Photolysis of caged cytokinin in single cells of Arabidopsis thaliana

Abstract Background Cytokinins are a class of phytohormone that play a crucial role in the development of plants. They are involved in the regulation of nearly every aspect of plant growth, from germination to senescence. The role of cytokinins in many developmental programs is complex and varies both spatially and temporally. Current techniques used to investigate the functions of cytokinins in plant development lack this spatial and temporal resolution required to observe cell-type specific effects. Results To this end, we present a method of activating a caged cytokinin in single cells. A caged benzyladenine was synthesized, along with caged adenine as a negative control. In vitro testing confirmed ultraviolet light-mediated uncaging, and subsequent root growth assays demonstrated that uncaging produced a cytokinin phenotype. This uncaging was confined to single cells using multiphoton confocal microscopy. Using an Arabidopsis thaliana cytokinin reporter line expressing TCSn::GFP, the resulting GFP expression was confined to the uncaging region, including in single cells. This study presents a novel cell-targeted method of cytokinin delivery, which has the potential to elucidate a broad range of processes in plant development. Conclusions We combined multiphoton confocal microscopy and a caged cytokinin treatment, allowing cell type-specific uncaging of a cytokinin in Arabidopsis roots.

摘要 背景：细胞分裂素（cytokinin）是一类在植物生长发育中发挥关键作用的植物激素（phytohormone），参与调控植物从萌发到衰老的几乎所有生长进程。细胞分裂素在诸多发育程序中的作用机制复杂，且呈现时空特异性差异。当前用于探究细胞分裂素在植物发育中功能的研究技术，均缺乏观测细胞类型特异性效应所需的时空分辨率。
结果：为此，我们开发了一种可在单个细胞中激活笼型细胞分裂素（caged cytokinin）的方法。我们合成了笼型苄基腺嘌呤（caged benzyladenine），并同步合成笼型腺嘌呤作为阴性对照。体外实验验证了紫外光介导的解笼锁（uncaging）过程，后续根系生长实验证实，解笼锁后可诱导产生细胞分裂素相关表型。借助多光子共聚焦显微镜（multiphoton confocal microscopy），该解笼锁过程可被限定在单个细胞范围内。使用表达TCSn::GFP的拟南芥（Arabidopsis thaliana）细胞分裂素报告株系，我们观察到绿色荧光蛋白（GFP）的表达仅局限于解笼锁区域，包括单个细胞中。本研究提出了一种全新的细胞靶向性细胞分裂素递送方法，该方法有望解析植物发育中诸多关键进程。
结论：我们将多光子共聚焦显微镜与笼型细胞分裂素处理相结合，实现了拟南芥根系中细胞类型特异性的细胞分裂素解笼锁。