MCA: Understanding cellulose synthase complex in planta using single molecule methods

纤维素主要由植物产生，是植物细胞壁的主要承载成分，提供强度，在植物细胞生长发育中起关键作用。作为“地球上最丰富的生物聚合物”，纤维素也为生物燃料和可再生生物材料提供了巨大的潜在资源，从而实现碳负经济。尽管它很重要，但植物中纤维素生物合成的详细分子机制在很大程度上仍然难以捉摸。该项目通过植物生物学家和光学物理学家之间的协同合作，通过应用先进的显微成像方法来捕捉活植物中纤维素合酶的动态活性，从而解决了这一知识差距。该研究团队将开发新的显微镜技术，以实现植物生长过程中细胞壁生物合成的实时可视化，特别是在亚细胞和分子水平上负责纤维素合成的酶。该结果将为纤维素生物合成提供新的见解，并为进一步合理设计和生产生物基材料提供关键信息。该项目还旨在促进密歇根州立大学和南达科他州矿业与技术学院当前教育和外展项目中年轻研究人员和学生的合作和多学科团队培训，以探索他们对植物科学的兴趣，并发现他们对科学和工程的热情。纤维素的生物合成被描述为由含有多种纤维素合成酶（CESAs）的纤维素合成酶复合物（CSC）在质膜（PM）中进行的空间和时间控制的过程。在拟南芥基因组中，鉴定了10个假定的cesa基因，生化和遗传学研究表明，至少有三种不同的cesa亚型以1:1:1的比例在植物中合成纤维素。然而，CSC的结构及其在合成纤维素中的动态功能在很大程度上是难以捉摸的。本项目旨在利用显微镜技术对植物中CSCs的组装和动态与纤维素微纤维的原位结构进行相关成像。在纤维素生物合成过程中，可能在CSC组装和运输中起关键作用的CESA结构域已经被设计成在相应的CESA敲除背景下表达荧光蛋白标签，用于超分辨率显微镜下的植物成像。具体而言，采用具有增强光子效率的晶格光片（LLSM）和倾斜选择平面照明显微镜系统进行3D/4D单分子跟踪，以提高定位精度和深部组织成像，并使用荧光共振能量转移（FRET）和时间相关单光子计数方法测量CESA-CESA相互作用。此外，还分析了3D/4D轨迹数据与其他初步成像结果的相关性，如AFM成像的纤维素微原纤维的超微结构和受激拉曼散射显微镜成像的细胞壁的物理化学性质。这个项目的发现将使我们能够检验我们的工作假设，并制定未来的研究方向。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Cellulose is predominantly produced by plants as the main load-bearing component of the plant cell wall to provide strength and play critical roles in plant cell growth and development. As “the most abundant biopolymer on earth,” cellulose also provides a great potential resource for biofuels and renewable biomaterials towards a carbon-negative economy. Despite its importance, the detailed molecular mechanism underlying cellulose biosynthesis in plants remains largely elusive. This project addresses this knowledge gap by applying advanced microscopic imaging methods to capture the dynamic activity of cellulose synthase in living plants, achieved through a synergistic collaboration between a plant biologist and an optical physicist. The research team will exploit and develop new microscopy techniques to allow real-time visualization of cell wall biosynthesis in growing plants, specifically the enzymes that are responsible for cellulose synthesis at the subcellular and the molecular levels. The results will provide new insights into cellulose biosynthesis and critical information required for further rational design and production of bio-based materials. The project also aims to foster collaboration and multidisciplinary team training of young researchers and students in the current education and outreach programs at Michigan State University and South Dakota School of Mines and Technology to explore their interests in plant science and discover their passion in science and engineering.The biosynthesis of cellulose has been described as a spatially and temporally controlled process carried out in the plasma membrane (PM) by a cellulose synthase complex (CSC) containing multiple cellulose synthases (CESAs). In the Arabidopsis genome, ten putative cesa genes are identified and biochemical and genetic studies have revealed that at least three different CESA isoforms at a 1:1:1 ratio are required for cellulose synthesis in planta. However, the architecture of CSC and its dynamic function in synthesizing cellulose has been largely elusive. This project aims to exploit and develop microscopy approaches to correlatively image the assembly and dynamics of CSCs in planta and cellulose microfibril structure in situ. The putative CESA domains that may play critical roles in CSC assembly and trafficking during cellulose biosynthesis have been engineered to express fluorescence protein tags in corresponding cesa knockout backgrounds for in planta imaging using super resolution microscopy. Specifically, the lattice light-sheet (LLSM) and oblique selective plane illumination microscopy systems with enhanced photon efficiency are used for 3D/4D single molecule tracking to improve localization accuracy and deep tissue imaging, and fluorescence resonance energy transfer (FRET) and time correlated single photon counting methods are used to measure CESA-CESA interactions. Furthermore, the 3D/4D trajectory data are analyzed to correlate with other preliminary imaging results, such as ultrastructure of cellulose microfibrils imaged by AFM and physicochemical properties of cell walls imaged by stimulated Raman scattering microscopy. The findings from this project will allow us to test our working hypotheses and formulate future research directions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.