Institutional Repository of Xishuangbanna Tropical Botanical Garden
| 蒺藜苜蓿复叶发育的分子机制研究 | |
| Alternative Title | The Molecular Mechanism of the Compound Leaf Development in Medicago truncatula |
| 贺亮亮 | |
| Thesis Advisor | 陈江华 |
| 2020 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 中国科学院西双版纳热带植物园 |
| Degree Name | 理学博士 |
| Degree Discipline | 植物学 |
| Keyword | 蒺藜苜蓿，复叶发育，形态建成，BEL1-like homeodomain protein，GRAS transcriptional factor，YUCCA1，LOB Medicago truncatula, compound leaf development, morphogenesis, BEL1-like homeodomain protein, GRAS transcriptional factor,YUCCA1, LOB |
| Abstract | 叶片是植物最重要的光合作用器官，叶的形态为植物的分类提供重要信息。因此，叶发育一直是植物研究领域的热点。基于形态的差异，叶可以分为单叶（Simple leaf）（一个叶片单元构成）和复叶（Compound leaf）（多个叶片单元即小叶），而最吸引人注意的就是千姿百态的复叶了。目前国内外对于单叶发育的研究有许多重要进展，但是复叶发育中的很多重要问题还没有回答，近年来，越来越多的国外植物科学家开始关注这些问题。复叶相比于单叶在形态上要复杂很多，这主要归因于复叶的发育过程中包含了一个由复叶原基干细胞介导的特殊形态建成过（morphogenetic process），即小叶的起始和排列（leaflet initiation and arrangment）根据小叶的排列方式不同，复叶可以分为羽状复叶和掌状复叶两种基本的类型。如建立和调节相关的形态发生活性（morphogenetic activity）、以形成适当的小叶模（即小叶数目和排列），是复叶发育研究的核心问题。豆科模式植物蒺藜苜（Medicago truncatula）具有典型的三出复叶，远轴端由一对侧小叶和一片顶小叶构成，近端为一对托叶包裹叶柄，侧小叶和顶小叶叶片基部均有感应昼夜节律变化的运动器称为叶枕。前期的研究表明：FLORICAULA (FLO)/LEAFY（LFY）的同源基SINGLELEAFLET1（SGL1）是维持复叶形态发生活性的核心因子，为侧部小叶始和发育所必须的；一个C2H2 锌指蛋白PALMATE-LIKE PENTAFOLIAT（PALM1）是关键的决定因子（determinancy factor），负调控SGL1 的时空表达，从而确立蒺苜蓿的三出复叶形态；sgl1 突变体变为单叶，而palm1 突变体具有五个小叶，以类状聚集在叶柄的顶部。本研究通过正向遗传学筛选到一类新的五叶突变体pinna（pinnate-like pentafoliata1）和pinna2，不同于palm1 突变体中小叶以掌状聚集pinna1 和pinna2 突变体中五个小叶以羽状方式排列，额外增生的两片小叶对生于端小叶的基部，形成类羽状复叶模式。首先，我们通过基于Tnt1 侧翼序列的分析，克隆到PINNA1 基因编码一个BEL1-like 亚家族同源异型结构域（homeodomain）蛋白，转基因互补实验验证了其在复叶发育上的功能。亚细胞定位分析表明PINNA1 基因编码的蛋白定位在细核。原位杂交显示PINNA1 基因特异地在不同时期叶原基中表达，在侧小叶和顶小叶原基中均有表达，而在顶端分生组织SAM 区和托叶原基检测不到明显信号。进一步通过遗传杂交构建多基因突变体表明，SGL1 基因为PINNA1 基因的遗传上位，pinna1 突变体增生的小叶主要但不是完全依赖于SGL1 基因的功能，pinna1 sgl1 双突变表型类似于sgl1 表型；pinna1 突变体中SGL1 的表达量和空间得到显著的增加和扩大，体内外生化实验表明PINNA1 蛋白能与SGL1 启动子特异结合抑制SGL1 基因的表达。与palm1 elp1 双突变体杂交（elp1 突变体使叶枕突变，不影响小叶数目），构建多基因突变体的表型分析显示，pinna1 palm1 双突变体和pinna1 palm1 elp1 三突变体产生最多可多达13 片小叶的二级复叶。综合多方面的数据显示：在顶小叶区域PINNA1 单独发挥作用，而侧小叶区域PALM1 作为主效调控因子（master regulator）、PINNA1 作为次级调控因子（secondary regulator），通过协同作用（synergy）来调节SGL1 基因在复叶发育过程中的时序表达，从而实现对复叶形态发生活性的精细控制，最终决定蒺藜苜蓿的三出复叶模式。另一方面，我们通过图位克隆分离到PINNA2 基因，发现它编码一类新的GRAS 蛋白。35S::PINNA2 转基因能够互补pinna2 突变体的表型；而在野生型中过表达PINNA2 会引起植株矮化。PINNA2-GFP 蛋白定位于细胞核，但是在酵母中的转录激活实验表明PINNA2 蛋白单独不具有转录激活活性；酵母双杂交实验表明PINNA2 蛋白可以通过自身的GRAS 结构域形成同源二聚体。原位杂交结果显示PINNA2 特异地在器官边界（boundary）区域表达。通过遗传杂交分析表明，SGL1 是PINNA2 遗传上位基因，pinna2 突变体增生的小叶完全依赖于SGL1基因的功能，pinna2 sgl1 双突变表型为sgl1 表型。pinna2 与已知的小叶边界控制基因FCL1、MtNAM 的功能缺失突变体进行遗传杂交表明，双突变pinna2 fcl1和pinna2 mtnam 的表型分别类似于单突变体fcl1 和mtnam，说明FCL1 和MtNAM都是PINNA2 遗传上位基因。通过酵母文库筛选与PINNA2 互作的关键蛋白，发现多个重要的转录因子如WOX、AP2 等。构建pinna1 pinna2 双突变体，发现其表型与pinna1 和pinna2 单突变体表型类似，都是类羽状五叶表型；但是与pinna1和pinna2 单突变体均有60~75%的五叶频率相比，pinna1 pinna2 双突变体的五叶频率达到100%（都是营养期统计）；qRT-PCR 和原位杂交揭示PINNA1 和PINNA2两个基因的表达相互独立，无论表达量上还是表达区域上，一个基因的突变都不会影响到另一个基因。三突变体pinna2 palm1 elp1 的叶模式和pinna1 palm1 elp1有类似之处，但也有显著不同；而四突变体pinna1 pinna2 palm1 elp1 在三突变体的基础上进一步增加了小叶数目。综合原位杂交、生化和分子实验结果初步揭示，PINNA2 与SGL1、FCL1、MtNAM、PINNA1 等基因间均没有直接的基因调控关系和蛋白互作关系；但是PINNA2 蛋白能与PALM1 蛋白互作。因此，不同于PINNA1 直接调控SGL1 的表达，PINNA2 是通过一种新的机制来控制复叶的形态建成，进一步解析PINNA2 的工作机理将是未来研究的核心问题。