K. KIKUCHI¹, K.T. YOSHIDA¹, M. UEKUCHI-TANAKA², M. MATSUOKA², Y. NAGATO¹ and H.-Y. HIRANO¹

1) Graduate School of Agricultural and Life Sciences, University of Tokyo. Tokyo. 113 Japan
2) BioScience Center. Nagoya University. Nagoya. 464-01 Japan

Plant development involves key regulatory genes encoding characteristic domains such as homeo and MADS domains. Genes with the NAC domain, which was newly identified in the No Apical Meristem (NAM) gene of Petunia (Souer et al. 1996) and the Cup-shaped cotyledon 2 (CUC2) gene of Arabidopsis (Aida et al. 1997), participate in the initiation and maintenance of the shoot apical meristem and in flower development. As the first step to elucidate the function of the NAC genes in monocotyledonous plants, we characterized rice cDNAs, which encoded protein with the NAC domain.

Database search detected many clones containing the NAC domain in randomly isolated cDNAs (expression sequence tags) from rice. After excluding redundant clones. we obtained eight independent clones from Rice Genome Project and designated them as OsNac1-OsNAC8 (O. sativa gene with the NAC domain). Nucleotide sequences of eight cDNA clones were determined. All clones contained the putative initiation codon (ATG) for protein synthesis, suggesting that cDNAs were almost full length. Deduced amino acid sequence indicated that the NAC domains were located at the N-terminal part in all OsNAC proteins as in NAM and CUC2 proteins. In contrast to high conservation in the N-terminal part, the amino acid sequences of C-terminal part were variable among OsNAC proteins. However, in some cases., such as those between OsNAC1 and OsNAC2 and between OsNAC5 and OsNAC6, some homologous regions were detected as some blocks of short sequence in their C-terminal part.

Fig. 1. Southern blot analysis with OsNAC6. Left panel: The 5' conserved region including the NAC domain of OsNAC6 was used as a probe. Right panel: The 3' region specific to OsNAC6 was used as a probe. Restriction enzymes used were as follows; 1, BamHI, 2, EcoRI, 3, HindIII.

Southern blot analysis was carried out using two kinds of probes: one consisting of the 5' conserved region corresponding to the NAC domain and the other the 3' region 5' specific to each clone. The use of latter probes showed one or two discrete band(s) (Fig. 1B), suggesting that each member of OsNAC genes existed as a single copy in rice genome. On the other hand, the smear pattern was found when the 5' conserved region was used as a probe (Fig. 1A). This result indicates that nucleotide sequences encoding the NAC domain are highly redundant and the OsNAC genes formed a multigene family in rice genome.

Phylogenetic tree was constructed by comparing the NAC domains from rice and other plants. The NAC proteins were divided into several subgroups. Rice NAC proteins most similar to NAM and CUC2 was OsNAC1 and OsNAC2. The homologous sequences in the C-terminal part as mentioned above were detected in these four proteins even among species, suggesting that this region may play a role specific to this subgroup. OsNAC5 and OsNAC6 were homologous to ATAF1 and ATAF2, which were isolated from Arabidopsis by their capability to activate a cauliflower mosaic virus 35S promoter. This indicates that proteins with the NAC domain have some roles for transcriptional control. Thus, these data provide the possibility that the OsNAC genes may be also involved in developmental regulation in rice, probably through gene regulation of other genes.

References

Aida, M., T. Ishida, H. Fukaki, H. Fujisawa and M. Tasaka, 1997. Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9: 841-857.

Souer, E., A. van Houwelingen, D. Kloos, J. Mol and R. Koes, 1996. The No Apical Meristem gene of petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell 85:159-170.

Vol. 14