene produces several transcripts with varying sizes (data not shown), suggesting apossibility of alternative splicing events. The alternative splicing might produce such novel transit peptides for the EPSP synthase. We have attempted to use 5� RACE PCR to extend the sequence for the transit peptide, but no extension product has been produced. This fact could lead to another interpretation that the EPSP synthase in monocot plants (at least rice and maize) may utilize different transit peptides from those of dicot plants. The transcripts of EPSP synthase in rice anthers have been observed to accumulate in relatively small amounts compared to those of the other organs such as shoot, root and inflorescent.

The two enzymes, Wx protein and EPSP synthase, function in plastids, and both genes reside in close vicinity to each other in the Japonica rice genome. Further investigation will be focused on identification of other plastid-targeted genes present in the vicinity of this region and the colinearity of this region in the other grass species.

The nucleotide sequence data for the rice EPSP synthase cDNA was reported to the DDBJ, GenBank and EMBL Nucleotide Sequence Databases under the accession number AB016765.

References

Gasser, C.S., J.A. Winter, C.M. Hironaka, D.M. Shah, 1988. Structure, expression, and evolution of the 5- enolpyruvylshikimate-3-phosphate synthase genes of petunia and tomato. J. Biol. Chem. 263: 4280- 4289.

Nagano, H., L-H. Wu, S. Kawasaki, Y. Kishima andY. Sano, 1999. Genomic organization of the 260 kb surrounding the waxy locus in aJaponica rice. Genome 42: 1121-1126.

Steinrucken, H.C., and N. Amrhein, 1980. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phospate syntase. Biochem. Biophys.Res. Commun. 94: 1207-1212..

Wu, L., H. Nagano, S. Kawasaki, Y. Kishima andY. Sano, 1997. Identification of the DNA fragments located in the vicinity of waxy locus respoosible for galnate eliminator. RGN. 14: 123-124.