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.
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.