Keywords: Other (Epigenetic regulation & Method development)
Multipotent tissues in plants comprised of cells with self-renewal and flexible differentiation capacity, which collectively form different types of meristems. New single cell genomics techniques can provide an unbiased approach for mapping differentiation programs within meristem tissues over time, and for monitoring the impact of genetic perturbations at native cellular resolution. We have recently described the application of sensitive MARS-seq protocol for profiling transcription in developing meristems, which resulted in rich temporal model for tissue-level transcriptional trends. This model can be used for “aligning” mutant data over the wildtype reference, e.g., dissecting florigen dependent and independent signaling in the shoot apical meristem. In another experimental system for plants cellular plasticity and commitment, we will describe a new approach for single cell RNA-seq in callus tissue, demonstrating how several parallel transcriptional programs emerge over time within epigenetically reprogrammed calli. Thus far, we characterized the gain and loss of pluripotency in callus tissue driven by injury of mature tomato hypocotyls by single-cell RNA analysis of >40K callus cells. This revealed a surprisingly rich heterogeneity of cellular programs within calli, despite the lack of distinct morphological features. A precise map of cell states in the callus and its combination with massively parallel single-callus bulk RNA-seq profiling pointed at multiple gene programs that are activated during reprograming, where only few of them are associated with a hierarchical regulatory cascade related to shoot differentiation. For example, we identified a large sub-population of cells expressing the photosynthetic apparatus that show no clear similarity to any known plant cell-type. The fraction of these photosynthetic cells increased over time and was coupled with gradual of loss proliferative capacity and meristematic features in the callus. Finally, a functional screen pointed at the transcription factor WOX4 as an important gene for callus reprogramming and differentiation and a potential regulator of the callus meristematic niche. Interestingly, wox4 mutated calli show altered kinetics of exit from multipotent state and failed to form the reprogrammed photosynthetic sub-population. This high-resolution analysis of the injury-driven callus provides a new quantitative framework for defining mechanisms of de-differentiation, fate determination and commitment in plants.