, Volume 230, Issue 4, pp 649–658 | Cite as

Identification and characterization of HTD2: a novel gene negatively regulating tiller bud outgrowth in rice

  • Wenzhen LiuEmail author
  • Chao Wu
  • Yaping Fu
  • Guocheng Hu
  • Huamin Si
  • Li Zhu
  • Weijiang Luan
  • Zhengquan He
  • Zongxiu Sun
Original Article


Tiller number is highly regulated by controlling the formation of tiller bud and its subsequent outgrowth in response to endogenous and environmental signals. Here, we identified a rice mutant htd2 from one of the 15,000 transgenic rice lines, which is characterized by a high tillering and dwarf phenotype. Phenotypic analysis of the mutant showed that the mutation did not affect formation of tiller bud, but promoted the subsequent outgrowth of tiller bud. To isolate the htd2 gene, a map-based cloning strategy was employed and 17 new insertions-deletions (InDels) markers were developed. A high-resolution physical map of the chromosomal region around the htd2 gene was made using the F2 and F3 population. Finally, the gene was mapped in 12.8 kb region between marker HT41 and marker HT52 within the BAC clone OSJNBa0009J13. Cloning and sequencing of the target region from the mutant showed that the T-DNA insertion caused a 463 bp deletion between the promoter and first exon of an esterase/lipase/thioesterase family gene in the 12.8 kb region. Furthermore, transgenic rice with reduced expression level of the gene exhibited an enhanced tillering and dwarf phenotype. Accordingly, the esterase/lipase/thioesterase family gene (TIGR locus Os03g10620) was identified as the HTD2 gene. HTD2 transcripts were expressed mainly in leaf. Loss of function of HTD2 resulted in a significantly increased expression of HTD1, D10 and D3, which were involved in the strigolactone biosynthetic pathway. The results suggest that the HTD2 gene could negatively regulate tiller bud outgrowth by the strigolactone pathway.


HTD2 High tillering Dwarf Map-based cloning T-DNA Rice (Oryza sativa L.) 



High tillering and dwarf 2


Thermal asymmetric interlaced PCR


RNA interference





We thank Dr. Qifa Zhang (Huazhong Agricultural University, China) for providing us with the pSMR-J18R plasmid. This project was supported by the grants from the National Basic Research Priorities (973) Programmes of China (G19990116-1 and 2005CB120801), the National High Technology Research and Development Program of China (2006AA10Z1E8), National Natural Science Foundation of China (30623006), Zhejiang Natural Science Foundation (Y307070 and 2008C22077), and Hubei Natural Science Foundation for Distinguished Young Scholar (2008CDB096). We are grateful to Mrs. Honglan Yan (China National Rice Research Institute, China) for taking pictures for the article.


