54. Characterization and expression of GASR1, a root-specific GAST1-like protein in rice


1) Meijo University, Tenpaku, Nagoya, 468 Japan
2) Bioscience Center, Nagoya University, Chikusa, Nagoya, 464-01 Japan

GAST (gibberellin (GA)-stimulated transcript) gene family has been reported in tomato (Shi et al. 1992; Taylor and Scheuring 1994) and Arabidopsis thaliana (Herzog et al. 1995), although its physiological function is still unknown. Shi et al. (1992) originally characterized the GAST1 gene from tomato as related to a GA-stimulated transcript encoding an RNA whose abundance increases 20-fold in shoots of the GA-deficient gib1 mutant following spraying with GA3. Taylor and Scheuring ( 1994) also reported a distinct GAST gene family, RSI-1, an auxin inducible and root-specific transcript. The corresponding peptides among GAST1, RSI-1 and Arabidopsis CAST gene family (GASA1 to GASA5) display comparable structural features; (1) a putative signal peptide of 18 to 23 residues; (2) a highly divergent hydrophilic region of about 22 amino acids; (3) a conservative 60 amino acid C-terminal domain containing 12 cysteines. In this communication, we report the characterization and expression pattern of rice GAST gene, GASR1.

Fig. 1. Alignment of predicted peptide sequences encoded by related cDNAs. GASR1 (this study) from rice: GAST1 and RSI-1 from tomato, GASA1 to GASA5 from Arahidopsis thaliana. Conserved cysteine residues are boxed.

Fig. 2. Immunoblot of protein extracts from rice seedling roots using GASR1 antibody. Eight day-old seedlings of rice (cultivar, Nipponbare) were treated with phytohormones for 3 days. Proteins were extracted with SDS sample buffer (ratio of 1:3: fresh weight: buffer) and applied to SDS-PAGE. Immunoblot were performed following a standard procedure. Lane 1, no treatment: lanes 2-4, GA3 treatment; lanes 5-7, NAA treatment; lanes 8-10, TIBA treatment. Treatment at the concentration of 0.1 μM (lanes 2, 5, 8), 1 mM (lanes 3, 6, 9), 10 μM (lanes 4, 7, 10). Arrows indicate 13 kDa and 40 kDa band signal. Molecular mass of standard proteins is indicated at the left side.

The cDNA clone pGASR1 used in this study was identified during random sequencing of a rice cDNA library constructed from the developing seeds of rice (Oryza sativa L. cultivar, Taichung 65). To prepare the GASR1 antigen, coding region for the mature protein (removing the putative signal peptide) of pGASR1 was ligated to pET32 (Novagen) BamH I and Hind III sites. An affinity-purified protein of size 13 kDa from the transformed Escherichia coli was identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The over-expressed protein was excised from the gel and used as the antigen to raise rabbit antibody. Immunoblots of protein extract from various tissues and phytohormone-treated roots were performed by using this rabbit antibody.

GASR1, GAST-like cDNA in rice is 708 bp long, bearing an ORF coding for 93 amino acids. This sequence shows complete identity with a partial cDNA identified in rice callus (the dbEST accession number D15611). The deduced peptide sequences from GAST family were aligned, as shown in Fig. 1, with the amino acids of GAST1 (Shi et al. 1992). RSI-1 (Taylor and Scheuring 1994) and GASA1 to GASA5 (Herzog et al. 1995). Significant homology was limited to their C-terminal end. Twelve cysteine residues among their alignment were completely conserved at the predicted positions, indicating that the cysteine residues may play an important role in their physiological function to maintain globular conformation through disulfide bridges. The presence of putative signal peptide in the N-terminal domain may indicate that these proteins are secreted.

Immunoblot of protein extract from various rice organs revealed that GASR1 is localized in roots of the seedlings, but not in shoots. The GASR1 protein is also undetected in all organs of the mature plants (data not shown). Fig. 2 shows the effect of GA, NAA α-naphthalene acetic acid) and TIBA (2,4,5-triiodobenzoic acid, an inhibitor of auxin transport) on the amount and molecular size of GASR1 in the roots. None of those effectors could alter the amount of GASR1 protein in the root. However, NAA showed an apparent effect on the molecular size of the antigen protein (from 13 kDa to 40 kDa on SDS-PAGE data), whereas GA and TIBA did not. These results indicate that NAA is a trigging molecule to induce molecular change(s) on GASR1 protein. It is unlikely that the molecular size shift of the GASR1 on SDS-PAGE consequent from the NAA treatment is due to the formation of disulfide bridges between conserved cysteine residues, since those bridges would be destroyed by a reducing reagent during preparation of SDS-PAGE sample. Further studies will be needed to clarify the molecular mechanism(s) involved in the NAA-induced molecular size change of the GASR1 protein.

GASR1 did not respond to exogenous GA3(Fig. 2), indicating distinct expression patterns from those of the GAST1, a typical GA inducible gene. The pattern of tissue localization and the effects of phytohormones on the expression for GASR1 resemble those of RSI-1, i.e., both are root-specific and auxin-modulated. RSI-I is an auxin inducible transcript in tomato roots and the promoter is expressed in the early lateral root primordia (Taylor and Scheuring 1994). Further studies on the GASR1 molecular weight shift mediated by auxin treatment and in situ detection in the roots may reveal the physiological roles of the protein in root formation.


Herzog, M., A.-M. Dome and F. Grellet, 1995. GASA, a gibberellin-regulated gene family from Arabidopsis thaliana related to the tomato GAST1 gene. Plant Mol. Biol. 257: 743-752.

Shi, L., R.T. Cast, M. Gopalraj and N.E. Olszewki, 1992. Characterization of a shoot-specific, GA3-and ABA- regulated gene from tomato. Plant J. 2: 153-159.

Taylor, B.H. and C.H Scheuring, 1994. A molecular marker for lateral root initiation: The RSI-1 gene of tomato (Lycopersicon esculentum Mill) is activated in early lateral root primordia. Mol. Gen. Genet. 243: 148-157.