53. Temporal and spatial expression pattern of rice α-amylase gene RAmy1A during seed development

N. SUGIMOTO1, G. TAKEDA2, Y. NAGATO1 and J. YAMAGUCTI3

1) Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo. 113 Japan
2) Faculty of Agriculture, Tamagawa University. Machida. 194 Japan
3) Bioscience Center, Nagoya University, Nagoya. 464-01 Japan
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During the germination of cereal grains, α-amylases (EC 3.2.1.1) play a key role in the mobilization of the energy reserves constituted by insoluble starch granules. This enzyme catalyzes the hydrolysis of the α-1,4 glucan bonds of the starch molecule and is de novo synthesized during the germination of cereal seeds. The expression of cereal α-amylases is regulated by gibberellic acid and abscisic acid, with the hormonal regulation operating mainly at the transcriptional level. Several techniques have been employed to localize α-amylase biosynthesis and gene expression in cereals. In rice, hydrolysis of starch films and in situ detection of mRNA and functional promoter analysis using transgenic rice seeds show that biosynthesis and expression of α-amylase is initiated in the scutellar epithelium and continues at higher levels in the aleurone layer at later stages of germination. In contrast to the well-known expression pattern during germination, it is almost unknown how α-amylase gene expression is regulated during seed development. We undertook detailed studies on the location and timing of the expression of rice α-amylase gene Ramy1A during seed maturation by in situ hybridization technique.

Developing rice seeds of Oryza sativa L., cv. Taichung 65, were harvested at various stages after pollination. They were fixed in 3% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 20 h at 5°C, and then dehydrated in a graded ethanol series. They were then embedded in Paraplast Plus (Fisher Scientific), sectioned (8 μm thick) by rotary microtome, and applied on slide glasses treated with Vectabond (Vector Lab.). Digoxygenin-labeled RNA probes of rice were prepared from cDNA clone of α-amylase gene, Ramy1A (O'Neill et al. 1992). Probes were degraded to a mean length of 100 bp by incubating in alkali at 60°C. In situ hybridization and the detection of hybridization signals were performed according to the methods of Kouchi and Hata (1993).

We have carried out in situ hybridization experiments using both sense and antisense probes. Only results with antisense probes are described in the following in situ hybridization experiments, since specific hybridization signals were not detected when sense probes were used (data not shown).

The transcripts of the rice α-amylase gene Ramy1A are known to be abundant during germination (O'Neill et al. 1992), but much less detectable in the developing seeds. The expression of the RAmy1A gene was first detected in the apical portion of scutellar epithelium at six days after pollination (6 DAP), when the first leaf primordium was already formed (Fig. 1A). Hybridization signals in the scutellar epithelium became stronger at 8 DAP (Fig. 1B). At 12 DAP when embryo was morphologically differentiated, the signals were weakly detected in the apical region of the scutellar epithelium (Fig. 1C), and almost no signals were detected at 20 DAP (Fig. 1D). Interestingly, no signals of Ramy1A transcripts were observed in the aleurone layer throughout the seed development (data not shown). These results show that Ramy1A gene is expressed in the scutellar epithelium during curly maturation stage, but not at the later stages.

The expression of α-amylase gene Ramy1A is known to be promoted by gibberellic acid in germinating cereal seeds. It is interesting how α-amylase gene expression is regulated in developing seeds. Hoecker et al. ( 1995) have reported that Viviparous-1 (VP1), a transcriptional activator of maize, inhibits the induction of a-amylase genes in aleurone cells of the developing maize seeds. It is likely that VP1 protein and endogenous levels of abscisic acid play a crucial role in the repression of α-amylase gene expression in developing seeds of rice. In fact, rice VP1 (OSVP1) transcripts were detected in the embryo of developing seed by in situ hybridization technique (Miyoshi et al. unpublished data). Further experiments will reveal regulatory mechanism(s) of α-amylase gene expression in developing seeds.

Fig. 1. In situ hybridization pattern of RAmy1A in developing rice seeds.
(A): 6, (B): 8, (C): 12 and (D): 20 DAP. Hybridization signals are observed only in scutellar epithelium
but not in the aleurone layer. Arrow: scutellar epithelium, arrowhead: aleurone layer. Scale bar = 0.5 mm.

References

Hoecker, U., I.K. Vasil and D.R. McCarty. 1995. Integrated control of seed maturation and germination programs by activator and represser functions of Viviparous-1 of maize. Gene & Development 9: 2459-2469.

Kouchi, H. and S. Hata, 1993. Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Mol. Gen. Genet. 238: 106-119.

O'Neill, S.D., M. Kumagai, A. Majumdar, N. Haung, T.D. Sutliff and R.L. Rodriguez, 1990. The α-amylase genes in Oryza sativa: characterization of cDNA clones and mRNA expression during seed germination. Mol. Gen. Genet. 221: 235-244.