第三，发现一组命名为lateral leaflet suppression 1（lls1）的突变体，呈现出两个侧部小叶无规律的生长缺陷，大部分侧小叶发育畸形或生长停滞，而顶部小叶发育则相对正常。扫描电镜分析显示lls1 突变体的叶片发育早期过程（即叶片起始及初级形态建成过程）相对正常，但次级形态建成（即叶片的膨大、生长及成熟等过程）出现发育缺陷。通过对突变体回交后代混合样的全基因组重测序，利用已报道的转座子插入位点鉴定算法（Identification of Transposon InsertionSites）定位了LLS1 基因。序列比对及进化树分析显示，LLS1 编码生长素合成通路中一个重要的黄素单加氧酶MtYUCCA1，与拟南芥、玉米、矮牵牛、豌豆中已报道的YUCCA1 同源。LLS1 早期在叶的起始部位、随后在小叶基部、后期在叶维管组织表达。进一步分析显示，LLS1/MtYUCCA1 基因参与生长素的合成，并通过影响侧小叶的维管组织发育来调节侧小叶的生长过程。通过对野生型植株外源施加YUCCA 酶的化学抑制剂yucasin，也重现了lls1 突变体的表型，进一步证实了LLS1 在蒺藜苜蓿复叶发育中的功能。遗传杂交结果显示LLS1 需要SGL1 基因来参与建立侧小叶，随后作用于侧小叶的次级形态建成过程；而LLS1与PALM1 可能通过不同的机制共同影响侧小叶发育。最后，我们初步解析了跳舞草叶片运动器官叶枕的结构和形态，克隆了叶枕属性相关基因。CmLOB1 和CmLOB2 为两个高度同源的LOB 基因，它们与其他豆科植物叶枕属性基因如ELP1、APU 和SLP 序列高度相似。原位杂交揭示，CmLOB2 在早期叶原基的叶枕部位特异表达，而CmLOB1 在叶枕部位检测不到明显表达。该工作为进一步解析跳舞草叶枕发育奠定了基础。综上所述，基于PINNA1 的研究，我们建立了蒺藜苜蓿复叶模式建成的模型。对pinna2 突变体的初步研究我们确定PINNA2 是一个边界特异表达基因，预示着在复叶的形态建成过程中，小叶数目的调控和小叶边界划分间存在着千丝万缕的联系。PINNA2 代表一类特殊的GRAS 蛋白，它广泛存在于水稻、番茄和豆科蒺藜苜蓿、碗豆和百脉根，但是在十字花科拟南芥和碎米荠中缺失；今后如果能进一步阐明不同物种中PINNA2 在复叶模式建成中的作用，将能更好的帮助我们理解复叶形态多样性的分子机理。 |
| Other Abstract | Leaves are primary organs of plants to capture the light for photosynthesis. The leaf shape provides essential information for plant taxonomy. Therefore, leaf development has always been a hotspot for plant scientists. Two basic leaf shapes in nature are the simple form (having a single blade) and the compound form (having multiple units termed leaflets). Significant progress has been made in understanding the simple leaf development. However，several key questions for compound leaf development had not been addressed. Recent years, more and more plant biologists focus on the molecular mechanism behind the diversification of compound leaf shapes. Compound leaves show a more complex pattern than simple leaves, and this is mainly attributed to a specific morphogenetic process (leaflet initiation and arrangement) that occurred during their development. How the relevant morphogenetic activity is established and modulated to form a proper pattern of leaflets is a central question. Two main compound leaf patterns are: palmate, where leaflets are all connected at the tip of the petiole; and pinnate, with leaflets forming two rows along an extension of the petiole called the rachis. The molecular mechanism underlying the different compound leaf patterns remains unclear. Medicago truncatula serves as one of key models in the research of compound leaf development, with leaves having a typical trifoliate pattern. The FLORICAULA (FLO)/LEAFY (LFY) orthologue SGL1 is key to the control of this pattern formation, and necessary for leaflet initiation. The sgl1 mutants produce simple leaves with a single leaflet, due to failure in initiating lateral leaflet primordia. Previously, we characterized a palmate-like pentafoliata1 (palm1) mutant that developed leaves with five leaflets arranged in a palmate pattern. Identification of the C2H2 zinc finger protein PALM1 as a key repressor of SGL1 transcription strongly suggests that the variation of compound leaf pattern is tightly correlated with the genetic modificationin morphogenetic activity. Here, we identified a novel type of M. truncatula leaf pattern mutants pinnate-like pentafoliata1 (pinna1) and pinna2 which form five leaflets on compound leaves arranged pinnately.At first, based on a Tnt1 insertion site-based mapping strategy, we successfullycloned the candidate gene corresponding to a putative BEL1-like homeodomain (BLH)gene Medtr3g112290. Both 35S::PINNA1-GFP and pPINNA1::GFP-PINNA1 cancomplement the mutant phenotype. GFP-PINNA1 and PINNA1-GFP were mainly localized to the nucleus, consistent with the putative role of PINNA1 as a transcriptional regulator. PINNA1 is expressed in all leaflet primordia and promotes the determinate growth mainly by directly repressing the transcription of the LEAFY(LFY) ortholog SINGLE LEAFLET1(SGL1). Analysis of the sgl1 pinna1 double mutant indicates that SGL1 is required for the proliferation of all the lateral leaflets in the pinna1 mutant. And more amazingly, the palm1 pinna1 double mutant produces higher-ordered compound leaves consisting of two orders and up to 13 leaflets in total. Further data demonstrate that, as a determinacy factor of leaf morphogenesis, PINNA1 not only functions alone in the terminal leaflet region but also synergizes with another key determinacy factor, the C2H2 zinc finger PALM1, in the lateral leaflet regions to define the spatiotemporal expression of SGL1, leading to an elaborate control of morphogenetic activity. The research of PINNA1 not only reveals a framework for the trifoliate leaf pattern formation but also sheds light on mechanisms generating diverse leaf forms. Secondly, by using a map-based approach, we identified the PINNA2 gene that encodes a novel type of GRAS transcriptional factors. The 35S::PINNA2 transgenic plants showed a full complementation of the leaf pattern phenotype, while several lines of 35S::GFP-PINNA2 overexpression transgenic plants exhibited a dwarf phenotype. PINNA2-GFP was mainly localized to the nucleus. Further results showed that PINNA2, differently from many known GRAS proteins (i.e. DELLA and MIG1), was unable to activate yeast growth when fused to the GAL4 DNA-binding domain. However, PINNA2 can form homodimers through its GRAS domain in yeast cells.RNA in situ hybridization indicated that PINNA2 was specially expressed at the boundaries between the SAM and lateral organs, stipules and leaflets, and leaflets and leaflets. Genetic analysis showed that SGL1, FCL1 and MtNAM were epistatic to PINNA2 in controlling leaf pattern. However, we have not identified a direct regulatory interaction or protein-protein interaction between PINNA2 and SGL1, FCL1 or MtNAM. Compared to pinna1 or pinna2 single mutants which produce approximate 60-75% pinnate pentafoliate leaves, pinna1 pinna2 double mutants produce almost 100% pentafoliate leaves. QRT-PCR and RNA in situ hybridization demonstrated that PINNA1 and PINNA2 were expressed independently of each other. There are similarities, but also significant differences between the compound leaf patterns of the triple mutant pinna2 palm1 elp1 and pinna1 palm1 elp1. However, the compound leaves of the tetrad mutant pinna1 pinna2 palm1 elp1 showed a significantly increased number of leaflets. A direct protein-protein interaction was not observed between PINNA1 and PINNA2, but was