HDACs repress runaway stress and cell identity to promote reprogramming in root regeneration

The widespread regenerative capacity of plants is due, in large part, to the ability of specialized cells to reverse their developmental course and reprogram to new cell identities. The path to reprogramming in plants has been termed dedifferentiation or transdifferentiation, but the sequential cellular states of regenerating plant cells remain an open question. To dissect the cellular progression of regenerating plant cells, we use a root regeneration system in the Arabidopsis root that does not require exogenous treatment, allowing us to follow endogenous reprogramming via single cell RNA/ATAC-seq, imaging, and mutant analysis. The earliest events were dependent on repressive chromatin modification, where Multiome analysis showed that Class I histone deacetylases (HDACs) HDA9 and HDA19 were needed to shut down old identities and to prevent a runaway stress response. Cell division mediates a second step needed for the acquisition of many new identity markers, where division rates were tuned by DOF transcription factor OBP1 accelerating and SMR5, 7, and 10 decelerating division rates in later hours. The results show how plants actively mediate the collapse of old identities within hours of injury and then tune cell division rates to rapidly reprogram cells to new identities. Overall design: Arabidopsis root tips were cut at 130um from the tip to induce regeneration. The roots were then collected, uncut (CTR), or at 4h, 9h, 14h, 24h and 36h post cut. Certain replicates involve treatment with TSA, roscovitin and curcumin. Roots were protoplasted and scRNAseq was performed using 10xgenomics 3'UTR kit as per protocol. Data was then processed using CellRanger and Seurat. Additionaly, one dataset was obtained from Arabidopsis root nuclei treated or not with TSA 0 or 2h post cut. Multiome analysis was performed using 10x genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit. Data was then processed using CellRanger and Signac.