Plant Cell Physiol. 46(1): 79���86 (2005) doi:10.1093/pcp/pci022, available online at www.pcp.oupjournals.org JSPP �� 2005 79 Rapid Paper Suppression of Tiller Bud Activity in Tillering Dwarf Mutants of Rice Shinji Ishikawa 1, 2, Masahiko Maekawa 3, Tomotsugu Arite 1, Kazumitsu Onishi 4, Itsuro Takamure 4 and Junko Kyozuka 1, 2, 5 1 Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657 Japan 2 CREST, Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012 Japan 3 Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama, 710-0046 Japan 4 Faculty of Agriculture, Hokkaido University, Sapporo, 060 Japan In this study, we analyzed five tillering dwarf mutants that exhibit reduction of plant stature and an increase in tiller numbers. We show that, in the mutants, axillary mer- istems are normally established but the suppression of tiller bud activity is weakened. The phenotypes of tillering dwarf mutants suggest that they play roles in the control of tiller bud dormancy to suppress bud activity. However, tillering dwarf mutants show the dependence of both node position and planting density on their growth, which implies that the functions of tillering dwarf genes are independent of the developmental and environmental control of bud activity. Map-based cloning of the D3 gene revealed that it encodes an F-box leucine-trich repeat (LRR) protein orthologous to Arabidopsis MAX2/ORE9. This indicates the conservation of mechanisms controlling axillary bud activity between monocots and eudicots. We suggest that tillering dwarf mutants are suitable for the study of bud activity control in rice and believe that future molecular and genetic studies using them may enable significant progress in understand- ing the control of tillering and shoot branching. Keywords: Axillary meristem ��� Bud activity ��� Bud dor- mancy ��� Rice ��� Tiller. Abbreviations: LLR, leucine-rich region RT���PCR, reverse tran- scriptase���polymerase cahin reaction SAM, shoot apical meristem. Introduction Shoot branching is ultimately important for the establish- ment of plant architecture (for reviews, see Schmitz and Theres 1999, Grbic 2001, Ward and Leyser 2004). The first step of shoot branching is the initiation of axillary meristems in the axils of leaves. The newly formed meristem starts to behave as a shoot apical meristem (SAM) and generates a few lateral leaves to form an axillary bud. Subsequently, the bud either remains active or goes dormant until outgrowth is triggered. Hence, shoot branching is regulated by two distinct steps, the establishment of axillary meristems and the outgrowth of axil- lary buds. The activity of axillary buds is controlled by multiple genetic, developmental and environmental signals (for reviews, see Shimizu-Sato and Mori 2001, Beveridge et al. 2003, Leyser 2003). The central mechanism, which is known as api- cal dominance, is the suppression of axillary bud growth by a growing apical meristem (Thimann and Skoog 1933). It is well known that the apical dominance is mediated by a network of hormonal signals. Apically-derived auxin inhibits bud activity and cytokinin promotes it. In addition to these well-established hormonal signals, recent studies with a series of bushy mutants in Arabidopsis, pea and petunia suggested that a novel graft- transmissible signal acts to inhibit bud growth (for reviews, see Napoli et al. 1999, Beveridge et al, 2003, Leyser 2003). The molecular analysis of three Arabidopsis mutants, max2 to max4, and pea RMS1, provided insights into understanding what the signaling molecule might be and how it works. MAX3 and MAX4/RMS1 encoding carotenoid cleavage dioxygenase and polyene dioxygenase, respectively, are predicted to func- tion in the production of a novel signaling molecule that might be derived from carotenoid (Sorefan et al. 2003, Booker et al. 2004). MAX2 encodes an F-box protein identical to previously reported ORE9 and is proposed to be required for the percep- tion of the signal (Woo et al. 2001, Stirnberg et al. 2002). Throughout rice development, three different types of branch shoots develop (Hoshikawa 1989). Axillary buds pro- duced during vegetative phase grow as tillers, while those pro- duced after transition to the reproductive phase grow to form panicle branches and spikelets. In most rice cultivars, vegeta- tive buds have the ability to go to dormancy, whereas axillary buds generated in the reproductive phase remain active and grow out immediately after the formation. As tillering is one of the major components determining rice yield, there is a long history of studies on tillering. How- ever, most were done from the agricultural point of view, and little attention has been paid to revealing the underlying mech- anisms controlling tiller bud activity. Recently, it was shown that FINE CULM 1 (FC1) is an ortholog of TEOSINTE 5 Corresponding author: E-mail, akyozuka@mail.ecc.u-tokyo.ac.jp Fax, +81-3-5841-5087.

Suppression of tiller bud activity in rice tillering dwarf mutants 80 BRANCHED1 (TB1) of maize (Takeda et al. 2003). In maize, TB1 acts as a repressor of axillary bud growth and an inducer of female inflorescence (Doebley et al. 1997, Hubbard et al. 2002). The dormancy of tiller buds was weakened in the rice fc1 loss-of-function mutants on the contrary, the ectopic expression of FC1 caused the suppression of tiller growth, indi- cating that FC1 functions in the repression of bud activity (Takeda et al. 2003). A large collection of rice mutants has accumulated in the course of breeding, and new ones have been established as resources for molecular and genetic studies, such as the tos17 mutant panel (Kinoshita and Takahashi 1991, Hirochika et al. 2004). Mutants that show altered patterns of tillering are included in these collections. In one group of tillering mutants, the outgrowth of tillers is repressed, leading to the reduction of tiller number. In another class, bud growth is not properly repressed, and the tiller number increases. As has been demon- strated in other plant species, such as Arabidopsis, petunia and pea, bushy mutants of rice are expected to be useful to study genetic and molecular mechanisms regulating bud activity because it is less likely that the defects are indirect conse- quences of other abnormalities affecting the vigor of buds and/ or plants. In this study, we analyzed five tillering dwarf mutants that show reduction of plant stature and increase in tiller numbers. Our analyses indicate that the establishment of axillary meris- tems is normal but subsequent suppression of tiller bud activ- ity is weakened in the tillering dwarf mutants. Map-based Table 1 Tillering dwarf mutants used in this study ND, not determined. Mutant line Gene Origin Chromosome No. back-crossed Id3 D3 bunketsuwaito 6 5 Id10 D10 kikeibanshinriki 1 9 Id14 D14 kamikawabunwai 3 10 Id17 D17 slender dwarf ND 8 Id27 D27 bunketsuto 11 9 Fig. 1 (A) Phenotype of tillering dwarf mutants 6 and 10 weeks after the germination. Because Shiokari is an early flowering cultivar, panicles have emerged 10 weeks after germi- nation. (B) The number of tillers in tillering dwarf mutants. (C) Culm length of tillering dwarf mutants.