found between PINNA2 and PALM1. Therefore, unlike PINNA1, which directly represses SGL1 transcription, PINNA2 controls the development of compound leaves through a new mechanism, and further analysis of its working mechanism will be the core of future research. The third, using genome resequencing approaches, we identified six mutant alleles of the LATERAL LEAFLET SUPPRESSION1 (LLS1) gene, encoding the auxin biosynthetic enzyme YUCCA1 in M. truncatula. Linkage analysis and complementation tests showed that the phenotypes of lls1 mutant were caused by the Tnt1 insertions that disrupted the LLS1 gene. The transcripts of LLS1 can be detected in primordia at early stages of leaf initiation and later in the basal regions of leaflets, and finally in vein tissues at late leaf developmental stages. The vein numbers and auxin level are reduced in lls1-1 mutant. Analysis of the lls1 sgl1 and lls1 palm1 double mutants uncovered that SGL1 is epistatic to LLS1, and LLS1 works with PALM1 in an independent pathway to regulate the growth of lateral leaflets. Our work demonstrates that the YUCCA1/YUCCA4 subgroup plays very important roles in the outgrowth of lateral leaflets during the compound leaf development of M. truncatula,in addition to leaf venation.Finally, the structure and morphology of the leaf motor organ (pulvinus) of dancing grass were analyzed, and the genes related to the motor organ identity control were cloned. CmLOB1 and CmLOB2 are two highly homologous LOB genes. Both encoded proteins share high sequence similarity with the motor organ specific LOB proteins of other legume species, such as ELP1, APU and SLP. In situ hybridization revealed that CmLOB2 was specifically expressed in the pulvinus region of the early leaf primordium, while the transcript of CmLOB1 was not detected in the pulvinus region. It laid a foundation for the further analysis of the development of dancing grass pulvinus. In summary, based on the research of pinna1, we have established a molecular framework underlying the compound leaf pattern of M. truncatula. The preliminary study on pinna2 mutant showed that the PINNA2 was a boundary specific gene, indicating tight and complex relationships between the maintenance of the leaflet number and the definition of boundaries between leaflets during leaf morphogenesis. PINNA2 represents a novel group of GRAS proteins, which was conserved in rice, tomato and legumes, but not not found in the Brassicaceae species Arabidopsis thaliana and Cardamine hirsuta. The future investigation of the role of PINNA2 in compound leaf development of different species will help to elucidate the molecular mechanism underlying diversification of compound leaf form during evolution. |
| Pages | 170 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | https://ir.xtbg.ac.cn/handle/353005/11701 |
| Collection | 西双版纳热带植物园毕业生学位论文 |
| Affiliation | 1.中国科学院大学; 2.中国科学院西双版纳热带植物园 |
| Recommended Citation GB/T 7714 | 贺亮亮. 蒺藜苜蓿复叶发育的分子机制研究[D]. 中国科学院西双版纳热带植物园. 中国科学院大学,2020. |
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| 贺亮亮.pdf（16028KB） | 学位论文 | 开放获取 | CC BY-NC-SA | Application Full Text | ||
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