杂交稻的推广和应用被誉为“第二次绿色革命”，但统计数据表明近些年普通籼型杂交稻单产潜力的提高已十分有限（Fang and Cheng, 2009）。为了解决这一难题，一方面，籼粳亚种间强大杂种优势的有效利用有望实现水稻单产的再次飞跃；但另一方面，由于籼粳交杂种普遍存在株高超亲等问题，而使得籼粳亚种间杂种优势的利用受到了极大的限制。研究显示，利用部分显性矮秆基因可有效解决籼粳杂种株高偏高的问题。此外，水稻的分蘖和株高被认为是水稻株型的重要组成部分，同时也是杂种产量形成的决定因素（Wang and Li, 2008）。 独脚金内酯（strigolactones，SLs）是由植物根分泌的一类信号物质，它可以刺激根寄生杂草种子的萌发并在植物和丛枝菌丝真菌建立的共生关系中起重要作用。最新的研究发现独脚金内酯可以参与调控植物的分枝（蘖），由此人们把这类来源于类胡萝卜素的小分子物质看作是一类新的植物激素（Gomez-Roldan et al., 2008；Umehara et al., 2008）。后续的许多研究表明独脚金内酯是优化植物生长发育的重要调节因子。到目前为止，研究发现独脚金内酯参与调节植物的多个发育进程，如种子的萌发、幼苗的光形态建成、芽的形态建成以及根的发育等（Xie et al., 2010）。尽管近期关于独脚金内酯生物合成途径的研究有了较快进展，但相对来说人们对其信号响应和传递机制还是知之甚少。 本研究报道了一个由于功能获得性突变而造成水稻矮化多分蘖表型的突变体dwarf 53（d53），它与其它水稻中已经报道的独脚金内酯合成或信号缺陷突变体——dwarf突变体（d突变体）具有非常相似的突变表型。野生型农林8号和d53突变体的杂交F1代植株具有介于两亲本的中间表型。结合它们F2代分离群体的遗传分析，我们认为d53基因突变表现为半显性遗传。研究发现，外源GR24（一种独脚金内酯的人工合成类似物）处理并不能有效抑制d53突变体中分蘖芽的生长。进一步对d53突变体根分泌物中的内源独脚金内酯含量的测定结果表明，相比于野生型，d53突变体中积累了更高水平的2’-epi-5-deoxystrigol（epi-5DS，一种天然的独脚金内酯活性成分）。综合上述实验证据表明，和d14突变体类似，d53是一个对独脚金内酯不敏感突变体。 利用图位克隆技术我们克隆了D53基因。遗传进化分析发现，该基因的编码产物与最近在拟南芥中发现的SMAX1蛋白具有一定的序列同源性。SMAX1蛋白参与karrikin（一类与独脚金内酯结构类似、来源于野火燃烧时产生的烟中并能促进种子萌发的活性小分子物质）的信号传导途径，它的发现源自于对拟南芥more axillary growth 2 (max2)突变体抑制子的筛选。尽管D53蛋白与I类Clp ATPase家族蛋白在一级氨基酸序列上相似度较低，但它们在蛋白高级结构预测中却有着十分相近的结构域组成，即都包含N、D1、M以及D2四个相对独立的结构域。随后，为了验证矮化多分蘖表型是由突变d53基因造成，我们在野生型背景下分别构建了表达正常D53基因和突变d53基因的转基因植株。结果表明，所有过表达突变d53基因的转基因植株都比过表达正常D53基因的植株具有更加显著的多分蘖表型。不仅如此，更有趣的是，与空载体对照转基因植株相比，过表达正常D53基因的转基因植株分蘖数也有适当的增加。此外，利用RNA干扰手段，降低d53突变体中d53基因的表达可以显著减少突变体的分蘖数。综上可知，D53蛋白是独脚金内酯介导的分枝抑制途径的抑制子且突变体的多分蘖表型是由d53基因的显性突变导致。 前人的研究显示，水稻中已经鉴定出两个参与独脚金内酯信号途径的组分，其中一个是F-box蛋白D3，另一个是α/β水解酶蛋白D14。为了阐明D53蛋白作为独脚金内酯信号途径抑制子的分子机制，我们检测了D53与D14和D3之间的蛋白互作。首先，多个实验结果表明，D53能够以依赖独脚金内酯的方式与D14蛋白互作，而且D53蛋白的D1结构域对它们之间的互作至关重要；其次，和前人利用酵母双杂交系统验证矮牵牛中DAD2（D14的同源蛋白）蛋白依赖GR24与PhMAX2A（D3的同源蛋白）蛋白互作结果一致，我们的体外pull-down结果显示D14蛋白可以和D3蛋白以依赖独脚金内酯的形式直接互作；然后，我们还发现D53与D3蛋白之间可能存在不依赖独脚金内酯的直接的本底互作；最后，来自体外pull-down蛋白样品的质谱分析结果进一步表明，独脚金内酯能够促进D53–D14–SCFD3蛋白复合体的形成。 为了进一步研究独脚金内酯对D53蛋白的调控机制，我们首先检测了与独脚金内酯信号相关的D53基因表达情况。荧光定量分析表明，D53基因在水稻各组织中呈普遍性表达模式，而且主要是在木质部的薄壁细胞中特异表达，这与前人报道的独脚金内酯合成、信号传导以及运输的作用位置一致。一方面，独脚金内酯处理可以诱导D53基因的上调表达；另一方面，相比于野生型，在水稻六个d突变体中D53基因下调表达。这些结果表明，D53基因的表达可能受独脚金内酯信号的负向反馈调控。进一步，其它的一系列实验结果表明，独脚金内酯能够以依赖D14蛋白和D3蛋白的方式诱导D53蛋白被蛋白酶体系统降解，而且更值得注意的结果是，与D53–GFP融合蛋白不同，突变d53–GFP蛋白对独脚金内酯的存在依然保持稳定而不被降解。 除了生化途径上的互作，为了进一步确定D53、D14、D3之间的遗传互作关系，我们又分别构建了d3d53和d14d53双突变体。分析结果表明由于双突变体在表型上与单突变体相比没有明显的加性效应，我们推测它们应该处于同一独脚金内酯信号途径中。此外，为了确定它们之间的上位性关系，我们分别在d3和d14突变体背景下敲除D53基因，结果显示下调D53基因的表达能够部分抑制d14和d3突变体的矮化多分蘖表型。这些结果暗示，从遗传上讲D53基因在D3和D14基因下游起作用，它是导致这些水稻独脚金内酯途径d突变体产生矮化多分蘖表型的主要因素，并再次证明它是独脚金内酯信号途径的负向调节因子。 我们的研究结果首次在遗传和生化层面证实D53蛋白是独脚金内酯信号途径的抑制子。激素诱导D53蛋白降解的分子机制将独脚金内酯的信号感知与响应有机的串联起来。这些发现不仅揭示了独脚金内酯和karrikins的信号传递机制，也为激素（生长素、细胞分裂素、赤霉素）、环境（光照、营养）、遗传等因素在调控单子叶和双子叶株型建成方面的复杂相互作用提供了新的视野和思路。不仅如此，更为重要的是，d53突变体的发掘与改良为籼粳交杂种优势在水稻育种栽培的有效利用提供了潜在的应用前景。

The creation and wide application of hybrid rice, so called “second green revolution”, make a great contribution to the food safety of our country. The indica hybrid rice yield accounts for 60% of the total rice production, but in recent thirty years, the increasing of which was attenuated (Fang and Cheng, 2009). Improving efficiency of heterosis between indica and japonica might further increase the rice yield. Given that a major barrier of the hybrid F1 progeny is the extraordinary plant height, which generally results in lodging, previously