  1. Arite T, Iwata H, Ohshima K, Maekawa M, Nakajima M, Kojima M, Sakakibara H, Kyozuka J (2007) DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice. Plant J 51:1019–1029PubMedCrossRefGoogle Scholar
  2. Bennett T, Sieberer T, Willett B, Booker J, Luschnig C, Leyser O (2006) The Arabidopsis MAX pathway controls shoot branching by regulating auxin transport. Curr Biol 16:553–563PubMedCrossRefGoogle Scholar
  3. Beveridge CA (2000) Long-distance signalling and a mutational analysis of branching in pea. Plant Growth Regul 32:193–203CrossRefGoogle Scholar
  4. Beveridge CA (2006) Axillary bud outgrowth: sending a message. Curr Opin Plant Biol 9:35–40PubMedCrossRefGoogle Scholar
  5. Beveridge CA, Symons GM, Murfet IC, Ross JJ, Rameau C (1997) The rms1 mutant of pea has elevated indole-3-acetic acid levels and reduced root-sap zeatin riboside content but increased branching controlled by graft-transmissible signals. Plant Physiol 115:1251–1258Google Scholar
  6. Booker J, Auldridge M, Wills S, McCarty D, Klee H, Leyser O (2004) MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule. Curr Biol 14:1232–1238PubMedCrossRefGoogle Scholar
  7. Booker J, Sieberer T, Wright W, Williamson L, Willett B, Stirnberg P, Turnbull C, Srinivasan M, Goddard P, Leyser O (2005) MAX1 encodes a cytochrome P450 family member that acts downstream of MAX3/4 to produce a carotenoid-derived branch-inhibiting hormone. Dev Cell 8:443–449PubMedCrossRefGoogle Scholar
  8. Brody MS, Vijay K, Price CW (2001) Catalytic function of an alpha/beta hydrolase is required for energy stress activation of the sigma(B) transcription factor in Bacillus subtilis. J Bacteriol 183:6422–6428PubMedCrossRefGoogle Scholar
  9. Cheng Y, Dai X, Zhao Y (2006) Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Dev 20:1790–1799PubMedCrossRefGoogle Scholar
  10. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016PubMedCrossRefGoogle Scholar
  11. Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pages V, Dun EA, Pillot JP, Letisse F, Matusova R, Danoun S, Portais JC, Bouwmeester H, Becard G, Beveridge CA, Rameau C, Rochange SF (2008) Strigolactone inhibition of shoot branching. Nature 455:189–194PubMedCrossRefGoogle Scholar
  12. Ishikawa S, Maekawa M, Arite T, Onishi K, Takamure I, Kyozuka J (2005) Suppression of tiller bud activity in tillering dwarf mutants of rice. Plant Cell Physiol 46:79–86PubMedCrossRefGoogle Scholar
  13. Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52:761–773PubMedCrossRefGoogle Scholar
  14. Li X, Qian Q, Fu Z, Wang Y, Xiong G, Zeng D, Wang X, Liu X, Teng S, Hiroshi F, Yuan M, Luo D, Han B, Li J (2003) Control of tillering in rice. Nature 422:618–621PubMedCrossRefGoogle Scholar
  15. Liu Y, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463PubMedCrossRefGoogle Scholar
  16. Liu W, Fu Y, Hu G, Si H, Zhu L, Wu C, Sun Z (2007) Identification and fine mapping of a thermo-sensitive chlorophyll-deficient mutant in rice (Oryza sativa L.). Planta 226:785–795PubMedCrossRefGoogle Scholar
  17. Ljung K, Bhalerao RP, Sandberg G (2001) Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J 28:465–474PubMedCrossRefGoogle Scholar
  18. McCouch SR, Teytelman L, Xu YB, Lobos KB, Clare K, Walton M, Fu B, Maghirang R, Li Z, Xing Y, Zhang Q, Kono I, Yano M, Fjellstrom R, DeClerck G, Schneider D, Cartinhour S, Ware D, Stein L (2002) Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.). DNA Res 9:199–207PubMedCrossRefGoogle Scholar
  19. McSteen P, Leyser O (2005) Shoot branching. Annu Rev Plant Biol 56:353–374PubMedCrossRefGoogle Scholar
  20. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325PubMedCrossRefGoogle Scholar
  21. Napoli C (1996) Highly branched phenotype of the petunia dad1–1 mutant is reversed by grafting. Plant Physiol 111:27–37PubMedGoogle Scholar
  22. Napoli CA, Ruehle J (1996) New mutations affecting meristem growth and potential in Petunia hybrida Vilm. J Hered 87:371–377Google Scholar
  23. Ongaro V, Leyser O (2008) Hormonal control of shoot branching. J Exp Bot 59:67–74PubMedCrossRefGoogle Scholar
