• Rgn13

94 Rice Genetics Newsletter Vol. 1.3

expression of OSEM is restricted to seed, and is undetectable in the seedlings. Globulin genes are also expressed in maturing embryos and are known to be induced by ABA. One of rice globulin genes, REG2, has recently been isolated, whose deduced amino acid sequence is 68% identical to that of maize GLB2 (Sun et al. 1996).

Since seed maturation must be a coordinated process involving a large number of genes, investigations on in situ expression pattern of ABA-related genes are highly important. In this study, we characterized the temporal and spatial expression patterns of two rice ABA-responsive genes, OSEM and REG2, in developing seeds by in situ hybridization analysis.

In situ hybridization was performed at various developmental stages according to the methods of Kouchi and Hata (1993) using cultivar Taichung 65. Developing seeds were fixed with 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M sodium phosphate

Fig. 1. ln situ hybridization of OSEM transcripts during seed development.

buffer (pH 7.2) for 20h at 5°C, and then dehydrated in a graded ethanol series. After substitution with xylene, they were embedded in Palaplast Plus (Fisher Scientific).

Samples were sectioned at 8mm by rotary microtome, and applied to slide glasses treated with Vectabond (Vector Lab.). RNA probes labeled with digoxygenin were prepared from the coding region without polyA-region of OSEM (ca. 0.7kb) and REG2 (ca. 1.6kb). Detection of hybridization signals was done according to Kouchi and Hata (1993).

The expression of OSEM in embryo was first detected at six days after pollination (6 DAP) in the radicle apex (Fig. 1 A). At 10 DAP, hybridization signals were observed in the radicle, shoot and vascular bundles (Fig. 1B). Then, signals became stronger through 15 DAP (Fig. 1C, D). Strong expression was observed in the shoot, radicle, root cap and vascular bundles, and weak expression in the scutellar epithelium and the epidermis of epiblast. In the shoot, OSEM was expressed uniformly in the shoot apex.

96 Rice Genetics Newsletter Vol. 13

leaves and the outer region of coleoptile (Fig. 1E). In contrast , highly tissue-specific expression was observed in the radicle (Fig. 1F). The apical meristem, central stele, outermost two or three cell files of cortex and root cap showed strong signals, whereas signals were absent in the epidermis and weak in most of the cortex. At 10 DAP and later, OSEM was also expressed in the aleurone layer, more strongly in the dorsal region than in the ventral region (Fig. 1G). The expression in the aleurone layer was much weaker than that in embryo.

The expression pattern of REG2 was both spatially and temporally different from that of OSEM. In embryo, weak signals of REG2 were observed in the scutellum at 6 DAP (Fig. 2A). At 10 DAP, whole embryo showed a low level expression (Fig. 2B). Organ- or tissue-specific expression was obvious at 15 and 20 DAP (Fig. 2C,D). The spatial expression pattern of REG2 (Fig. 2D) was exactly opposite to that of OSEM (Fig. 1D). Strong signals were detected in the scutellum, epiblast and coleorhiza, and no signal was detected in the shoot, vascular bundles, root cap and radicle except inner region of cortex (Fig. 2E). In the coleoptile, weak signals were observed in the inner region in contrast to OSEM expression in the outer region. Interestingly, REG2 was not expressed in the scuteller epithelium and the epidermis of epiblast, where OSEM was expressed. In the aleurone layer, strong signals were observed in the basal region as early as 6 DAP (Fig. 2A). The strong expression was maintained through 20 DAP in both dorsal and ventral regions (Fig. 2F).

In the present study, it was found that two genes, OSEM and REG2, have different organ- and tissue-specificities, although both are commonly activated by ABA. Wheat Em, homologous to OSEM, is known to be expressed in shoot by northern hybridization analysis. The present study shows that OSEM is also expressed in the radicle and epithelium as well as in the shoot and aleurone layer of endosperm. The tissue specificity suggests that OSEM is mainly activated in indeterminate cells such as shoot and radicle apical meristems and in determinate but metabolically active cells such as young leaves, epithelium and aleurone cells. In the scutellar epithelium and aleurone layer, it is known that many genes are expressed encoding enzymes such as a-amylase and various proteases. In radicle, most of the cortex cells with no OSEM signals are considered to be metabolically inactive because these cells soon undergo programed cell death and form aerenchyma (air space) after germination.

Hattori et al. (1995) identified several cis-regulatory elements responsible for the regulation by ABA or VPI in the promoter of OSEM. The present results indicate that the promoter has cis-elements addressing tissue specificity in addition to these identified elements. The onset of OSEM expression in the epithelium and aleurone cells is retarded than that in the shoot and radicle. Accordingly, different mechanisms of temporal regulation must be operating among the tissues.

Interestingly, the tissue specificity of REG2 is opposite to that of OSEM, although both are activated by ABA. Since REG2 encodes embryo storage protein, 46kDa globulin, it is conceivable that indeterminate cells and metabolically active cells do not accumulate storage substances. REG2 is expressed in aleurone layer already at 6

• Cite Gramene
• Feedback
• About
• Site Map
• Home

 

SPECIES: