DIVISION OF PLANT DEVELOPMENTAL GENETICS

Associate Professor: TSUKAYA, Hirokazu
Research Associate: HORIGUCHI, Gorou
Technical Staffs: KONDO, Makiko (- Nov. 30)
  TAKABE, Eriko
  YAMAGUCHI, Chinami
  SAKAI, Keiko
Postdoctoral Fellows: CHO, Kiuhyon
  ISHIKAWA, Naoko
  ALI, Ferjani
  YAMAGUCHI, Takahiro
  YANO, Satoshi
Graduate Students: KOZUKA, Toshiaki
  NARITA, Noriyuki
  MANO, Eriko
  FUJIKURA, Ushio
Secretaries: KOJIMA, Yoko
  KABEYA,Kazuko (- Mar. 30)

The leaf is the fundamental unit of the shoot system, which is composed with leaf and stem. Diversity of plant form is mostly attributable to variation of leaf and floral organs, which are modified leaves. Moreover, leaf shape is sensitive to environmental stimuli. So the leaf is the key organ for a full understanding of plant morphogenesis. However, the genetic control of development of these shapes had remained unclear. Recently, studies of leaf morphogenesis have been in a turning point, after our successful application of the techniques of developmental and molecular genetics to it, using model plants, Arabidopsis thaliana (L.) Heynh. (reviewed in Tsukaya, 2003).

Focusing on mechanisms that govern polarized growth of leaves in Arabidopsis thaliana we have identified genes for polar-dependent growth of leaf lamina: the

Fig. 1. Intracellular localization of ROT4::GFP in onion epidermal cells before (upper) and after (lower) osmosis.

ANGUSTIFOLIA (AN) gene regulates width of leaves and the ROTUNDIFOLIA3 (ROT3) gene regulates length of leaves. Both AN and ROT3 genes control leaf cell shape.

How have these genes evolved the function of leaf-shape control? The AN is a homolog of animal CtBP/BARS gene family which has varied roles in morphogenesis and organelle control. To understand the common role(s) of AN subfamily in plant kingdom, we analyzed a homolog of AN from Ipomoea nil, IAN, and showed that the IAN exhibits the same function with the AN on the control of leaf shape when introduced to Arabidopsis thaliana (Cho et al., in press). On the other hand, the ability of the IAN to control branching pattern of trichomes was less efficient than the AN. Further comparative analyses of the AN homologs from various plants are now on going.

In addition to the above factors, in this year, we added ROT4 gene to the list of the genes responsible for the polarity-dependent control of leaf shape (Narita et al., 2004). ROT4 is a member of novel peptide family (RTFL: RoT-Four-Like) which is specific to seed plants. Overexpression of the ROT4 results in stunted leaves with normal width, due to decrease of number of leaf cells specifically to the longitudinal direction. All RTFL peptide conserve RTFL domain and even truncated forms of ROT4 that have RTFL domain show the same effect on leaf shape with full-length ROT4, the RTFL family is thought to have common role in control of leaf cell proliferation to the leaf-length direction. Interestingly, the ROT4 peptide appears to localize on plasma membrane when fused with GFP marker (Fig. 1). The role of the RTFL in the control of leaf-cell proliferation is under investigation.

How cell proliferation and cell enlargement are coordinated in leaf morphogenesis? In a determinate organ, a leaf, number of leaf cells is not necessarily reflected on leaf shape or, in particular, leaf size. Genetic analyses of leaf development in Arabidopsis shows that a compensatory system(s) act in leaf morphogenesis and an increase of cell volume might be triggered by a decrease in cell number (Tsukaya, 2003). Thus, leaf size is, at least to some extent, uncoupled from the size and number of cells by the compensatory system(s). Recently, we have revealed that ANGUSTIFOLIA3 (AN3) gene iinvolved in maintenance/establishment of activity of cell proliferation in leaf primordia. AN3 encodes a co-activator, and is speculated to control cell cycling in leaf primordia (Horiguchi et al., submitted). Interestingly, the an3 shows clear “compensation”, namely, accelerated cell expansion in relation to decrease of number of leaf cells. By using various mutants with altered number and/or size of leaf cells, we are currently analyzing genetic system of the compensation.

On the other hand, we also focused on the effects of environmental factors on leaf morphogenesis. In darkness, expansion of leaf lamina is inhibited, while at the same time, petiole elongation is enhanced. This phenomenon is termed the shade-avoidance syndrome. We analyzed the nature of the shade-avoidance syndrome and found that phytochromes and cryptochromes specifically regulate the contrasting growth patterns of the leaf blade and petiole in shade (Kozuka et al., in press; Fig. 2). Differed from photomorphogenesis of hypocotyl, cell elongation was stimulated in the petiole in dark conditions without an increase in the ploidy level. By examining the effects of sucrose on the growth of the leaf blade and petiole, we revealed that growth promotional effects of sucrose are highly dependent on the light conditions.

Recently, we found that the ROT3 gene encodes a cytochrome P450 that catalyzes the conversion of typhasterol to castasterone, an activation step in the biosynthesis pathway of brassinosteroids (BRs) (Kim et al., in press). Differed from already known mutants of genes for biosynthesis of BRs, loss-of-function mutant of ROT3 has specific defect in the length of leaves, suggesting importance of fine tuning of levels of BRs on the polarized growth of leaves. Interestingly, CYP90D1, the most closely related cytochrome P450 to the ROT3/CYP90C1 enzyme, was suggested to catalyze the other conversion steps of BR biosynthesis (Kim et al., in press). Double mutant for the ROT3/CYP90C1 and for the CYP90D1 exhibited extreme dwarf that is observed for the other known mutants of genes for biosynthesis of BRs. Since the loss-of-function mutant of ROT3 has defect in response of petioles to dark, the ROT3 might have specific role(s) in the shade-avoidance syndrome. In relation to this topics, adaptive responses of arabidopsis leaves against gravity and other environmental factors were also analyzed and interaction between light signal and gravity-response in leaves were suggested (Tsukaya, in press).

Fig. 2. Shade-avoidance syndrome in Arabidopsis thaliana. Left, under white light; right, under dark. Bar: 5 mm.

On the other hand, we are also interested in environmental adaptation of leaf size in wild plants. In the course of field research of natural evolution of leaf shape/size, we have revealed some aspects of biodiversity of plant forms (Tsukaya, 2004; in press; Tsukaya et al., 2004; Yokoyama et al., in press). Interestingly, many plants are known to have evolved small-sized leaves in some islands, such as Yakushima, Kinkazan and Miyajima islands. Similar phenomenon is also known in plants inhabited in precincts of shrines and temples in Japan. Typical example of the evolution of small-sized leaves in these environments is known in Plantago asiatica. To understand what factors have accelerated the evolution of the small-sized leaves, we collected P. asiatica and P. major from a number of localities in and around Japan and established more than thirty imbred lines to analyze the genetic background of the evolution. To nurse study of this field, co-organized with Dr. Araki of Kyoto University, we held a domestic meeting on Plantago Research in Okazaki Conference Center, in July 3, 2004, inviting twelve researchers from various universities. So called "Evo/Devo" study of leaf morphogenesis is also one of our research project in NIBB.

Publication list:

Cho, K.-H., Shindo, T., Kim, G.-T., Nitasaka, E. and Tsukaya, H. (2005) Characterization of a member of the ANsubfamily, IAN, from Ipomoea nil. Plant Cell Physiol. (in press)

Kozuka, T., Horiguchi, G., Kim, G.-T., Ohgishi, M., Sakai, T. and Tsukaya, H. (2005) The different growth responses of the Arabidopsis thaliana leaf blade and the petiole during shade avoidance are regulated by photoreceptors and sugar. Plant Cell Physiol. (in press)

Narita, N. N., Moore, S., Horiguchi, G., Kubo, M., Demura, T., Fukuda, H., Goodrich J., and Tsukaya, H. (2004) Over-expression of a novel small peptide ROTUNDIFOLIA4 decreases of cell proliferation and alters leaf shape in Arabidopsis. Plant J. 38: 699-713.

Tsukaya, H. (2004) Gene flow between Impatiens radicans and I. javensis (Balsaminaceae) in Gunung Pangrango, central Java, Indonesia. Amer. J. Bot. 91: 2119-2123.

Tsukaya, H. (2005) Leaf shape: genetic controls and environmental factors. Int. J. Dev. Biol. (in press).

Tsukaya, H. (2005) Molecular variation of Spiranthes sinensis (Orchidaceae) in Japan, with special reference to systematic treatment of seasonally differentiated groups and a dwarf form, f. gracilis, from Yakushima Island. J. Plant Res. (in press).

Tsukaya, H., Iokawa, Y., Kondo, M. and Ohba, H. (2005); Large-scale general collection of DNA of wild plants in Mustang, Nepal. J. Plant Res. (in press).

Tsukaya, H., Okada, H. and Mohamed, M. (2004) A novel feature of structural variegation in leaves of tropical plant, Schismatoglottis calyptrata. J. Plant Res. 117: 477-480.

Yokoyama, J., Fukuda, T. and Tsukaya, H. (2005) Molecular identification of the mycorrhizal fungi of the epiparasitic plant Monotropastrum humile var. glaberrimum (Ericaceae). J. Plant Res. (in press)