- Email: [email protected]
Identification and functional analysis of the MOC1 interacting protein 1
JOURNAL OF
GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 6977
www.jgenetgenomics.org
Identification and functional analysis of the MOC1 interacting protein 1 Fengli Sun a, Weiping Zhang a, Guosheng Xiong a, Meixian Yan b, Qian Qian b, Jiayang Li a, Yonghong Wang a, * a
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China b State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China Received for publication 12 September 2009; revised 2 December 2009; accepted 6 December 2009
Abstract Rice tillering is one of the most important agronomic traits that determine grain yields. Our previous study has demonstrated that the MONOCULM1 (MOC1) gene is a key component that controls the formation of rice tiller buds. To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering, we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins (MIPs). Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo. The overexpression of MIP1 resulted in enhanced tillering and reduced plant height. In-depth characterization of the context of MIP1 and MOC1 would further our understanding of molecular regulatory mechanisms of rice tillering. Keywords: rice; MOC1; MIP1; tillering; protein-protein interaction
Introduction Tillering or branching is one of the most important agronomic traits that determine plant architecture and ultimately the grain yields. Tillering generally comprises two distinct steps: the initiation of axillary buds and their outgrowth. Tremendous efforts have been made to elucidate the molecular mechanisms underlying the genetic control of tillering in grass crops and significant progresses have been achieved over the last decade (Wang and Li, 2008). MOC1 was the first characterized gene that controls the initiation and outgrowth of rice tiller buds (Li et al., 2003). MOC1 belongs to the plant-specific GRAS transcription factor family and the orthologs of MOC1 have also been identified in tomato and Arabidopsis, which were sug* Corresponding author. Tel: +86-10-6480 6008; Fax: +86-10-6488 3428. E-mail address: [email protected] DOI: 10.1016/S1673-8527(09)60026-6
gested to be essential genes controlling shoot branching (Schumacher et al., 1999; Greb et al., 2003). The loss-of-function of MOC1 leads to the defect in the tiller bud initiation. Further analysis on expression patterns of the SAM-determined marker gene OSH1 suggested that MOC1 may participate in the very early step of axillary meristem initiation (Sato et al., 1996; Li et al., 2003). The LAX PANICLE1 (LAX1) is another essential gene controlling rice branching at both vegetative and reproductive developmental stages (Komatsu et al., 2003). The latest study suggested that LAX1 may affect later stages after MOC1 in the initiation of tiller buds (Oikawa and Kyozuka, 2009). However, the context of LAX1 and MOC1 has not been explained. After the initiation of axillary meristems of rice plants, OsTB1, an ortholog of TB1 in maize, has been proven to be responsible for the subsequent outgrowth of axillary buds (Doebley et al., 1995; Hubbard et al., 2002; Takeda et
70
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
al., 2003). Moreover, studies on a serials of more branching mutants in diverse species have revealed a conserved carotenoid-derived MAX/RMS/D (more axillary branching) pathway that inhibits the outgrowth of axillary buds (Wang and Li, 2008). Recently, two groups have independently reported that the MAX/RMS/D pathway is involved in the production and signaling of strigolactones, which are a group of terpenoid lactones that have been found in root exudates of diverse plant species and synthesized from carotenoids (Cook et al., 1972; Humphrey and Beale, 2006; Gomez-Roldan et al., 2008; Lopez-Raez et al., 2008; Umehara et al., 2008). In rice, five genes have been identified in the MAX pathway, in which D10, HTD1/D17, D27 are suggested to be responsible for the biosynthesis of strigolactones, and D14 and D3 for the perception of strigolactones-related signals (Zou et al., 2006; Arite et al., 2007, 2009; Yan et al., 2007; Lin et al., 2009). Although several important regulators have been identified in controlling rice tillering, our understanding on the tillering regulatory mechanism is still fragmental due to the lack of further studies on their actions. We therefore try to dissect the MOC1-medieated rice tillering regulation by identifying MOC1 interacting proteins (MIPs) through yeast-two-hybrid screening. In this paper, we showed that the MIP1 can interact with MOC1 and promote rice tillering.
Materials and methods Yeast-two-hybrid screening The full length cDNA of MOC1 were used as a bait to screen cDNA library for MOC1 interacting proteins by PROQUESTTM Two-Hybrid System (Invitrogen, USA). Interactions were tested on SD medium minus Leu, Trp, and His, and containing 30 mmol/L 3-Amino-1, 2, 4Triazole (3AT), using the yeast strain MaV203 according to the manufacturer’s manual.
Co-immunoprecipitation in vitro MOC1 was subcloned into the Nde I-EcoR I digested pGBKT7 vector and MIP1 was subcloned into the Sma I-Spe I digested pGADT7. Then c-MYC tagged MOC1 and HA tagged MIP1 were translated using the TNT T7 Coupled Reticulocyte Lysate Kit (Promega, USA) with
35S-labeled methionine, according to the manufacturer’s instructions. Co-immunoprecipitation between MOC1 and MIP1 was conducted using MATCHMAKER Co-IP Kit (Clontech, USA) according to manufacturer’s manual. The primer sequences used are listed in Supplemental Table 1.
Overexpression of MIP1 Full-length of MIP1 cDNA was subcloned into the BamH I-Spe I digested pTCK303 vector to generate the plasmid MIP1OE, which was subsequently introduced into Agrobacterium tumefaciens stain EHA105 by electroporation and then into Nipponbare as previous report (Hiei et al., 1994). The phenotypes of the transgenic plants were examined in the T2 progeny.
RT-PCR Total RNA was prepared by a TRIzol Kit according to the user’s manual (Cat. No. 15596-026, Invitrogen). One microgram of total RNA was treated with DNase I (Ambion) and used for cDNA synthesis using the Superscript RT Kit (Invitrogen). The primer sequences used for RT-PCR are listed in Supplemental Table 1.
