Rice Genetics Newsletter, Vol. 18

Rgn18

8.	GYPSY EMBRYO gene controls embryo position by regulating integument development
	S.YAMAKI and Y. NAGATO Graduate School of Agricultural and Life Sciences, University of Tokyo, 113-8657 Japan

In grasses, the embryo lies in the lemma side base of the seed suggesting that its position in the seed is conserved phylogenetically. However, little is known about the regulation of embryo position. Here, we describe a mutant of rice exhibiting altered position of embryo in seed, and discuss how the embryo position is regulated developmentally.

We have identified a single recessive mutant, gypsy embryo (gem), from M2 plants of rice cv. Taichung 65 mutagenized with methyl-nitrosourea. In approximately 6% of mature gem seeds, embryos shifted to more apical position (Fig. 1A, F). The position was highly variable along the lemma side of the seed, from the normal basal position to the opposite end (top of seed).

To determine if the abnormal embryo position was derived from the ectopic onset of embryogenesis, we examined the early stage of seed development. In 5-day-old seeds after pollination, ectopically developing embryos were observed at a frequency comparable to that in mature seeds (Fig. 1B, G). Thus, the positional abnormality of mature embryos was caused by the ectopic onset of embryogenesis. Interestingly, some ectopic embryos differentiated shoot apical meristem opposite of the normal position, i. e. dorsal side (Fig. 1C, H). Since the relative position of organs was normal, it is suggested that the position of embryo affects the orientation of dorsoventral axis.

We examined the orientation of embryo sac. In the wild-type anatropous ovules, egg-antipodal cell axis is oriented vertically. However, ca. 10% of gem ovules showed embryo sacs with aberrant orientation (Fig. 1D, I). In these ovules, egg cell was positioned in the upper region, and antipodal cells in its opposite end. Accordingly, ectopic embryo formation in gem was resulted from the ill-orientation of embryo sac. Since the relative positions of egg apparatus, central cell, and antipodal cells were normal, we conclude that ectopic formation of egg cell in gem is resulted from the incomplete curvature of ovule, i.e. not anatropous but nearly orthotropous ovule was formed.

In the orthotropous ovule of gem, the apical region of inner integument did not develop well (Fig. 1E, J). On the other hand, apparently normal

anatropous ovules of gem had normal inner integument. The correlation between the degree of inner integument development and the degree of ovule curvature leads us to the idea that inner integument development contributes to the degree of ovule curvature, egg cell position and then embryo position.

The frequencies of ovules with underdeveloped inner integument and of orthotropous ovules were as low as ca. 10%. In contrast, most ovules showed abnormal outer integument. The outer integument frequently elongated into the style and sometimes passed through it to form cell mass on the ovary. In other cases, the boundary between inner and outer integuments was unclear, and the growth of both integuments was suppressed (Fig. 1J). Therefore, GEM gene is considered to regulate outer integument development.

In order to reveal the initial step at which gem mutant phenotype appeared, we observed the early stage of ovule development. The ovule primordia of gem were larger than those of the wild type (Fig. 2A, D), and at the later stage, gem showed the retarded growth of carpel relative to ovule growth (Fig. 2B, E).

The present study has revealed that the embryo position in seed depends on the degree of ovule curvature, which in turn is affected by the development of inner integument. The GEM gene is involved in the determination of embryo position by regulating ovule development.