In plants, the aboveground parts are produced due to the continuous activity of shoot apical meristem (SAM). Thus, the elucidation of mechanisms required in the establishment and maintenance of SAM is essential for understanding the whole plant developmental processes.

In previous study (Satoh et al. 1999), we have identified four SHOOTLESS genes namely SHL1, SHL2, SHL3 and SHL4, whose loss-of-function mutations cause a complete loss of SAM. Thus, the SHL genes are indispensable for the initiation of SAM. In shl1, shl2 and shl4 embryos, coleoptile and epiblast are also absent, but radicle and scutellum are normally formed. in situ hybridization analysis using a rice homeobox gene, OSH1, as a probe indicates that SHL1 and SHL2 function upstream of OSH1, whereas SHL3 functions downstream or independently of OSH1. Although the SHL genes play an important role in SAM initiation, their specific function in the SAM after initiation is not clear. For analyzing the role of SHL genes in the SAM, we need to identify weak allelic mutants of SHL genes in which SAM is more or less produced. Therefore the objective of this report is to characterize a weak allele of SHL2.

Aiming to obtain weak alleles of SHL genes, we have identified several mutants that were lethal at the seedling stage from the M2 population of rice cv. Taichung 65 mutagenized with MNU. One of the mutants was found to be an allele of SHL2 locus, and designated as shl2-6.

Previously identified five alleles (shl2-1 - shl2-5) showed the same embryo phenotypes, lacking SAM, coleoptile and epiblast. On the other hand, shl2-6 formed SAM in the embryo 5-6 days after pollination, and finally produced one or two leaves and epiblast without coleoptile (Fig. 1). However, in the mature shl2-6 embryo, the SAM was not detected, suggesting that it was consumed in the course of embryo development (Fig. 1). Interestingly, epiblast was produced in shl2-6 but not in other strong alleles, although

coleoptile was deleted in all the alleles. This indicates that the coleoptile and epiblast differ in the degree of dependence on SAM. The coleoptile may require complete activity of SAM.

Upon germination, the seedlings of shl2-6 mutant withered after the emergence of one to three narrow and small leaves. The SAM was not present in the seedlings. Accordingly, it is considered that the shl2-6 does initiate SAM but fails to maintain it. Thus, SHL2 gene functions in both the initiation and maintenance of SAM. To investigate the general function of SHL2 in shoot development, we regenerated adventitious shoots from scutellum-derived calli of shl2-6. The adventitious shoots were more vigorous than the seedlings, and produced about 15 malformed leaves before they died. The regenerants produced narrow and small leaves with irregular phyllotaxy. This indicates that SHL2 is also associated with the regulation of leaf initiation pattern.

The above results suggest that in shl2-6, the SAM is differently organized from that of the wild type. Then we examined the expression pattern of OSH1 that is expressed in indeterminate cells but down-regulated in the leaf founder cells of SAM. In the strong

alleles (shl2-1 - shl2-5) lacking SAM, the expression domain of OSH1 was extremely reduced to a few cells (Fig. 2). In shl2-6, the expression domain 4-6 days after pollination was larger than that in the strong alleles, but much narrower than that in the wild type (Fig. 2). The expression of OSH1 was gradually reduced and became undetectable at the first- or second-leaf-primordium stage, when the wild-type embryo retained the strong expression, indicating that the cells in SAM were consumed up by leaf primordia. Thus, the SAM of shl2-6 is composed of a large number of cells competent for developing the leaf primordia and a small number of indeterminate cells.

In conclusion, SHL2 gene is indispensable for the initiation and maintenance of SAM. Moreover, SHL2 functions to maintain the proper ratio of indeterminate versus determinate cells, which is necessary for the regular pattern of leaf initiation and leaf morphogenesis.

Reference

Satoh, N., S.-K. Hong, A. Nishimura, M. Matsuoka, H. Kitano and Y. Nagato, 1999. Initiation of shoot apical meristem in rice: characterization of four SHOOTLESS genes. Development 126: 3629-3636.