48. A cDNA clone from rice accelerated overgrowth (ao) mutant encoding xyloglucan-related protein homolog

T. AOKI1, N. KAMEYA2 and I. NAKAMURA2

1) Dept. of Appl. Biol. Sci., Nihon Univ., Kameino, Fujisawa, 252 Japan.
2) Iwate Biotech. Res. Cent., 22-174-4 Narita, Kitakami, 024 Japan.
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Ao mutant of rice, showing overgrowth characteristics, has been isolated from the progeny of γ-ray irradiated seeds of a japonica cultivar Koshihikari (Nakamura 1992). Although the seedlings of ao mutant show a phenotype similar to that of the seedlings infected by the foolish seedling disease, caused by Gibberella fujikuroi, the mutant phenotype is not due to the overproduction of an endogenous gibberellic acid and is characteristic of accelerated elongation of the internodes at seedling stage.

Xyloglucan is a major component of structural polysaccharide and acts as a cross-linker between cellulose-microfibrils within cell wall of plants. Thus, the cleavage and rejoining of the xyloglucan linkers are likely to be involved in the extension of cell wall during cell growth, differentiation and hence morphogenesis in plants (Nishitani 1995), such as internode elongation in rice plant. Endoxyloglucan transferase (EXT) is a class of glycosyltransferase purified from Vigna angularis (Nishitani and Tominaga 1992) and it catalyzes both cleavage and grafting of xyloglucan molecules. cDNA clones encoding EXTs have been isolated from Arabidopsis thaliana, Zea mays, Triticum aestivum, Lycoperisicon esculentum, and Glycine max (Okazawa et al. 1993). By searching GenBank database, some plant genes were found to encode EXT homologs and xyloglucan-related proteins (XRPs), such as nasturtium (Tropaeolum majus) xyloglucan-specific glucanase (NXG) (de Silva et al. 1993), Arabidopsis Meri5, identified as meristem specific protein (Medford et al. 1991), and soybean BRU-1, as brassinosteroid-regulated gene product (Zurek and Clouse 1994). The amino acid sequences of EXTs and XRPs share conserved regions and may play an important role in regulation of changing cell shape through the reconstruction of xyloglucan network.

We prepared a cDNA library from elongating internodes excised from seedlings of ao mutant (fourth leaf stage) and cDNA clones were sequenced partially and analyzed their homology using blastn program in Blast e-mail server of NCBI. We found one clone, ASL321, that was similar to maize EXT cDNAs. Northern blot analysis was carried out to compare the amount of ASL321 mRNA in shoots and roots between normal and ao mutant. The accumulation of ASL321 message was higher in the culm of ao mutant than normal plant, whereas the amounts of ASL321 messages were similar in the etiolated leaves. Relatively low level of mRNA was observed in the roots of ao mutant and normal plants (Fig. 1).

Fig. 1. Comparison of the expression of OSXRP mRNA in etiolated leaf ( 1,2), roots (3, 4) and culm (5, 6) of seedlings between ao mutant (1, 3, 5) and normal (2, 4. 6). Upper: EtBr stain, Lower: Northen blot using OSXRP cDNA as a probe.

Fig. 2. Alignment of amino acid sequences of end-xlyloglucan transferases from A. thaliana (EXT, 296 a.a.), xyloglucan-related proteins (XRPs) from A. thaliana (Meri5,269 a.a.), G.max(BRU1, 283 a.a.) and L. esculentum (XET, 289 a.a.), OSXRP (277 a.a.) isolated from rice in this study and xyloglucan specific glucanase from T. majus (NXG, 295 a.a.) using UPGMA program. Six xyloglucan related proteins share some conserved domains in their central parts and a solid line indicates proposed catalytic center conserved among EXTs, XRPs, OSTXRP and NXG. The sequences of catalytic center, DEIDFEFLG, is conserved among EXT, Meri5, BRU1 and XET, but both in OSXRP and NXG, phenylalanine residue of 5 th position (arrow) is replaced by isoleucine residue. Asterisks indicate the position at which the amino acid residues are identical among five xyloglucan related proteins.