DNA and RNA gel blotting analysis Rice genomic DNA (20 ȝg) was completely digested with indicated restriction endonucleases respectively and separated in a 0.8% agarose gel. After electrophoresis, DNA was transferred onto a nylon membrane (Hybrid N+; Amersham, USA), and hybridization was performed as previously described (Mou et al., 2000). A 1,023 bp fragment of the MIP1 gene amplified from rice cDNA was labeled with 32P-dCTP (Amersham) for hybridization. Total RNA was isolated with TRIzol reagent (Invitrogen) and the RNA gel blot analysis was performed as previously described (Hu et al., 2000). RNA (20 μg per lane) was separated in a 0.8% agarose gel containing 10% (v/v) formaldehyde, then transferred onto a Hybond N+ membrane (Amersham) and probed with the PCR-amplified DNA fragments with a specific primer pair of MIP1 (Supplemental Table1)
Subcellular localization of MIP1 To generate CaMV35S-GFP, an Xba I-Apa I fragment
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
containing GFP without stop codon was amplified by primers GFPF and GFPR using SGFP (S65T) as template. The Xba I-Apa I sites containing GFP fragment was then subcloned into the Xba I-Apa I digested vector pSPYNE (R) 173 (Waadt et al., 2008). A BamH I-Xho I fragment containing the coding region of MIP1 amplified by the primers of MIP1F and MIP1R was subcloned into the BamH I and Xho I sites of CaMV35S-GFP to generate CaMV35S:GFP-MIP1. The plasmids CaMV35S- GFP and CaMV35S:GFP-MIP1 were introduced into rice leaf protoplasts as described (Bart et al., 2006). After overnight incubation in the dark, the GFP signals were observed under a confocal microscope (FluoView 1000, Olympus, Japan) at an excitation wavelength of 488 nm.
BiFC assay in tobacco leaves BiFC assays were performed as described previously (Waadt et al., 2008). Briefly, MOC1 was subcloned into the BamH I-Kpn I digested binary BiFC vector SCYNE (R), which was fused with N-terminal fragment of CFP. MIP1 was subcloned into the BamH I-Xho I digested SCYCE (R) vector, which was fused with the C-terminal of CFP. These constructs were transformed into A. tumefaciens strain EHA105 and co-expressed transiently in Nicotiana benthamiana leaves (Voinnet et al., 2003). Equal volume of transformed EHA105 culture (OD600 = 1.0) was mixed before infiltration. The CFP signals were observed after 40–48 h infiltration under a confocal microscope (FluoView 1000, Olympus) at an excitation wavelength
71
of 405 nm.
Results Identification and confirmation of MIP1 Our previous study has identified the rice MOC1 gene, which encodes a putative GRAS family nuclear protein. Functional analysis revealed that MOC1 controls not only the formation of rice tiller buds but also the outgrowth of tillers (Li et al., 2003). To investigate the molecular events involved in the MOC1-mediated rice tillering, we screened for MOC1 interacting proteins (MIPs) using the yeast two-hybrid system with a rice cDNA library. The MIP1 protein is one of 15 candidate proteins that exhibit interactions with MOC1. To verify the interaction between MOC1 and MIP1 in vitro, we performed an X-Gal assay after co-transformation of MOC1-BD and MIP1-AD into yeast cells. The results showed that MOC1 could interact with MIP1 in yeast, and their interaction strength is comparable to the yeast control strain B (Fig. 1A), indicating a weak interaction strength between MOC1 and MIP1. We therefore further confirmed their interaction using 35S labeled Methionine in a Rabbit Reticulocyte Lysate system, in which MOC1 fused with the c-MYC tag and MIP1 with the HA tag was translated in vitro. The Co-IP result showed that MOC1 could be precipitated by Į-HA antibody and MIP1 by Į-c-Myc (Fig. 1B), indicating that MOC1 did interact with MIP1 in vitro.
Fig. 1. Confirmation of the interaction between MOC1 and MIP1 in vitro. A: confirmation of the interaction between MOC1 and MIP1 by the X-Gal essay in yeast. Controls A–E: yeast control strains, indicating different levels of interaction strength bewteen two proteins. Control A, pPC97 and pPC86; control B, pPC97-RB and pPC86-E2F1; control C, pPC97-CYH2 -dDP and pPC86-dE2F; control D, pPC97-Fos and pPC86-Jun; control E, pCL1 (encoding full length GAL4) and pPC86. B: confirmation of the interaction between MOC1 and MIP1 by Co-IP. Lane 1, MOC1 + MIP1; lane 2, MOC1 + MIP1+ HA-Tag antibody; lane 3, MOC1 + c-Myc antibody; lane 4, MIP1 + HA-Tag antibody; lane 5, MOC1 + HA-Tag antibody; lane 6, MIP1 + c-Myc antibody.
72
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
Characterization of MIP1
Subcellular localization of MIP1
The Blast analysis revealed that MIP1 is a member of the Bre1 family, which include Sc_Bre1 (Yeast), Ce_Bre1 (Caenorhabditis elegans), Dm_Bre1 (Drosophila melanogaster), Hu_Bre1A and Hu_Bre1B (Human), At_HUB1and At_HUB2 (Arabidopsis thaliana), Zm_Bre1 (Zea mays), OsBre1A and OsBre1B (Oryza sativa). The Bre1 family proteins contain a C3HC4 RING finger domain, which is also included in MIP1. In addition, MIP1 contains three conserved sumoylation consensus motifs (Fig. 2). Amino acid sequence alignment showed that MIP1 shared highest similarity with AtHUB1. Sequence comparison between genomic and complementary DNAs revealed that MIP1 is composed of 19 exons and encodes an 884 amino acid polypeptide. MIP1 is a single copy gene in the rice genome (Fig. 3A) and is a ubiquitously expressed gene at a very low level (Fig. 3B).
The MIP1 protein was predicted to contain a putative nuclear localization signal (Fig. 2). To determine its subcellular localization, we performed a transient expression experiment of MIP1 in rice leaf protoplasts. The N-terminal of MIP1 was fused with GFP under the control of cauliflower mosaic virus (CaMV) 35S promoter and the construct was transferred into rice leaf protoplasts by the PEG-mediated method. In contrast to the control that was ubiquitous in protoplast cells (Fig. 4A), the GFP-MIP1 fusion protein was predominantly localized in small discrete foci in the nucleus (Fig. 4B) and the GFP-MIP1 and RFP-MOC1 fusion proteins were co-localized in the nucleus (Fig. 4C). However, the GFP-MIP1 protein was diffusely localized in the nucleus in the moc1 protoplast cells (Fig. 4D). These results demonstrate that MOC1 is required for the normal localization of MIP1 in the cell.
Fig. 2. Alignment of deduced amino acid sequence of MIP1 with its homologs in Arabidopsis. The underlined letters refer to the putative NLS domain, the triangle-labeled letters to sumoylation consensus motifs, and asterisks to the C3HC4 RING finger domain.
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
73
Fig. 3. DNA and RNA gel blot analysis. A: DNA gel blot analysis indicated that MIP1 is a single copy gene in the rice genome. B: the MIP1 expression pattern revealed by RNA gel blot analysis. L, mature leaves ; R, seedling roots; S, stems after heading ; P, young panicles.
