微丝是植物细胞骨架的重要组成成分之一，对于细胞运动、细胞分裂、细胞极性建立 和维持以及细胞形态发生等多种生理过程都具有非常重要的作用。微丝的功能高度依赖于 其动态变化，即肌动蛋白单体与微丝之间不停的动态转换。微丝的动态在时间和空间上受 到多种不同的肌动蛋白结合蛋白的精细调控，同时消耗大量储能化合物 ATP。目前对于微 丝骨架的研究主要集中于微丝的动态特性和肌动蛋白结合蛋白对微丝的调控作用，而对于 细胞的能量水平如何影响微丝动态的研究还非常有限。 本研究利用微丝解聚药物红海海绵素 B (Latrunculin B, LatB) 筛选拟南芥 T-DNA 突变 体库，发现雷帕霉素靶蛋白 (Target of Rapamycin, TOR) 复合物 1 (TORC1) 中的关键亚基 RAPTOR1B 的突变体 (raptor1b，简写为 rb) 下胚轴具有对于 LatB 处理敏感性降低的表 型。利用微丝解聚药物 LatB 和细胞松弛素 (Cytochalasin D, CD) 分别处理 rb 的不同突变 位点的突变体，确认 rb 突变体下胚轴确实对于微丝解聚药物处理敏感性下降。与此结果一 致的是，TOR 活性被特异性药物 AZD8055 和 Torin2 抑制的植株以及 TOR 表达量下调的植 株均展现出对微丝抑制剂药物敏感性降低的表型。这些结果表明 RAPTOR1B 和 TOR 作为 一个复合体调控植株对于微丝解聚药物的敏感性。接着，利用活细胞成像技术检测野生型、 rb 突变体以及 TOR 抑制剂处理的植株中 mNeonGreen-fABD2 标记的微丝形态和动态，结 果表明 rb 突变体和 TOR 抑制剂处理的植株中的微丝形态均与对照无显著差异，但微丝的 整体动态性相比对照显著降低；进一步对单根微丝动态的分析表明，rb 突变体和 TOR 抑 制剂处理的植株中微丝的延伸速率和切割频率相比对照显著下降、存留时间显著增加。这 些结果表明，TOR 复合体功能受损的植株中微丝变得更加稳定。 为了解析植物 TORC1 的潜在功能，对 TOR 和 RAPTOR1B 的亚细胞定位展开探索。 为此，构建自身启动子驱动、荧光蛋白标记的融合蛋白的表达载体 pRAPTOR1B::EGFPRAPTOR1B 和 pRAPTOR1B::mNeonGreen-RAPTOR1B，利用浸花法获得 rb 突变体背景下的 转基因材料。这些载体回补了 rb 的表型，表明融合蛋白具有正确的功能。亚细胞定位结果 表明，EGFP-RAPTOR1B 定位于胞质和一类细胞器，并且不与高尔基体、胞内体、TGN 的 荧光蛋白标记共定位，表明 RAPTOR1B 不定位于这些细胞器中；相反，RAPTOR1B 与线 粒体染料 mito-tracker 标记的线粒体呈现出高度共定位，表明蛋白定位于线粒体。同时，构 建自身启动子驱动、荧光蛋白标记的融合蛋白的表达载体 pTOR::TOR-3×mNeonGreen，利 用浸花法获得转基因材料。TOR 与线粒体染料 mito-tracker 标记的线粒体也呈现出共定位。 由于线粒体为产能单位，对野生型、rb 突变体以及经 TOR 抑制剂处理的植株的 ATP 水平 展开检测。ATP 检测试剂盒和 ATP 传感器株系的检测结果均表明，rb 突变体和 TOR 抑制剂处理的细胞的 ATP 含量相比对照显著降低；对线粒体膜电位和 Ca2+含量的分析进一步表 明，rb 突变体和 TOR 抑制剂处理的细胞中线粒体的膜电位和 Ca2+含量相较于对照显著下 降。这些数据表明拟南芥 TORC1 定位于线粒体，并通过影响线粒体膜电位和 Ca2+含量调 控细胞的 ATP 水平。 为了探究细胞的 ATP 水平是否影响微丝的动态，使用线粒体抑制剂抗霉素 (antimycin, AA)、寡霉素 (Oligomycin) 和氰氯苯腙 (Carbonyl cyanide m-chlorophenyl hydrazine, CCCP) 分别处理 Col-0 植株、并检测植株对微丝解聚药物的敏感性以及细胞中微丝的动态。结果 表明，经线粒体抑制剂处理的植株对 LatB 的敏感性相比对照显著降低，微丝的形态相比对 照无显著变化，但微丝的整体动态性以及单根丝的延伸速率和切割频率相比对照显著下降。 这些结果表明，线粒体功能的抑制导致植物表现出类似于 rb 突变体和 TOR 抑制剂处理的 植株的表型。 为了探究 TORC1 是否通过控制细胞 ATP 水平来调控微丝的动态，通过外源施加线粒 体抑制剂和 ATP 合成的底物腺嘌呤来改变 TORC1 功能受损植株的 ATP 水平，并检测植株 对微丝解聚药物的敏感性和细胞中微丝的动态。外源施加线粒体抑制剂显著降低了 rb 突变 体、TOR 表达量下调植株、以及 AZD8055 抑制剂处理的植株的 ATP 含量，显著加剧了这 些植株对 LatB 处理敏感性下降的表型，细胞中微丝的整体动态性也进一步降低。这些结果 表明，线粒体抑制剂处理降低微丝动态性的表型不依赖于 TORC1 活性，暗示着 ATP 水平 对微丝的调控作用可能位于 TORC1 下游。与线粒体抑制剂处理结果相反的是，外源施加 腺嘌呤显著提高了 TORC1 功能受损植株的 ATP 水平，突变体植株对 LatB 处理的敏感性 下降以及突变体细胞微丝动态性下降的的表型均得到部分恢复。这些结果表明 TORC1 功 能受损细胞中微丝动态下降的表型至少部分是由于细胞 ATP 水平下降造成的。 综上所述，本研究综合利用药理学、遗传学和细胞生物学等技术手段深入分析了 TORC1 功能受损植株耐受微丝解聚药物和微丝动态下降的表型，揭示了 TOR 和 RAPTOR1B 在胞质和线粒体的定位，提出了植物 TORC1 通过控制细胞 ATP 水平来调控微 丝动态的工作模型。这些实验结果不仅从新的视角即细胞能量水平的角度解析了微丝动态 的调控机制，也揭示了降低微丝动态是植物细胞应对能量短缺条件的策略之一。

As one of the important components of plant cytoskeleton, actin filaments play a very important role in various physiological processes including cell movement, cell division, establishment and maintenance of cell polarity, and cell morphogenesis. The function of actin filaments is highly dependent on their dynamic changes, the continuous dynamic conversion between actin monomers and filaments. The actin dynamics is finely regulated in time and space by