在植物细胞中，微丝通常组装成更高级的结构例如微丝束来作为胞质环流和囊泡运输的轨道。微丝束的形成和维持需要微丝成束蛋白质的参与，这些蛋白质结合在微丝的端部或者侧面将微丝聚集起来形成微丝束。Formin和villin蛋白质家族是两类具有多种肌动蛋白调节活性的微丝成束蛋白质，我们分别选择了这两个蛋白质家族的一个成员AtFH16和AtVLN4作为研究对象，对它们的生化性质和体内功能进行了研究。植物formin根据结构域组成上的不同分为I型和II型两大类，目前关于植物formin的研究大都集中在I型formin上，还没有关于高等植物II型formin的研究报道。AtFH16属于一个典型的II型formin，只具有FH1和FH2结构域，在N端不含其它结构域。我们原核表达并纯化了两个包含AtFH16功能结构域的截短蛋白质：FH2和FH1FH2。AtFH16(FH2)在溶液中形成二聚体和三聚体。AtFH16(FH1FH2)能促进肌动蛋白成核，但其成核能力弱于AtFH8(FH1FH2)；AtFH16的FH1结构域对于其成核能力是必需的，缺少FH1结构域的AtFH16(FH2)丧失了成核能力。AtFH16(FH1FH2)促进形成由混合方向微丝组成的微丝束；单独的FH2结构域就具有成束能力，FH1结构域增强了这种作用。除了调节肌动蛋白的动态变化外，AtFH16(FH1FH2)还能够直接结合并使微管成束；当同时存在微丝和微管时，其优先结合微管。此外，RFP标记的AtFH16(FH1FH2)在洋葱表皮细胞中与微管共定位。AtFH16主要在拟南芥植株发生分枝的部位表达，特异地在组织原基基部的细胞中表达。当在拟南芥中过量表达AtFH16时，其通过促进细胞分裂使转基因植株具有更长的主根和更大的叶片；AtFH16过量表达植株也具有更多的侧根数目，并且在根表皮细胞中具有更多的微丝束。基于这些结果，我们推测AtFH16可能通过同时作用于微丝和微管从而参与细胞分裂而在各种组织或器官的发生过程中发挥作用。Villin属于villin/gelsolin/fragmin蛋白质超家族，该家族蛋白质是目前已知的唯一一类活性与Ca2+相关的肌动蛋白结合蛋白。拟南芥基因组中存在5个编码villin的基因AtVLN1-AtVLN5，其中只有AtVLN1得到了研究。我们原核表达并纯化了AtVLN4的全长蛋白质，对其生化性质进行了系统研究。AtVLN4在高浓度和低浓度Ca2+条件下都能够结合微丝；其以Ca2+浓度依赖性的方式成核、解聚和封端微丝，但以Ca2+浓度不依赖性的方式诱导形成微丝束。在烟草花粉中过量表达AtVLN4后，AtVLN4-GFP与微丝共定位，诱导形成更多的微丝束，减慢花粉管的生长速率和胞质环流速率。此外，AtVLN4在烟草花粉管中抵抗LatB对微丝的解聚作用；并在用Ca2+载体A23187处理时，促进点状肌动蛋白聚集体的形成。AtVLN4在拟南芥根毛细胞中表达，atvln4突变体表现出根毛变短、根毛细胞中微丝骨架异常的表型。atvln4突变体根毛细胞中丧失了长的微丝束，并且微丝束的数量也降低了。基于这些结果，我们认为AtVLN4是拟南芥根毛细胞中负责形成和维持长微丝束的必需组分，同时可能通过解聚和封端作用在根毛细胞顶端调节微丝的动态变化。综上所述，本文研究了两种微丝成束蛋白质AtFH16和AtVLN4的生化性质和生理功能。AtFH16和AtVLN4都能够在体外和细胞内诱导形成微丝束，但它们也具有不同的性质，例如AtFH16能同时作用微管、而AtVLN4的生化性质受到Ca2+的调节等。我们的工作拓展了对植物微丝成束蛋白质的认识。

In plant cells, actin filaments are often organized into higher-order structures, such as cables and bundles, which serve as tracks for cytoplasmic streaming and intracellular transport of organelles and vesicles. Actin bundling proteins are thought to be responsible for the actin bundle formation. Formin and villin are two classes of actin filament bundling proteins that possess multiple actin regulatory activities. Here, we studied the biochemical activities and in vivo functions of AtFH16 and AtVLN4, which belongs to formin and villin family, respectively.Plant formins were grouped into two subclasses: clade I and clade II, based on their domain organization. Several clade I formins have been studied. However, the functional roles of clade II formins have not yet been well characterized in higher plants. AtFH16 is a typical clade II formin. It possesses only FH1 and FH2 domain and has no known daomain at its N-terminal. We generated and purified two recombinant proteins containing the FH1FH2 and the FH2 domain of AtFH16, respectively. AtFH16(FH2) partially formed dimers and trimers in solution. AtFH16(FH1FH2) promoted the polymerization of actin filament from G-actin monomers, while AtFH16(FH2) did not have the activity. Both AtFH16(FH2) and AtFH16(FH1FH2) generated actin bundles where the actin filaments were of mixed orientation. In