studies showed that dominant dwarf genes of hybrid parents could recover this negative effect. Therefore, isolation of new dominant dwarf genes is crucial for improving rice yield. In addition to plant height, tillering is another important component of plant architecture and key determinant of rice yield (Wang and Li, 2008). With the discovery of strigolactones (SLs) as root exudate signals that trigger parasitic weed seed germination and play a role in symbiotic plant-arbuscular mycorrhizal fungi interactions, and then as a shoot branching inhibitor, a newly discovered class of carotenoid-derived phytohormones, the next phase of SL research has quickly revealed this hormone class as a major player in optimizing plant growth and development (Gomez-Roldan et al., 2008, Umehara et al., 2008). Until now, their versatile roles in modulating seed germination, seedling photomorphogenesis, shoot morphology, and root development have been widely explored (Xie et al., 2010). Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signaling mechanisms of SL remain poorly understood. In this study, we found that the rice (Oryza sativa) dwarf 53 (d53) mutant, which produces an exaggerated number of tillers mimicking several other rice dwarf (d) mutants defective in SL biosynthesis or signaling, is caused by a gain-of-function mutation. The intermediate phenotype of F1 heterozygous plants and genetic analyses of an F2 population derived from a cross of d53 and the wild-type parent indicated that the d53 mutation behaved in a semi-dominant manner. Exogenous application of a SL analogous, GR24, didn’t effectively inhibit the outgrowth axillary buds of d53. Measurement of SLs produced in the root exudates showed that d53 accumulated markedly higher levels of 2’-epi-5-deoxystrigol (epi-5DS), a native SL of rice, than the wild-type cultivar Norin 8, providing further evidence that like d14, d53 is a rice SL-insensitive mutant. Map-based cloning revealed that D53 encodes a protein sharing a certain similarity with a newly found class of SMAX1 family proteins relating to karrikin signaling emerged from a screen for suppressors of more axillary growth 2 (max2) in Arabidopsis. Sequence analysis revealed that D53 shares a similar secondary structure composition, despite low primary sequence homology, to proteins of the class I Clp ATPase family, which are characterized by an N-terminal domain, a D1 ATPase domain, an M domain and a D2 ATPase domain. Further, to verify that d53 mutation caused the tillering dwarf phenotype, we generated transgenic plants expressing the wild-type or mutant D53 gene in a wild-type background. Notably, all transgenic plants expressing the mutant d53 gene showed a more exaggerated tillering phenotype than those expressing the wild-type D53 gene. Interestingly, overexpression of the wild-type D53 gene also caused a moderate increase in tillering, compared to the vector control plants. Besides, reducing d53 gene expression using an RNA interference (RNAi) approach in a d53 background markedly reduced the tiller number. Taken together, these observations support the proposition that the D53 protein acts as a repressor in the SL-mediated branching-inhibition pathway and that the dominant tillering phenotype of the d53 mutant was most likely caused by a gain-of-function mutation