a variety of different actin-binding proteins, and at the same time consumes a large amount of the energy storage compounds ATP. At present, the research on actin filaments mainly focuses on the dynamic characteristics of actin filaments and the regulation of actin binding proteins on actin filaments, while the research on how the energy level of cells affects actin dynamics is very limited. In this study, the actin filament-depolymerizing drug Latrunculin B (LatB) was used to screen the homozygous SALK T-DNA collections of Arabidopsis. A mutant of raptor1b, a key subunit of the Target of Rapamycin (TOR) complex 1 (TORC1), was found to be less sensitive to LatB treatment compared to the wild-type plants. LatB and another specific actin cytoskeleton disruptor Cytochalasin D (CD) were used to treat additional independent T-DNA mutants of RAPTOR1B. All the four raptor1b mutants were less sensitive to LatB and CD treatment. Impairment of TOR function, either by genetically knocking down the expression level of TOR or by inhibiting TOR activity using the inhibitors, AZD8055 and Torin2, also resulted in significantly reduced sensitivity to LatB. These data suggest that TOR and RAPTOR1B, most likely as a complex, negatively regulate the sensitivity of seedlings to actin disruptors. The organization of actin filaments in rb mutants and AZD8055-treated Col-0 plants resembled that of WT seedlings under normal growth conditions, but that actin filament dynamics were attenuated. Further analysis of individual actin filament dynamics revealed that, the filament elongation rate and severing frequency were significantly reduced in both rb mutants and AZD8055-treated plants compared to their controls and the filament lifetime was significantly increased. These data suggested that actin filaments become more stable in TORC1-impaired cells. To examine the subcellular localization of TORC1 and hence deduce its possible function, two fluorescently-tagged RAPTOR1B constructs under control of its native promoter, pRAPTOR1B::EGFP-RAPTOR1B and pRAPTOR1B::mNeonGreen-RAPTOR1B were generated, and transformed into rb10 and rb22 mutant plants, respectively. The constructs were functional as they restored the phenotypes of rb mutants. EGFP-RAPTOR1B was localized in cytoplasm and organelles. The organelles were not Golgi apparatus, endosome and TGN. Instead, RAPTOR1B showed substantial co-localization with mito-tracker labelled mitochondria. These data indicate that a portion of RAPTOR1B is localized to mitochondria in plant cells. Fluorescently-tagged TOR constructs under control of its native promoter, pTOR::TOR-3×mNeonGreen was generated and transformed into mutant background. TOR-3×mNeonGreen also showed substantial colocalization with mito-tracker labelled mitochondria. Quantification of ATP concentration revealed that the ATP content was significantly reduced in rb mutants and AZD8055-treated plants compared to their controls. Quantification