microRNA control of stem cell reconstitution and growth in root regeneration

Plants display a remarkable regeneration capacity, which allows them to replace damaged or lost cells, tissues and organs, and thus recover from a broad spectrum of injuries. Even lost stem cells can be regenerated from non-stem cells after competence acquisition, highlighting the enormous plasticity of plant cells. However, the molecular mechanisms underlying this process are still poorly understood. In the root, the highly conserved microRNA miR396 and its targets, the GROWTH REGULATING FACTORs (GRFs), control the transition from stem cells to proliferative cells. miR396 promotes stem cell activity by repressing the GRFs, which in turn promote cell proliferation. Here we show that the miR396-GRF regulatory module guides stem cell reconstitution after root tip excision, playing a dual role: while miR396 promotes competence, the GRFs control regeneration speed. Moreover, plants with ectopic miR396 expression have defined stem cell niches before the excision, but do not reconstitute them afterwards, remaining in an open state despite continuing to grow. We propose that this phenomenon is caused by dispersed stem cell activity, which supports growth after root tip excision without reconstituting the organized stem cell niche typical of Arabidopsis roots. Overall design: Protoplasts from cut (5 days after cut) or uncut (control) roots, from wild-type and miR396-overexpressing plants, were isolated per quadruplicate, and scRNA-seq was performed.