Fig. 4. Subcellular localization of GFP-MIP1 fusion proteins in rice protoplasts. A: subcellular localization of 35S:GFP. B: subcellular localization of 35S:GFP-MIP1. C: co-localization of 35S:GFP-MIP1 and 35S:RFP-MOC1 in rice protoplasts. D: subcellular localization of 35S:GFP-MIP1 in moc1 protoplasts. Left panels: dark filed (GFP); Middle panels: bright field in (A), (B) and (D), dark field (RFP) in (C). Right panels: merged images of left and middle panels. Bars = 5 ȝm.
74
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
MIP1 may interact with MOC1 in vivo To verify whether MOC1 could interact with MIP1 in vivo, we performed bimolecular fluorescence complementation (BiFC) assays in Nicotiana leaf epidermal cells. In BiFCs, MOC1 and MIP1 are fused to the N- and C-terminal regions of the CFP, respectively. A physical association between bait and prey brings the N- and C-terminal fragments of the split CFP together, and allows functional reassembly of the fluorescent protein (Waadt et al., 2008). Our results showed that when YNE-MOC1 and YCE-MIP1were co-expressed, positive CFP signals were detected mainly in the nuclei (speckles). No CFP fluorescence signal was detected in the negative control MOC1-YNE and empty vector-YCE (Fig. 5). These observations confirmed that MOC1 and MIP1 are able to physically interact in vivo.
Overexpression of MIP1 leads to an increase in tiller number To understand the context of MIP1 and MOC1 in controlling rice tillering, the full length cDNA of MIP1 under the control of the maize ubiquitin promoter was transferred into a rice variety Nipponbare. The transgenic plants having an increased expression level of MIP1 displayed a significantly enhanced rice tillering phenotype at both vegetative and reproductive stages (Fig. 6, C–E). In addition, the transgenic plants also exhibited semi-dwarf, delayed head-
ing, and sterility. The phenotypes of MIP1-overexpressing transgenic plants are quite similar to those of MOC1overexpressing plants (Li et al., 2003).
Discussion Tillering in rice is not only an important agronomic trait for grain yield, but also an ideal system for studying branching in higher plants, especially for monocotyledonous species. In this study, we identified a MOC1 interacting protein, MIP1, via the yeast two-hybrid system. The interaction between MIP1 and MOC1 was confirmed by both Co-IP in vitro and BiFC assays in vivo (Figs. 1 and 5). MIP1 belongs to the Bre1 family which play roles in regulation of monoubiquitination of H2B (Fleury et al., 2007; Liu et al., 2007). The overexpression of MIP1 resulted in an increase in tiller number and a decrease in plant height, suggesting an essential role of MIP1 in determining the plant architecture of rice. In yeast, Bre1 regulates cell morphology through H2B monoubiquitination (Hwang et al., 2003). In Drosophila, Bre1 affects the development of wings and legs through the Notch signaling pathway (Bray et al., 2005). In Arabidopsis, HUB1 was shown to act as an E3 ligase involved in the H2B monoubiquitination (Fleury et al., 2007; Liu et al., 2007). MIP1 shared the highest identity with HUB1 (Fig. 2). As one kind of chromatin modification, monoubiquitination of H2B is crucial for gene expression
Fig. 5. Confirmation of the interaction between MOC1 and MIP1 by BiFC. A–C: BiFC experiments between MIP1 and MOC1. MOC1 fused with N-terminal fragment of CFP and MIP1 fused with C-terminal fragment of CFP were co-infiltrated into tobacco leaves. D–F: BiFC of MOC1 fused with N-terminal fragment of CFP and C-terminal CFP vector co-infiltrated into tobacco leaves as the control. Bars = 20ҏ ȝm.
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
75
Fig. 6. Phenotypes of MIP1-overexpressing transgenetic plants. A: examination of MIP1 transcripts by RT-PCR. Actin transcripts were used as controls. B: the semi-quantitive RT-PCR analysis of MIP1 transcripts in the wild-type and MIP1-overexpressing plants. Data are means ± SE (n = 3). C: the phenotype of the wild type (left) and MIP1-overexpressing plants (right) at the tillering stage. D: the phenotype of the wild type (left) and MIP1-overexpressing plants (right) at the matured stage. Bars = 10 cm. E: comparison of tiller numbers between the wild type and MIP1-overexpressing plants. Data are means ±SE (n = 15).
(Eckardt, 2007). HUB1 has also been suggested to be a key regulator that controls seed dormancy, cell-cycle, flowering time, and plant defense (Liu et al., 2007; Fleury et al., 2007; Cao et al., 2008; Dhawan et al., 2009; Gu et al., 2009; Xu et al., 2009). MIP1 can interact with MOC1, however, the relationship between MOC1 and MIP1 remains to be elucidated. As putative transcriptional regulators, GRAS family proteins play important roles in regulating plant development and growth by affecting the expression of their target genes (Bolle, 2004). However, the expression level of MIP1 was unchanged in the moc1 mutant (data not shown), implicating that MOC1 may regulate MIP1 at the posttranscriptional level or through an unknown mechanism. It appears that the interaction between MOC1 and MIP1 may
regulate the subcellular localization or modification of MIP1, thus affects the monoubiquitination of H2B and then regulates the expression of downstream genes of H2B and promotes tillering in rice. The transgenic plants overexpressing MIP1 displayed an increased tiller number and reduced plant height (Fig. 6), which is similar to that of MOC1-overexpressing plant (Li et al., 2003). MIP1 was nuclear localized and distributed mainly in some speckles. Since MOC1 interacts with MIP1, the enrichment of MOC1 at speckles in the nucleus may involve in the regulation of downstream genes. When MIP1 is overexpressed in plants, the subcellular localization of MOC1 may redistribute in the nucleus and trigger the downstream events to control rice tillering.
76
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
Acknowledgements This work was supported by the grant from the Ministry of Science and Technology of China (No. 2005CB1208).
Supplemental data Supplemental Table 1 associated with this article can be found in the online version at www.jgenetgenomics.org.