  24. Rameau C, Murfet IC, Laucou V, Floyd RS, Morris SE, Beveridge CA (2002) Pea rms6 mutants exhibit increased basal branching. Physiol Plant 115:458–467PubMedCrossRefGoogle Scholar
  25. Ryu CH, You JH, Kang HG, Hur J, Kim YH, Han MJ, An K, Chung BC, Lee CH, An G (2004) Generation of T-DNA tagging lines with a bidirectional gene trap vector and the establishment of an insertion-site database. Plant Mol Biol 54:489–502PubMedCrossRefGoogle Scholar
  26. Schwartz SH, Qin X, Loewen MC (2004) The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching. J Biol Chem 279:46940–46945PubMedCrossRefGoogle Scholar
  27. Simons JL, Napoli CA, Janssen BJ, Plummer KM, Snowden KC (2007) Analysis of the DECREASED APICAL DOMINANCE genes of petunia in the control of axillary branching. Plant Physiol 143:697–706PubMedCrossRefGoogle Scholar
  28. Sorefan K, Booker J, Haurogne K, Goussot M, Bainbridge K, Foo E, Chatfield S, Ward S, Beveridge C, Rameau C, Leyser O (2003) MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea. Genes Dev 17:1469–1474PubMedCrossRefGoogle Scholar
  29. Stirnberg P, Chatfield SP, Leyser HM (1999) AXR1 acts after lateral bud formation to inhibit lateral bud growth in Arabidopsis. Plant Physiol 121:839–847PubMedCrossRefGoogle Scholar
  30. Stirnberg P, van De Sande K, Leyser HM (2002) MAX1 and MAX2 control shoot lateral branching in Arabidopsis. Development 129:1131–1141PubMedGoogle Scholar
  31. Stirnberg P, Furner IJ, Ottoline Leyser HM (2007) MAX2 participates in an SCF complex which acts locally at the node to suppress shoot branching. Plant J 50:80–94PubMedCrossRefGoogle Scholar
  32. Takeda T, Suwa Y, Suzuki M, Kitano H, Ueguchi-Tanaka M, Ashikari M, Matsuoka M, Ueguchi C (2003) The OsTB1 gene negatively regulates lateral branching in rice. Plant J 33:513–520PubMedCrossRefGoogle Scholar
  33. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  34. Tanaka M, Takei K, Kojima M, Sakakibara H, Mori H (2006) Auxin controls local cytokinin biosynthesis in the nodal stem in apical dominance. Plant J 45:1028–1036PubMedCrossRefGoogle Scholar
  35. Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S (2001) Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length, variation, transposon associations, and genetic marker potential. Genome Res 11:1441–1452PubMedCrossRefGoogle Scholar
  36. Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-Kamiya N, Magome H, Kamiya Y, Shirasu K, Yoneyama K, Kyozuka J, Yamaguchi S (2008) Inhibition of shoot branching by new terpenoid plant hormones. Nature 455:195–200PubMedCrossRefGoogle Scholar
  37. Vain P, Thole V (2009) Gene insertion patterns and sites. Methods Mol Biol 478:203–226PubMedCrossRefGoogle Scholar
  38. Wang MB, Waterhouse PM (1997) A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance genes. Plant Mol Biol Rep 15:209–215CrossRefGoogle Scholar
  39. Ward SP, Leyser O (2004) Shoot branching. Curr Opin Plant Biol 7:73–78PubMedCrossRefGoogle Scholar
  40. Xu M, Zhu L, Shou H, Wu P (2005) A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. Plant Cell Physiol 46:1674–1681PubMedCrossRefGoogle Scholar
  41. Zhu ZG, Xiao H, Fu YP, Hu GC, Yu YH, Si HM, Zhang JL, Sun ZX (2001) Construction of transgenic rice populations by inserting the maize transponson Ac/Ds and genetic analysis for several mutants. Chin J Biotech 17:288–292 (in Chinese with English abstract)Google Scholar
  42. Zou J, Zhang S, Zhang W, Li G, Chen Z, Zhai W, Zhao X, Pan X, Xie Q, Zhu L (2006) The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds. Plant J 48:687–698PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Wenzhen Liu
    • 1
    Email author
  • Chao Wu
    • 1
  • Yaping Fu
    • 1
  • Guocheng Hu
    • 1
  • Huamin Si
    • 1
  • Li Zhu
    • 1
  • Weijiang Luan
    • 1
  • Zhengquan He
    • 1
    • 2
  • Zongxiu Sun
    • 1
  1. 1.State Key Laboratory of Rice BiologyChina National Rice Research InstituteHangzhouChina
  2. 2.Biotechnology Research CenterChina Three Gorges UniversityYichangChina

Personalised recommendations