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Time lapse microscopy is a transformative technique for plant cell and developmental biology. Light sheet microscopy, which manipulates the amount of light a sample is exposed to in order to minimize phototoxicity and maximize signal intensity, is an increasingly popular tool for time lapse imaging. However, many light sheet imaging systems are not designed with the unique properties of plant samples in mind. Recent advances have decreased the cost and increased the technical accessibility of light sheet microscopy, but plant samples still require special preparation to be compatible with these new systems. Here, we apply a novel light sheet microscopy system to regenerating Arabidopsis roots damaged via laser ablation. To adapt this system for Arabidopsis roots we establish a new protocol for sample mounting, as well as an automated root tip tracking system that requires no additional proprietary software. The methods presented here can be used to increase researcher access to long-term time-lapse imaging in Arabidopsis biology. 

Most plant roots have multiple cortex layers that make up the bulk of the organ and play key roles in physiology, such as flood tolerance and symbiosis. However, little is known about the formation of cortical layers outside of the highly reduced anatomy of Arabidopsis . Here, we used single-cell RNA sequencing to rapidly generate a cell-resolution map of the maize root, revealing an alternative configuration of the tissue formative transcription factor SHORT-ROOT (SHR) adjacent to an expanded cortex. We show that maize SHR protein is hypermobile, moving at least eight cell layers into the cortex. Higher-order SHR mutants in both maize and Setaria have reduced numbers of cortical layers, showing that the SHR pathway controls expansion of cortical tissue to elaborate anatomical complexity.