We screened for full length cDNA clone from ao library using ASL321 clone as a probe. The isolated cDNA clone, designated as OSXRP, was 1,239 bp in length and contained the deduced open reading frame encoding 31 kDa protein of 277 amino acid residues. OSXRP protein showed 49% identity with Arabidopsis EXT, 49% with Arabidopsis Meri5, soybean BRUI, 54% with tomato XET and 41% with nasturtium NXG. Alignment of amino acid sequences of EXT from Arabidopsis (296 a.a.), Arabidopsis Meri5 (269 a.a.), soybean BRU1 (283 a.a.) and tomato XET (289 a.a.), rice OSXRP (this study), and NXG (295 a.a.) was generated. Six xyloglucan-related proteins share several conserved motifs and a solid line in Figure 2 indicates a proposed catalytic center conserved among EXTs, XRPs, NXG and B. subtilis glucanases. The sequence of catalytic center, DEIDFEFLG motif, is conserved among EXT, Meri5, BRU1, XET, NXG and OSXRP, except that phenylalanine residue of 5th position (arrow) is replaced by isoleucine residue in OSXRP, NXG and fungal glucanases (Fig. 2). Dendrogram drawn by the UPGMA method showed the relationships among deduced amino acid sequences of EXTs and XRPs in plants (Fig. 3). All plant xyloglucan-related proteins except OSXRP and NXG were separated into two different sub-groups, EXTs and XRPs and OSXRP protein was found to stand between a group of EXTs and XRPs, and NXG .

These results suggest that OSXRP gene isolated in this study might encode a unique xyloglucan-related protein or xyloglucan-specific glucanase, which is involved in unknown aspects of matrix polysaccharides in plant cell wall and may play a role of cell elongation during shoot development in rice plant.

Fig. 3. Dendrogram showing the relationships among deduced amino acid sequences of endo-xyloglucan transferases (EXTs) and xyloglucan related proteins (XRPs) in plants. Sequences were obtained from Gen Bank, EXT-L (D 16456), EXT-T (D 16457), EXT-A (D 16454), EXT-V (D 16458), EXT-G (D 16455), wusl (U15781), TCH4 (U27609), Meri5 (X82683), BRU1 (L22162), XET (X82685) and NXG (X68254). @

References

de Silva. J., C.D. Jarman, D.A. Arrowsmith, M.S. Stronach, S. Chengappa, C. Sidebottom and J.S. Reid, 1993. Molecular characterization of a xyloglucan-specific endo-( 1-- >4)-beta-D-glucanase (xyloglucan endo-transglycosylase) from nasturtium seeds. Plant J. 3: 701-711.

Medford, J.I., J.S. Elmer and H.J. Klee. 1991. Molecularcloning and characterization of gene expressed in shoot apical meristems. Plant Cell 3: 359-370.

Nakamura.I., 1992. Accerelated internode overgrowth mutant in rice. RGN 9: 61-62.

Nishitani, K. and R. Tominaga, 1992. Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecules to another xyloglucan molecules. J. Biol. Chem. 267: 21058-21064.

Nishitani, K., 1995. Endo-xyloglucan transferase. a new class of transferase involved in cell wall construction. J. Plant Res. 108:137-14S.

Okazawa, K., Y. Sato, T. Nakagawa, K. Asada. I. Kato, E. Tomita and K. Nishizawa, 1993. Molecular cloning and cDNA sequencing of endoxyloglucan transferase, a novel class of glycosyltransferase that mediates molecular grafting between matrix polysaccharides in plant cell walls. J. Biol. Chem. 268: 25364-25368.

Zurek, D. M. and S.D. Clouse. 1994. Molecular cloning and characterization of a brassinosteroid-regulated gene from elongating soybean {Glycine max L.) epicotyls. Plant Physiol. 104: 161-170.