Differential Expression of the Diverse Plant Actins

描述（由申请人提供）：我们提出有两类基于肌动蛋白的细胞骨架系统，营养（VEG）和生殖（REP），它们在维管植物中相对独立地发挥作用和进化超过3.5亿年，类似于脊椎动物中的细胞质和肌肉肌动蛋白系统。为了了解基因调控和蛋白质同源序列差异的相对重要性，我们研究了编码8个肌动蛋白（ACT），5个profilins（PRF），11个肌动蛋白解聚因子（ADF）和6个核肌动蛋白相关蛋白（阿普）的基因家族。ACT，ADF和PRF细胞骨架家族包含具有VEG或REP表达模式的基因的古老亚类，而ARPs组成型表达。ACT、ADF、PRF和阿普基因之间的敲除和敲低等位基因揭示了多细胞发育的每个阶段中的许多不同的表型缺陷。我们构建了高度合成的健康植物与一个单一的VEG肌动蛋白同工酶，以证明VEG基因调控的重要性。我们能够用REP抑制异位REP肌动蛋白基因表达，但不能用VEG、ADFs和PRFs，这证明了同种变体特异性相互作用的重要性。虽然ACT，ADF和PRF缺陷导致细胞骨架的改变，但一些等位基因共享转录因子基因表达的表观遗传变化，导致阿普突变体的发育改变。由于已知核ARPs主要在含有常规肌动蛋白亚基的染色质重塑复合物中起作用，这些结果导致了一个新的提议：发育的主调节因子的转录表达由在染色质重塑复合物中起作用的常规肌动蛋白控制，细胞核中的肌动蛋白水平由ADF和PRFs控制。我们在下一个资助期的具体目标是：1）表征ACT，ADF和PRF突变体的表型; 2）表征基因和同种变体特异性相互作用及其在多细胞发育中的作用; 3）探索这些蛋白质的作用如何在核表观遗传和细胞质细胞骨架功能之间平衡。蛋白质异构体之间的差异及其差异调节在动物多细胞发育中起着重要作用。毫无疑问，我们的实验设计和研究成果为分子医学的此类研究勾勒出了一条道路（例如，人线状肌动蛋白突变）。

DESCRIPTION (provided by applicant): We propose there are two classes of actin-based cytoskeletal systems, vegetative (VEG) and reproductive (REP), which have functioned and evolved with relative independence for more than 350 million years in vascular plants, similar to the cytoplasmic and muscle actin systems in vertebrates. To understand the relative importance of differences in gene regulation and protein isovariant sequence, we examined the gene families encoding eight actins (ACT), five profilins (PRF), eleven actin depolymerizing factors (ADF), and six nuclear actin-related proteins (ARP). The ACT, ADF, and PRF cytoskeletal families contained ancient subclasses of genes with VEG or REP expression patterns, while the ARPs were constitutively expressed. Knockout and knockdown alleles among the ACT, ADF, PRF, and ARP genes revealed numerous and diverse phenotypic defects in every stage of multicellular development. We constructed highly synthetic healthy plants with a single VEG actin isovariant to demonstrate the importance of VEG gene regulation. We were able to suppress ectopic REP actin gene expression with REP, but not VEG, ADFs and PRFs demonstrating the importance of isovariant-specific interactions. While ACT, ADF, and PRF defects resulted in alterations in the cytoskeleton, a few alleles shared epigenetic changes in transcription factor gene expression leading to altered development with ARP mutants. Because nuclear ARPs are known to function primarily in chromatin remodeling complexes containing conventional actin subunits, these results led to a new proposal: the transcriptional expression of master regulators of development is controlled by conventional actins functioning in chromatin remodeling complexes, with the level of actin in the nucleus controlled by ADFs and PRFs. Our Specific Aims in the next grant period are: 1) to characterize the phenotypes of ACT, ADF, and PRF mutants; 2) to characterize gene and isovariant specific interactions and their roles in multicellular development; and 3) to explore how the roles of these proteins are balanced between nuclear epigenetic and cytoplasmic cytoskeletal functions. Differences among protein isovariants and their differential regulation are now widely recognized to play an essential role in animal multicellular development. Undoubtedly, our experimental designs and research outcomes outline a path for such studies in molecular medicine (e.g., human nemaline actin mutations).