Developmental trajectory of pluripotent stem cell establishment in Arabidopsis callus guided by a quiescent center-related gene network.

In plant tissue culture, callus formation is induced by a high auxin concentration. Among the three cell layers (the outer, middle and inner cell layers) of the callus, pluripotency acquisition in the middle cell layer is required for the potential ability of the callus to regenerate organs. Here, we reveal the developmental trajectory of middle cell layer initiation and maintenance in callus tissue originating from Arabidopsis thaliana hypocotyls. The S phase of the cell cycle is essential for the expression of quiescent center-related SCARECROW (SCR), PLETHORA1 (PLT1) and WUSCHEL-RELATED HOMEOBOX5 (WOX5) genes during the division of callus founder cells to initiate the callus primordium. After callus initiation, SHOOT-ROOT (SHR) proteins move from the inner to the middle cell layer and act together with SCR to promote the expression of PLT1 and WOX5. WOX5 represses the expression of VASCULAR-RELATED NAC-DOMAIN (VND) genes, thereby preventing callus tissue from differentiating into xylem cells. PLT1 and PLT2 directly activate JACKDAW (JKD), which is necessary for pluripotency acquisition in the middle cell layer. We hypothesize that the middle cell layer could have pluripotent stem cell activity and its establishment requires the quiescent center-related SCR-SHR-WOX5-PLT1/2-JKD gene network.