References Arite, T., Umehara, M., Ishikawa, S., Hanada, A., Maekawa, M., Yamaguchi, S., and Kyozuka, J. (2009). d14, a strigolactone insensitive mutant of rice, shows an accelerated outgrowth of tillers. Plant Cell Physiol. 50: 14161424 Arite, T., Iwata, H., Ohshima, K., Maekawa, M., Nakajima, M., Kojima, M., Sakakibara, H., and Kyozuka, J. (2007). DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice. Plant J. 51: 10191029. Bart, R., Chern, M., Park, C.J., Bartley, L., and Ronald, P.C. (2006). A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2: 13. Bolle, C. (2004). The role of GRAS proteins in plant signal transduction and development. Planta 218: 683692. Bray, S., Musisi, H., and Bienz, M. (2005). Bre1 is required for Notch signaling and histone modification. Dev. Cell 8: 279286. Cao, Y., Dai, Y., Cui, S., and Ma, L. (2008). Histone H2B monoubiquitination in the chromatin of FLOWERING LOCUS C regulates flowering time in Arabidopsis. Plant Cell 20: 25862602. Cook, C.E., Whichard, L.P., Wall, M.E., Egley, G.H., Coggon, P., Luhan, P.A., and McPhail, A.T. (1972). Germination stimulants. II. The structure of strigol-a potent seed germination stimulant for witchweed (Striga lutea Lour.). J. Amer. Chem. Soc. 94: 61986199. Dhawan, R., Luo, H., Foerster, A.M., Abuqamar, S., Du, H.N., Briggs, S.D., Mittelsten Scheid, O., and Mengiste, T. (2009). HISTONE MONOUBIQUITINATION1 interacts with a subunit of the mediator complex and regulates defense against necrotrophic fungal pathogens in Arabidopsis. Plant Cell 21: 10001019. Doebley, J., Stec, A., and Gustus, C. (1995). teosinte branched1 and the origin of maize: evidence for epistasis and the evolution of dominance. Genetics 141: 333346. Eckardt, N.A. (2007). Two tales of chromatin remodeling converge on HUB1. Plant Cell 19: 391393. Fleury, D., Himanen, K., Cnops, G., Nelissen, H., Boccardi, T.M., Maere, S., Beemster, G.T., Neyt, P., Anami, S., Robles, P., Micol, J.L., Inze, D., and Van Lijsebettens, M. (2007). The Arabidopsis thaliana homolog of yeast BRE1 has a function in cell cycle regulation during early leaf and root growth. Plant Cell 19: 417432. Gomez-Roldan, V., Fermas, S., Brewer, P.B., Puech-Pages, V., Dun, E.A., Pillot, J.P., Letisse, F., Matusova, R., Danoun, S., Portais,
J.C., Bouwmeester, H., Becard, G., Beveridge, C.A., Rameau, C., and Rochange, S.F. (2008). Strigolactone inhibition of shoot branching. Nature 455: 189194. Greb, T., Clarenz, O., Schafer, E., Muller, D., Herrero, R., Schmitz, G., and Theres, K. (2003). Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Genes Dev. 17: 11751187. Gu, X., Jiang, D., Wang, Y., Bachmair, A., and He, Y. (2009). Repression of the floral transition via histone H2B monoubiquitination. Plant J. 57: 522533. Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271282. Hu, Y., Bao, F., and Li, J. (2000). Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway in Arabidopsis. Plant J. 24: 693701. Hubbard, L., McSteen, P., Doebley, J., and Hake, S. (2002). Expression patterns and mutant phenotype of teosinte branched1 correlate with growth suppression in maize and teosinte. Genetics 162: 19271935. Humphrey, A.J., and Beale, M.H. (2006). Strigol: biogenesis and physiological activity. Phytochemistry 67: 636640. Hwang, W.W., Venkatasubrahmanyam, S., Ianculescu, A.G., Tong, A., Boone, C., and Madhani, H.D. (2003). A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol. Cell 11: 261266. Komatsu, K., Maekawa, M., Ujiie, S., Satake, Y., Furutani, I., Okamoto, H., Shimamoto, K., and Kyozuka, J. (2003). LAX and SPA: major regulators of shoot branching in rice. Proc. Natl. Acad. Sci. USA 100: 1176511770 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., and Li, J. (2003). Control of tillering in rice. Nature 422: 618621. Lin, H., Wang, R., Qian, Q., Yan, M., Meng, X., Fu, Z., Yan, C., Jiang, B., Su, Z., Li, J., and Wang, Y. (2009). DWARF27, an iron-containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant Cell 21: 15121525. Liu, Y., Koornneef, M., and Soppe, W.J. (2007). The absence of histone H2B monoubiquitination in the Arabidopsis hub1 (rdo4) mutant reveals a role for chromatin remodeling in seed dormancy. Plant Cell 19: 433444. Lopez-Raez, J.A., Charnikhova, T., Gomez-Roldan, V., Matusova, R., Kohlen, W., De Vos, R., Verstappen, F., Puech-Pages, V., Becard, G., Mulder, P., and Bouwmeester, H. (2008). Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation. New Phytol. 178: 863874. Oikawa, T., and Kyozuka, J. (2009). Two-step regulation of LAX PANICLE1 protein accumulation in axillary meristem formation in rice. Plant Cell 21: 10951108. Sato, Y., Hong, S.K., Tagiri, A., Kitano, H., Yamamoto, N., Nagato, Y., and Matsuoka, M. (1996). A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis. Proc. Natl. Acad. Sci. USA 93: 81178122. Schumacher, K., Schmitt, T., Rossberg, M., Schmitz, G., and Theres, K. (1999). The Lateral suppressor (Ls) gene of tomato encodes a new
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
member of the VHIID protein family. Proc Natl Acad Sci USA 96: 290295. Takeda, T., Suwa, Y., Suzuki, M., Kitano, H., Ueguchi-Tanaka, M., Ashikari, M., Matsuoka, M., and Ueguchi, C. (2003). The OsTB1 gene negatively regulates lateral branching in rice. Plant J. 33: 513520. Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N., Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K., Kyozuka, J., and Yamaguchi, S. (2008). Inhibition of shoot branching by new terpenoid plant hormones. Nature 455: 195200. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33: 949956. Waadt, R., Schmidt, L.K., Lohse, M., Hashimoto, K., Bock, R., and Kudla, J. (2008). Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK com-
77
plexes in planta. Plant J. 56: 505516. Wang, Y., and Li, J. (2008). Molecular basis of plant architecture. Annu. Rev. Plant Biol. 59: 253279. Xu, L., Menard, R., Berr, A., Fuchs, J., Cognat, V., Meyer, D., and Shen, W.H. (2009). The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana. Plant J. 57: 279288. Yan, H., Saika, H., Maekawa, M., Takamure, I., Tsutsumi, N., Kyozuka, J., and Nakazono, M. (2007). Rice tillering dwarf mutant dwarf3 has increased leaf longevity during darkness-induced senescence or hydrogen peroxide-induced cell death. Genes Genet. Syst. 82: 361366. Zou, J., Zhang, S., Zhang, W., Li, G., Chen, Z., Zhai, W., Zhao, X., Pan, X., Xie, Q., and 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: 687698.
GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 6977
www.jgenetgenomics.org
Identification and functional analysis of the MOC1 interacting protein 1 Fengli Sun a, Weiping Zhang a, Guosheng Xiong a, Meixian Yan b, Qian Qian b, Jiayang Li a, Yonghong Wang a, * a
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China b State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China Received for publication 12 September 2009; revised 2 December 2009; accepted 6 December 2009
Abstract Rice tillering is one of the most important agronomic traits that determine grain yields. Our previous study has demonstrated that the MONOCULM1 (MOC1) gene is a key component that controls the formation of rice tiller buds. To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering, we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins (MIPs). Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo. The overexpression of MIP1 resulted in enhanced tillering and reduced plant height. In-depth characterization of the context of MIP1 and MOC1 would further our understanding of molecular regulatory mechanisms of rice tillering. Keywords: rice; MOC1; MIP1; tillering; protein-protein interaction
Introduction Tillering or branching is one of the most important agronomic traits that determine plant architecture and ultimately the grain yields. Tillering generally comprises two distinct steps: the initiation of axillary buds and their outgrowth. Tremendous efforts have been made to elucidate the molecular mechanisms underlying the genetic control of tillering in grass crops and significant progresses have been achieved over the last decade (Wang and Li, 2008). MOC1 was the first characterized gene that controls the initiation and outgrowth of rice tiller buds (Li et al., 2003). MOC1 belongs to the plant-specific GRAS transcription factor family and the orthologs of MOC1 have also been identified in tomato and Arabidopsis, which were sug* Corresponding author. Tel: +86-10-6480 6008; Fax: +86-10-6488 3428. E-mail address: [email protected] DOI: 10.1016/S1673-8527(09)60026-6
gested to be essential genes controlling shoot branching (Schumacher et al., 1999; Greb et al., 2003). The loss-of-function of MOC1 leads to the defect in the tiller bud initiation. Further analysis on expression patterns of the SAM-determined marker gene OSH1 suggested that MOC1 may participate in the very early step of axillary meristem initiation (Sato et al., 1996; Li et al., 2003). The LAX PANICLE1 (LAX1) is another essential gene controlling rice branching at both vegetative and reproductive developmental stages (Komatsu et al., 2003). The latest study suggested that LAX1 may affect later stages after MOC1 in the initiation of tiller buds (Oikawa and Kyozuka, 2009). However, the context of LAX1 and MOC1 has not been explained. After the initiation of axillary meristems of rice plants, OsTB1, an ortholog of TB1 in maize, has been proven to be responsible for the subsequent outgrowth of axillary buds (Doebley et al., 1995; Hubbard et al., 2002; Takeda et
70
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
al., 2003). Moreover, studies on a serials of more branching mutants in diverse species have revealed a conserved carotenoid-derived MAX/RMS/D (more axillary branching) pathway that inhibits the outgrowth of axillary buds (Wang and Li, 2008). Recently, two groups have independently reported that the MAX/RMS/D pathway is involved in the production and signaling of strigolactones, which are a group of terpenoid lactones that have been found in root exudates of diverse plant species and synthesized from carotenoids (Cook et al., 1972; Humphrey and Beale, 2006; Gomez-Roldan et al., 2008; Lopez-Raez et al., 2008; Umehara et al., 2008). In rice, five genes have been identified in the MAX pathway, in which D10, HTD1/D17, D27 are suggested to be responsible for the biosynthesis of strigolactones, and D14 and D3 for the perception of strigolactones-related signals (Zou et al., 2006; Arite et al., 2007, 2009; Yan et al., 2007; Lin et al., 2009). Although several important regulators have been identified in controlling rice tillering, our understanding on the tillering regulatory mechanism is still fragmental due to the lack of further studies on their actions. We therefore try to dissect the MOC1-medieated rice tillering regulation by identifying MOC1 interacting proteins (MIPs) through yeast-two-hybrid screening. In this paper, we showed that the MIP1 can interact with MOC1 and promote rice tillering.
Materials and methods Yeast-two-hybrid screening The full length cDNA of MOC1 were used as a bait to screen cDNA library for MOC1 interacting proteins by PROQUESTTM Two-Hybrid System (Invitrogen, USA). Interactions were tested on SD medium minus Leu, Trp, and His, and containing 30 mmol/L 3-Amino-1, 2, 4Triazole (3AT), using the yeast strain MaV203 according to the manufacturer’s manual.
Co-immunoprecipitation in vitro MOC1 was subcloned into the Nde I-EcoR I digested pGBKT7 vector and MIP1 was subcloned into the Sma I-Spe I digested pGADT7. Then c-MYC tagged MOC1 and HA tagged MIP1 were translated using the TNT T7 Coupled Reticulocyte Lysate Kit (Promega, USA) with
35S-labeled methionine, according to the manufacturer’s instructions. Co-immunoprecipitation between MOC1 and MIP1 was conducted using MATCHMAKER Co-IP Kit (Clontech, USA) according to manufacturer’s manual. The primer sequences used are listed in Supplemental Table 1.
Overexpression of MIP1 Full-length of MIP1 cDNA was subcloned into the BamH I-Spe I digested pTCK303 vector to generate the plasmid MIP1OE, which was subsequently introduced into Agrobacterium tumefaciens stain EHA105 by electroporation and then into Nipponbare as previous report (Hiei et al., 1994). The phenotypes of the transgenic plants were examined in the T2 progeny.
RT-PCR Total RNA was prepared by a TRIzol Kit according to the user’s manual (Cat. No. 15596-026, Invitrogen). One microgram of total RNA was treated with DNase I (Ambion) and used for cDNA synthesis using the Superscript RT Kit (Invitrogen). The primer sequences used for RT-PCR are listed in Supplemental Table 1.