in d53. Previous studies have identified the F-box protein D3 and the α/β hydrolase D14 as two key components of SL signaling in rice. To unravel the underlying mechanism of D53 as a repressor in SL signaling, we examined whether D53 can interact with D14 or D3. Firstly, we showed that D53 could interact with D14 in a SL-dependent manner and the D1 domain of D53 was essential for the interaction. Secondly, consistent with the previously reported GR24-depedent interaction between DAD2 and PhMAX2A (an orthologue of D3 in petunia) in yeast, our in vitro pull-down assay also revealed a direct physical interaction between D14 and D3 in a SL-dependent manner. Thirdly, D53 protein could interact with D3 in the in vitro pull-down assay with or without the presence of GR24. Finally, based on the mass spectrum spectrometry analysis of the samples from in vitro pull-down assay, we concluded that SL could promote the formation of D53–D14–SCFD3 complex. To further investigate how SL regulates D53, we first examined the expression of D53 in relation to SL signaling. qPCR analysis revealed that D53 was widely expressed in the examined rice tissues, preferentially in the parenchyma cells surrounding the xylem, consistent with previous reported the functional sites of SL biosynthesis, response and transport. Moreover, D53 expression was up-regulated by SL treatment in wild-type plants, but down-regulated in six d mutants. These results suggested that D53 expression may be subjected to a negative feedback control of SL signaling. Further, performing a set of additional experiments, we demonstrated that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome. Notably, unlike the wild-type D53–GFP fusion protein, the mutant d53–GFP fusion protein appeared to be stable in the presence of GR24. To provide genetic support for the functional relationship between D53, D3 and D14, we generated d3 d53 and d14 d53 double mutants. The lack of obvious additive effects among these double mutants suggests that D3, D14 and D53 act in the same signaling pathway. To further test their epistatic relationship, we knocked down D53 gene expression in the d3 and d14 backgrounds. Transgenic plants results showed that reduced expression of D53 can partially suppress the dwarf and increased tillering phenotypes of either d14 or d3 mutant, suggesting that D53 functions downstream of D14 and D3 and is a negative regulator of the SL signaling pathway, and that accumulation of D53 protein is responsible for conferring the dwarf tillering phenotype in these mutants. These combined genetic and biochemical data firstly revealed that D53 acts as a repressor of the SL signaling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses. These findings not only shed fresh lights on the signaling mechanisms of SLs and karrikins (a group of SL-analogous seed germination promoting compounds), but also offer new opportunities to dissect the complex interactions among hormones (e.g. auxin, cytokinins, gibberellin), environment (e.g. light and nutrition) and genetics in shaping the form of plant architecture, for both monocots and dicots. Importantly, the discovery and improvement of the d53 mutant remarkably promote potentially practical application of the heterosis between indica and japonica in the breeding and cultivation of rice.