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2) Center for Molecular Biology and Genetics. Mie University, Tsu, 514 Japan

During the late phase of development, seeds acquire desiccation tolerance and become dormant. The phytohormone abscisic acid (ABA) plays a key role in the control of these events. The viviparous-1 (vp1) mutant of maize has been characterized as an ABA-insensitive mutant in which various events during the seed maturation are blocked. The VP1 was cloned and found to encode a transcriptional factor (McCarty et al. 1989). Homologues of VP1 gene have been cloned from several species such as AB13 from Arabidopsis and OSVP1 from rice (Hattori et al. 1994).

In this report, we describe the expression pattern of OSVP1 in developing rice seeds using in situ hybridization and immunohistochemical techniques. Developing seeds of rice cv. Taichung 65 were fixed with 4% paraformaldehyde and

Research Notes 139

0.25% glutalaldehyde in 0.1% sodium phosphate buffer (pH 7.2) for 12h at 5°C. Then they were dehydrated with a graded ethanol series, substituted with xylene, and embedded in Paraplast Plus. Microtome sections (7 m m thick) were applied to slide glasses treated with Vectabond. Digoxigenin-labeled RNAs prepared from full length cDNAs of OSVP1 were used as probes. Detection of the hybridized probes was carried out with TSA™-Indirect (ISH) kit according to the manufacturer's instructions (NEN Life Science Products). TSA™-Indirect (ISH) kit was also used for the immunohistochemical detection of monoclonal antibody against OSVP1 according to the manufacturer's instructions.

In the embryos at early two days after pollination (2DAP), OSVP1 mRNA was not detected, but was already observed at late 2DAP (Fig. 1B). Examination of independent seeds revealed that OSVP1 mRNA became detectable in the embryos, ca. 60 (m m long, which corresponded to the mid globular stage. Until 4DAP, when the coleoptile started to dilferentiate, OSVP1 transcripts were distributed rather uniformly in the embryo (Fig. 1B-D). At 6DAP, however, localized signals were observed in the regions where the shoot and radicle were developing (Fig. IE). A clear tissue/organ dependent distribution of OSVP1 mRNA was observed when all the embryonic organs were established (Fig. 1F-H). Localized signals of OSVP1 mRNA relative to the other parts of embryo were detected in the vascular tissues, abaxial surface of coleoptile, leaves, shoot apex, radicle and root cap. In the radicle, tissue specificity was remarkable. Strong signals were recognized in the subcpidcrmal layer of cortex and central stele. Epidermal layers of the scutellum and epiblast also showed stronger signals of OSVP1 mRNA appeared to be decreased at the mature stage (20DAP, Fig. 1H) as compared to those at 10-15DAP (Fig. 1F and G). As well as in the embryo, OSVP1 mRNA was detected in the aleurone layer, although the onset of OSVP1 expression in the aleurone layer (8DAP) was much delayed than that in embryo.

Localization of OSVP1 protein was essentially the same as that of mRNA throughout the embryogenesis (Fig. 2A-F). OSVP1 protein was also detected in the aleurone layer. The protein was first detected at early 3DAP. Thereafter the OSVP1 protein followed the same expression pattern as the mRNA. Examination with higher magnification confirmed that the signals of OSVP1 protein were localized in the nuclei irrespective of developmental stages. This observation provides an additional piece of evidence that OSVP1 is a transcription factor.

In the present study, we have clearly shown that OSVP1 is expressed in developmentally and spatially regulated manner. The expression pattern of OSVP1 after 6DAP will correspond to the known function on seed maturation and dormancy, since the transcripts are mainly localized in mctabolically active tissues such as shoot, radicle, vascular tissues, scutellar epithelium and aleurone layer. The early (2-3DAP) and uniform expression in embryo, however, will not be explained by the maturation-related function of OSVP1 leading to a question whether OSVP1 has any role in the early phase of embryogenesis. None of the genes under the control of OSVP1 are known, to date, to be expressed at such early stages. In addition, any abnormality in maize vp1 mutants during early to mid phase of embryogenesis is not known. Thus OSVP1 may not play any particular role in such

early stages. To be functionally active, OSVP1 may require a co-factor or some modification such as phosphorylation/dephosphorylation, which are not set up until the later stages of embryogenesis. Another possibility is that OSVP1 plays a distinct role in early embryogenesis which is different from that in maturation process. In this case, other gene(s) may be present, which are functionally redundant to OSVP1.

McCarty, D.R., C.B. Carson, P.S. Stinard and D.S. Robertson, 1989. Molecular analysis of viviparous-1: an