DNA and RNA gel blotting analysis Rice genomic DNA (20 ȝg) was completely digested with indicated restriction endonucleases respectively and separated in a 0.8% agarose gel. After electrophoresis, DNA was transferred onto a nylon membrane (Hybrid N+; Amersham, USA), and hybridization was performed as previously described (Mou et al., 2000). A 1,023 bp fragment of the MIP1 gene amplified from rice cDNA was labeled with 32P-dCTP (Amersham) for hybridization. Total RNA was isolated with TRIzol reagent (Invitrogen) and the RNA gel blot analysis was performed as previously described (Hu et al., 2000). RNA (20 μg per lane) was separated in a 0.8% agarose gel containing 10% (v/v) formaldehyde, then transferred onto a Hybond N+ membrane (Amersham) and probed with the PCR-amplified DNA fragments with a specific primer pair of MIP1 (Supplemental Table1)
Subcellular localization of MIP1 To generate CaMV35S-GFP, an Xba I-Apa I fragment
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
containing GFP without stop codon was amplified by primers GFPF and GFPR using SGFP (S65T) as template. The Xba I-Apa I sites containing GFP fragment was then subcloned into the Xba I-Apa I digested vector pSPYNE (R) 173 (Waadt et al., 2008). A BamH I-Xho I fragment containing the coding region of MIP1 amplified by the primers of MIP1F and MIP1R was subcloned into the BamH I and Xho I sites of CaMV35S-GFP to generate CaMV35S:GFP-MIP1. The plasmids CaMV35S- GFP and CaMV35S:GFP-MIP1 were introduced into rice leaf protoplasts as described (Bart et al., 2006). After overnight incubation in the dark, the GFP signals were observed under a confocal microscope (FluoView 1000, Olympus, Japan) at an excitation wavelength of 488 nm.
BiFC assay in tobacco leaves BiFC assays were performed as described previously (Waadt et al., 2008). Briefly, MOC1 was subcloned into the BamH I-Kpn I digested binary BiFC vector SCYNE (R), which was fused with N-terminal fragment of CFP. MIP1 was subcloned into the BamH I-Xho I digested SCYCE (R) vector, which was fused with the C-terminal of CFP. These constructs were transformed into A. tumefaciens strain EHA105 and co-expressed transiently in Nicotiana benthamiana leaves (Voinnet et al., 2003). Equal volume of transformed EHA105 culture (OD600 = 1.0) was mixed before infiltration. The CFP signals were observed after 40–48 h infiltration under a confocal microscope (FluoView 1000, Olympus) at an excitation wavelength
71
of 405 nm.
Results Identification and confirmation of MIP1 Our previous study has identified the rice MOC1 gene, which encodes a putative GRAS family nuclear protein. Functional analysis revealed that MOC1 controls not only the formation of rice tiller buds but also the outgrowth of tillers (Li et al., 2003). To investigate the molecular events involved in the MOC1-mediated rice tillering, we screened for MOC1 interacting proteins (MIPs) using the yeast two-hybrid system with a rice cDNA library. The MIP1 protein is one of 15 candidate proteins that exhibit interactions with MOC1. To verify the interaction between MOC1 and MIP1 in vitro, we performed an X-Gal assay after co-transformation of MOC1-BD and MIP1-AD into yeast cells. The results showed that MOC1 could interact with MIP1 in yeast, and their interaction strength is comparable to the yeast control strain B (Fig. 1A), indicating a weak interaction strength between MOC1 and MIP1. We therefore further confirmed their interaction using 35S labeled Methionine in a Rabbit Reticulocyte Lysate system, in which MOC1 fused with the c-MYC tag and MIP1 with the HA tag was translated in vitro. The Co-IP result showed that MOC1 could be precipitated by Į-HA antibody and MIP1 by Į-c-Myc (Fig. 1B), indicating that MOC1 did interact with MIP1 in vitro.
Fig. 1. Confirmation of the interaction between MOC1 and MIP1 in vitro. A: confirmation of the interaction between MOC1 and MIP1 by the X-Gal essay in yeast. Controls A–E: yeast control strains, indicating different levels of interaction strength bewteen two proteins. Control A, pPC97 and pPC86; control B, pPC97-RB and pPC86-E2F1; control C, pPC97-CYH2 -dDP and pPC86-dE2F; control D, pPC97-Fos and pPC86-Jun; control E, pCL1 (encoding full length GAL4) and pPC86. B: confirmation of the interaction between MOC1 and MIP1 by Co-IP. Lane 1, MOC1 + MIP1; lane 2, MOC1 + MIP1+ HA-Tag antibody; lane 3, MOC1 + c-Myc antibody; lane 4, MIP1 + HA-Tag antibody; lane 5, MOC1 + HA-Tag antibody; lane 6, MIP1 + c-Myc antibody.
72
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
Characterization of MIP1
Subcellular localization of MIP1
The Blast analysis revealed that MIP1 is a member of the Bre1 family, which include Sc_Bre1 (Yeast), Ce_Bre1 (Caenorhabditis elegans), Dm_Bre1 (Drosophila melanogaster), Hu_Bre1A and Hu_Bre1B (Human), At_HUB1and At_HUB2 (Arabidopsis thaliana), Zm_Bre1 (Zea mays), OsBre1A and OsBre1B (Oryza sativa). The Bre1 family proteins contain a C3HC4 RING finger domain, which is also included in MIP1. In addition, MIP1 contains three conserved sumoylation consensus motifs (Fig. 2). Amino acid sequence alignment showed that MIP1 shared highest similarity with AtHUB1. Sequence comparison between genomic and complementary DNAs revealed that MIP1 is composed of 19 exons and encodes an 884 amino acid polypeptide. MIP1 is a single copy gene in the rice genome (Fig. 3A) and is a ubiquitously expressed gene at a very low level (Fig. 3B).
The MIP1 protein was predicted to contain a putative nuclear localization signal (Fig. 2). To determine its subcellular localization, we performed a transient expression experiment of MIP1 in rice leaf protoplasts. The N-terminal of MIP1 was fused with GFP under the control of cauliflower mosaic virus (CaMV) 35S promoter and the construct was transferred into rice leaf protoplasts by the PEG-mediated method. In contrast to the control that was ubiquitous in protoplast cells (Fig. 4A), the GFP-MIP1 fusion protein was predominantly localized in small discrete foci in the nucleus (Fig. 4B) and the GFP-MIP1 and RFP-MOC1 fusion proteins were co-localized in the nucleus (Fig. 4C). However, the GFP-MIP1 protein was diffusely localized in the nucleus in the moc1 protoplast cells (Fig. 4D). These results demonstrate that MOC1 is required for the normal localization of MIP1 in the cell.
Fig. 2. Alignment of deduced amino acid sequence of MIP1 with its homologs in Arabidopsis. The underlined letters refer to the putative NLS domain, the triangle-labeled letters to sumoylation consensus motifs, and asterisks to the C3HC4 RING finger domain.
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
73
Fig. 3. DNA and RNA gel blot analysis. A: DNA gel blot analysis indicated that MIP1 is a single copy gene in the rice genome. B: the MIP1 expression pattern revealed by RNA gel blot analysis. L, mature leaves ; R, seedling roots; S, stems after heading ; P, young panicles.
