51. A gene associated with temporal regulation of vegetative development in rice J.I. itoh1 a .hasegawa2 H. kitano3 and Y. nagato1

1) Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113 Japan

2) Aichi University of Education, Kariya, 448 Japan

3) Faculty of Agriculture. Nagoya University, Nagoya, 464 Japan

 Heterochronic mutations affecting the timing of developmental events may be of major significance in developmental and evolutionary aspects. In maize, several dominant heterochronic mutations that affect vegetative development and markedly alter shoot architecture have been identified (Poethig 1988). However, almost no recessive heterochronic mutations have been reported.

Here, we describe a novel heterochronic mutation of rice which causes a short plastochron and ectopic expression of vegetative programs in the reproductive phase. The phenotype of this recessive mutant is very similar to leafy head reported by Hu (1961). Unfortunately the leafy head is no longer available and we cannot conduct allelism test. Accordingly, we designate the present mutant plastochron 1 (pla1) due to its characteristics during vegetative phase.

The pla1 was identified among M; plants of rice (Oryza sativa L.) cv. Fukei 71 mutagenized with 200Gy of g -rays. The shape of mature pla1 embryo was identical to that of the wild type, and both embryos differentiated three leaves. At the early stage after germination, we could not distinguish pla1 seedlings from wild type ones. Accordingly, pla1 did not affect embryonic development. At two weeks after germination, seven leaves emerged in pla1 seedlings as compared five leaves in the wild type (Fig.1). Since three leaves already differentiated in embryos of both pla1 and wild type, four leaves were produced and emerged in pla1 within two weeks' after germination, but only two leaves emerged in the wild type. At any stage, the number of leaves emerging after germination in pla1 was about twice that in the wild type. Therefore, plastochron of pla1 in vegetative phase is estimated to be reduced to half of that in the wild type. However, the duration of vegetative phase from germination through the emergence of flag leaf was the same in both types. Thus, pla1 did not affect the timing of reproductive phase initia-

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Fig. 1. Seedlings of wild type (left) and pla1 (right) at two weeks after germination.

tion, although the number of phytomers in the vegetative phase was doubled.

Next, we observed the shoot apical meristem. The apical meristem was much larger in pla1 than that in wild type, although meristem shape was similar (Fig. 2). In an in situ hybridization experiment using rice Histone 4 gene as a S-phase specific probe, pla1 meristem showed more hybridization signals than the wild type (Fig. 2). The mean number of cells expressing Histone 4 per median longitudinal section was 3.8 in pla1, whereas only 1.9 in the wild type. These results show that the shoot apical meristem of pla1 has higher cell division activity than that of wild type, corresponding to the rapid production of leaf primordia.

Leaf size was also reduced in pla1. The maximum length and width of the leaf blade were nearly half of those of the wild type. However, the changing pattern of leaf size was conserved between wild type and pla1, suggesting that the onset of adult phase is not altered in pla1, although twice the number of leaves were needed to pass some devel-

Research Notes 143

Fig. 2. Shape and cell division frequency in shoot apex. A,B: Nomarski image of shoot apex in wild type (A) and pla1 (B), C, D: in situ hybridization pattern of Histone 4 in wild type (C) and pla1 (D). Hybridization signals were presented by black color.

Bar = 50 mm

 opmental phase in pla1.

The development of shoot after the differentiation of flag leaf was marked in pla1. Following the emergence of flag leaf, several vegetative shoots were produced instead of panicle (Fig. 3A). These shoots showed spiral phyllotaxy instead of the 1/2 alternate in normal tillers. In addition, the bract was enormously elongated at each shoot (Fig. 3B). However, the differentiation of primary rachis branch primordia with a 2/5 phyllotaxy seemed apparently normal at the early stage (Fig. 3C). The cross section of the later stage of these primordia showed that shoots were differentiated in a 2/5 phyllotaxy, each shoot being surrounded with a large bract (Fig. 3D). This 2/5 phyllotaxy was observed in the wild type plant only at the differentiation of primary rachis branches of panicle. In each of these ectopic shoots, normal leaves were produced in 1/2 phyllotaxy. These findings indicate that in pla1, primordia of primary rachis branches were converted into vegetative shoots. As shown in Fig. 3E, hairs were produced from the main axis, a characteristic of panicle during the differentiation of primordia of secondary rachis branches. Therefore, both reproductive and vegetative programs were simultaneously in operation during the reproductive phase of pla1.

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Fig. 3. Phenotypes of pla1 in reproductive phase. A: Ectopic shoots produced after flag leaf was differentiated. B: Side view of young panicle showing enlarged bract. C: Scanning electron microscopy of primary rachis branch primordia produced in 2/5 phyllotaxy. D: Cross section of young panicle showing ectopic shoots arranged in 2/5 phyllotaxy. E: Enlarged view of the central portion of young panicle showing hairs (arrowheads) characteristic to panicle at the stage of secondary rachis branch differentiation. F: Side view of panicle showing elongated bracts (arrows) but apparently normal flowers. The ectopic shoot repeated the onset of reproductive growth (differentiation of primary rachis branch primordia) and the conversion into shoots. In many pla1 plants, ectopic shoots eventually differentiated panicles in November when grown under constant temperature (Fig. 3F). These panicles were truncated, and bracts were elongated. Floral organs were apparently normal, although most of them were sterile.

Since the pla1 showed no abnormalities in embryo and in the timing of reproductive phase initiation, it is a heterochronic mutation prolonging the vegetative phase. The wild type gene PLA1 is considered to regulate the duration of vegetative phase through controlling the plastochron.

References

Hu, C.H., 1961. An X-ray induced panicle-degenerating mutant in rice. Japan. J. Breed. II: 19-23. (in Japanese with English summary) Poethig, R.S., 19S8. Heterochronic mutations affecting shoot development in maize. Genetics 119: 959-973.