of mitochondrial membrane potential and Ca2+ content further showed that mitochondrial membrane potential and Ca2+ levels in TORC1-impaired cells were significantly decreased. These data suggest that Arabidopsis TORC1 was localized at mitochondria and regulates ATP levels by affecting mitochondrial membrane potential and Ca2+ content. In order to test whether altered ATP levels impacted the actin cytoskeleton in plant cells, the mitochondrial inhibitors, including antimycin (AA), Oligomycin and carbonyl cyanide mchlorophenyl hydrazone (CCCP), were applied to wild-type plants and their effects on Lat sensitivity were examined. In the presence of mitochondria inhibitors, the seedlings became less sensitive to LatB. The application of mitochondrial inhibitors did not cause obvious alteration of actin filament organization compared to the DMSO-treated plants. However, global actin filament dynamics and individual actin filament dynamics, including elongation rate and severing frequency were significantly reduced, resulting in increased lifetime of actin filaments in oligomycin-treated plants. These data demonstrated that inhibition of mitochondrial functions led to phenotypes mimicking those observed in TORC1-impaired seedlings at both plant growth and actin dynamics levels. To further explore the role of ATP in the reduced actin dynamics of TORC1-impaired plants, these plants were supplied with mitochondrial inhibitors or ATP precursor, adenine. Application of oligomycin significantly reduced ATP levels, LatB sensitivity and the overall actin filament dynamics in all the TORC1-impaired plants. These data hinted that inhibiting mitochondrial ATP synthesis impacted actin dynamics by reducing ATP levels independently of the effect on TORC1. In contrast, exogenous feeding of adenine significantly elevated ATP levels and also restored LatB sensitivity of the TORC1-impaired plants. More importantly, the overall actin filament dynamics and the single actin filament dynamics, including the filament elongation rate, the filament severing frequency and the filament lifetime, were also partially restored in TORC1-impaired cells by the application of adenine. These results together support that adenine can restore actin filament dynamics, most likely by increasing ATP levels, in TORC1-compromised cells. In summary, by pharmacological, genetic and cell biological techniques, this study investigated the mechanism underlying the phenotypes of TORC1-impaired plants, including resistance to actin filament inhibitors and reduced actin dynamics, based on which a working model of plant TORC1 regulating actin dynamics by controlling cellular ATP levels was proposed. These data not only shed light on the regulatory mechanism of actin dynamics from a new perspective, i.e., the cellular energy level, but also support the notion that reducing actin dynamics is one of the strategies of plant cells to overcome energy shortage.