Fig. 4. Subcellular localization of GFP-MIP1 fusion proteins in rice protoplasts. A: subcellular localization of 35S:GFP. B: subcellular localization of 35S:GFP-MIP1. C: co-localization of 35S:GFP-MIP1 and 35S:RFP-MOC1 in rice protoplasts. D: subcellular localization of 35S:GFP-MIP1 in moc1 protoplasts. Left panels: dark filed (GFP); Middle panels: bright field in (A), (B) and (D), dark field (RFP) in (C). Right panels: merged images of left and middle panels. Bars = 5 ȝm.
74
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
MIP1 may interact with MOC1 in vivo To verify whether MOC1 could interact with MIP1 in vivo, we performed bimolecular fluorescence complementation (BiFC) assays in Nicotiana leaf epidermal cells. In BiFCs, MOC1 and MIP1 are fused to the N- and C-terminal regions of the CFP, respectively. A physical association between bait and prey brings the N- and C-terminal fragments of the split CFP together, and allows functional reassembly of the fluorescent protein (Waadt et al., 2008). Our results showed that when YNE-MOC1 and YCE-MIP1were co-expressed, positive CFP signals were detected mainly in the nuclei (speckles). No CFP fluorescence signal was detected in the negative control MOC1-YNE and empty vector-YCE (Fig. 5). These observations confirmed that MOC1 and MIP1 are able to physically interact in vivo.
Overexpression of MIP1 leads to an increase in tiller number To understand the context of MIP1 and MOC1 in controlling rice tillering, the full length cDNA of MIP1 under the control of the maize ubiquitin promoter was transferred into a rice variety Nipponbare. The transgenic plants having an increased expression level of MIP1 displayed a significantly enhanced rice tillering phenotype at both vegetative and reproductive stages (Fig. 6, C–E). In addition, the transgenic plants also exhibited semi-dwarf, delayed head-
ing, and sterility. The phenotypes of MIP1-overexpressing transgenic plants are quite similar to those of MOC1overexpressing plants (Li et al., 2003).
Discussion Tillering in rice is not only an important agronomic trait for grain yield, but also an ideal system for studying branching in higher plants, especially for monocotyledonous species. In this study, we identified a MOC1 interacting protein, MIP1, via the yeast two-hybrid system. The interaction between MIP1 and MOC1 was confirmed by both Co-IP in vitro and BiFC assays in vivo (Figs. 1 and 5). MIP1 belongs to the Bre1 family which play roles in regulation of monoubiquitination of H2B (Fleury et al., 2007; Liu et al., 2007). The overexpression of MIP1 resulted in an increase in tiller number and a decrease in plant height, suggesting an essential role of MIP1 in determining the plant architecture of rice. In yeast, Bre1 regulates cell morphology through H2B monoubiquitination (Hwang et al., 2003). In Drosophila, Bre1 affects the development of wings and legs through the Notch signaling pathway (Bray et al., 2005). In Arabidopsis, HUB1 was shown to act as an E3 ligase involved in the H2B monoubiquitination (Fleury et al., 2007; Liu et al., 2007). MIP1 shared the highest identity with HUB1 (Fig. 2). As one kind of chromatin modification, monoubiquitination of H2B is crucial for gene expression
Fig. 5. Confirmation of the interaction between MOC1 and MIP1 by BiFC. A–C: BiFC experiments between MIP1 and MOC1. MOC1 fused with N-terminal fragment of CFP and MIP1 fused with C-terminal fragment of CFP were co-infiltrated into tobacco leaves. D–F: BiFC of MOC1 fused with N-terminal fragment of CFP and C-terminal CFP vector co-infiltrated into tobacco leaves as the control. Bars = 20ҏ ȝm.
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
75
Fig. 6. Phenotypes of MIP1-overexpressing transgenetic plants. A: examination of MIP1 transcripts by RT-PCR. Actin transcripts were used as controls. B: the semi-quantitive RT-PCR analysis of MIP1 transcripts in the wild-type and MIP1-overexpressing plants. Data are means ± SE (n = 3). C: the phenotype of the wild type (left) and MIP1-overexpressing plants (right) at the tillering stage. D: the phenotype of the wild type (left) and MIP1-overexpressing plants (right) at the matured stage. Bars = 10 cm. E: comparison of tiller numbers between the wild type and MIP1-overexpressing plants. Data are means ±SE (n = 15).
(Eckardt, 2007). HUB1 has also been suggested to be a key regulator that controls seed dormancy, cell-cycle, flowering time, and plant defense (Liu et al., 2007; Fleury et al., 2007; Cao et al., 2008; Dhawan et al., 2009; Gu et al., 2009; Xu et al., 2009). MIP1 can interact with MOC1, however, the relationship between MOC1 and MIP1 remains to be elucidated. As putative transcriptional regulators, GRAS family proteins play important roles in regulating plant development and growth by affecting the expression of their target genes (Bolle, 2004). However, the expression level of MIP1 was unchanged in the moc1 mutant (data not shown), implicating that MOC1 may regulate MIP1 at the posttranscriptional level or through an unknown mechanism. It appears that the interaction between MOC1 and MIP1 may
regulate the subcellular localization or modification of MIP1, thus affects the monoubiquitination of H2B and then regulates the expression of downstream genes of H2B and promotes tillering in rice. The transgenic plants overexpressing MIP1 displayed an increased tiller number and reduced plant height (Fig. 6), which is similar to that of MOC1-overexpressing plant (Li et al., 2003). MIP1 was nuclear localized and distributed mainly in some speckles. Since MOC1 interacts with MIP1, the enrichment of MOC1 at speckles in the nucleus may involve in the regulation of downstream genes. When MIP1 is overexpressed in plants, the subcellular localization of MOC1 may redistribute in the nucleus and trigger the downstream events to control rice tillering.
76
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
Acknowledgements This work was supported by the grant from the Ministry of Science and Technology of China (No. 2005CB1208).
Supplemental data Supplemental Table 1 associated with this article can be found in the online version at www.jgenetgenomics.org.