addition to regulating actin dynamics, AtFH16(FH1FH2) can also bind to and bundle MTs. It bound preferentially to MTs when added to the mixture of MFs and MTs. Moreover, AtFH16(FH1FH2)-RFP colocalized with GFP-MAP4 when transiently transformed into onion epidermal cells by microprojectile bombardment. Promoter/β-glucuronidase expression studies demonstrated that AtFH16 was mainly expressed in the tissues that had branches, including nodes and receptacles etc. AtFH16 was specially expressed in the cells in the bottom of primordium. Overexpression of AtFH16 in Arabidopsis resulted in increase of primary root length and leaf blade size which was attributable to a change in cell number rather than cell size and stimulated supernumerary actin cable formation in the cytoplasm. Taken together, these data suggest that AtFH16 may play an important role in organizing actin filaments and microtubules and facilitate the cell proliferation in plant cells.Villin belongs to villin/gelsolin/fragmin superfamily which are the only known actin binding proteins that regulated by Ca2+. Arabidopis genome has five villin genes: AtVLN1-5. Among them, only AtVLN1 have been detailedly studied in vitro. Here, the biochemical activity of AtVLN4 and its role on actin organization in plant cells were examined. AtVLN4 bound to actin filaments at both high and low concentration of Ca2+. It depolymerized actin filaments, capped the barbed ends and promoted actin nucleation in a Ca2+-dependent manner, but generated actin bundles in a Ca2+-independent manner. Overexpression of AtVLN4 in tobacco pollens induced the formation of more actin cables and caused the slowdown of pollen growth and cytoplasmic streaming rate. Moreover, AtVLN4 resisted the depolymerizing effect of LatB on actin organization in tobacco pollens and promoted the formation of punctate actin foci when treated with Ca2+ ionophore A23187. AtVLN4 is expressed in Arabidopsis root hair cells and AtVLN4-knockout plants exhibited a short-root-hair phenotype and an abnormal actin organization. The prominent long actin cables were almost completely absent in the shanks of atvln4 root hairs and were replaced with some discontinuous short cables. In addition, atvln4 root hairs had consistently less actin cables than wild-type hairs did. These data indicate that AtVLN4 is essential component of the long axial actin cables in Arabidopis root hairs, and suggest it might be involved in regulating actin dynamics through its capping and depolymerization activities in the apical region of the root hairs.In brief, the biochemical acitivities and in vivo functions of AtFH16 and AtVLN4 were investigated in this study. They both stimulated actin bundle formation in vitro and in vivo, and exhibited some different features. The work confirms and extends our understanding of the functions of plant actin bundling proteins.