References Arite, T., Umehara, M., Ishikawa, S., Hanada, A., Maekawa, M., Yamaguchi, S., and Kyozuka, J. (2009). d14, a strigolactone insensitive mutant of rice, shows an accelerated outgrowth of tillers. Plant Cell Physiol. 50: 14161424 Arite, T., Iwata, H., Ohshima, K., Maekawa, M., Nakajima, M., Kojima, M., Sakakibara, H., and Kyozuka, J. (2007). DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice. Plant J. 51: 10191029. Bart, R., Chern, M., Park, C.J., Bartley, L., and Ronald, P.C. (2006). A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2: 13. Bolle, C. (2004). The role of GRAS proteins in plant signal transduction and development. Planta 218: 683692. Bray, S., Musisi, H., and Bienz, M. (2005). Bre1 is required for Notch signaling and histone modification. Dev. Cell 8: 279286. Cao, Y., Dai, Y., Cui, S., and Ma, L. (2008). Histone H2B monoubiquitination in the chromatin of FLOWERING LOCUS C regulates flowering time in Arabidopsis. Plant Cell 20: 25862602. Cook, C.E., Whichard, L.P., Wall, M.E., Egley, G.H., Coggon, P., Luhan, P.A., and McPhail, A.T. (1972). Germination stimulants. II. The structure of strigol-a potent seed germination stimulant for witchweed (Striga lutea Lour.). J. Amer. Chem. Soc. 94: 61986199. Dhawan, R., Luo, H., Foerster, A.M., Abuqamar, S., Du, H.N., Briggs, S.D., Mittelsten Scheid, O., and Mengiste, T. (2009). HISTONE MONOUBIQUITINATION1 interacts with a subunit of the mediator complex and regulates defense against necrotrophic fungal pathogens in Arabidopsis. Plant Cell 21: 10001019. Doebley, J., Stec, A., and Gustus, C. (1995). teosinte branched1 and the origin of maize: evidence for epistasis and the evolution of dominance. Genetics 141: 333346. Eckardt, N.A. (2007). Two tales of chromatin remodeling converge on HUB1. Plant Cell 19: 391393. Fleury, D., Himanen, K., Cnops, G., Nelissen, H., Boccardi, T.M., Maere, S., Beemster, G.T., Neyt, P., Anami, S., Robles, P., Micol, J.L., Inze, D., and Van Lijsebettens, M. (2007). The Arabidopsis thaliana homolog of yeast BRE1 has a function in cell cycle regulation during early leaf and root growth. Plant Cell 19: 417432. Gomez-Roldan, V., Fermas, S., Brewer, P.B., Puech-Pages, V., Dun, E.A., Pillot, J.P., Letisse, F., Matusova, R., Danoun, S., Portais,
J.C., Bouwmeester, H., Becard, G., Beveridge, C.A., Rameau, C., and Rochange, S.F. (2008). Strigolactone inhibition of shoot branching. Nature 455: 189194. Greb, T., Clarenz, O., Schafer, E., Muller, D., Herrero, R., Schmitz, G., and Theres, K. (2003). Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Genes Dev. 17: 11751187. Gu, X., Jiang, D., Wang, Y., Bachmair, A., and He, Y. (2009). Repression of the floral transition via histone H2B monoubiquitination. Plant J. 57: 522533. Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271282. Hu, Y., Bao, F., and Li, J. (2000). Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway in Arabidopsis. Plant J. 24: 693701. Hubbard, L., McSteen, P., Doebley, J., and Hake, S. (2002). Expression patterns and mutant phenotype of teosinte branched1 correlate with growth suppression in maize and teosinte. Genetics 162: 19271935. Humphrey, A.J., and Beale, M.H. (2006). Strigol: biogenesis and physiological activity. Phytochemistry 67: 636640. Hwang, W.W., Venkatasubrahmanyam, S., Ianculescu, A.G., Tong, A., Boone, C., and Madhani, H.D. (2003). A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol. Cell 11: 261266. Komatsu, K., Maekawa, M., Ujiie, S., Satake, Y., Furutani, I., Okamoto, H., Shimamoto, K., and Kyozuka, J. (2003). LAX and SPA: major regulators of shoot branching in rice. Proc. Natl. Acad. Sci. USA 100: 1176511770 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., and Li, J. (2003). Control of tillering in rice. Nature 422: 618621. Lin, H., Wang, R., Qian, Q., Yan, M., Meng, X., Fu, Z., Yan, C., Jiang, B., Su, Z., Li, J., and Wang, Y. (2009). DWARF27, an iron-containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant Cell 21: 15121525. Liu, Y., Koornneef, M., and Soppe, W.J. (2007). The absence of histone H2B monoubiquitination in the Arabidopsis hub1 (rdo4) mutant reveals a role for chromatin remodeling in seed dormancy. Plant Cell 19: 433444. Lopez-Raez, J.A., Charnikhova, T., Gomez-Roldan, V., Matusova, R., Kohlen, W., De Vos, R., Verstappen, F., Puech-Pages, V., Becard, G., Mulder, P., and Bouwmeester, H. (2008). Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation. New Phytol. 178: 863874. Oikawa, T., and Kyozuka, J. (2009). Two-step regulation of LAX PANICLE1 protein accumulation in axillary meristem formation in rice. Plant Cell 21: 10951108. Sato, Y., Hong, S.K., Tagiri, A., Kitano, H., Yamamoto, N., Nagato, Y., and Matsuoka, M. (1996). A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis. Proc. Natl. Acad. Sci. USA 93: 81178122. Schumacher, K., Schmitt, T., Rossberg, M., Schmitz, G., and Theres, K. (1999). The Lateral suppressor (Ls) gene of tomato encodes a new
Fengli Sun et al. / Journal of Genetics and Genomics 37 (2010) 6977
member of the VHIID protein family. Proc Natl Acad Sci USA 96: 290295. Takeda, T., Suwa, Y., Suzuki, M., Kitano, H., Ueguchi-Tanaka, M., Ashikari, M., Matsuoka, M., and Ueguchi, C. (2003). The OsTB1 gene negatively regulates lateral branching in rice. Plant J. 33: 513520. Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N., Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K., Kyozuka, J., and Yamaguchi, S. (2008). Inhibition of shoot branching by new terpenoid plant hormones. Nature 455: 195200. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33: 949956. Waadt, R., Schmidt, L.K., Lohse, M., Hashimoto, K., Bock, R., and Kudla, J. (2008). Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK com-
77
plexes in planta. Plant J. 56: 505516. Wang, Y., and Li, J. (2008). Molecular basis of plant architecture. Annu. Rev. Plant Biol. 59: 253279. Xu, L., Menard, R., Berr, A., Fuchs, J., Cognat, V., Meyer, D., and Shen, W.H. (2009). The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana. Plant J. 57: 279288. Yan, H., Saika, H., Maekawa, M., Takamure, I., Tsutsumi, N., Kyozuka, J., and Nakazono, M. (2007). Rice tillering dwarf mutant dwarf3 has increased leaf longevity during darkness-induced senescence or hydrogen peroxide-induced cell death. Genes Genet. Syst. 82: 361366. Zou, J., Zhang, S., Zhang, W., Li, G., Chen, Z., Zhai, W., Zhao, X., Pan, X., Xie, Q., and 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: 687698.