Shoot stem cell specification in roots by the WUSCHEL transcription factor.

The WUSCHEL homeobox transcription factor is required to specify stem-cell identity at the shoot apical meristem and its ectopic expression is sufficient to induce de novo shoot meristem formation. Yet, the manner by which WUS promotes stem-cell fate is not yet fully understood. In the present research we address this question by inducing WUS function outside of its domain. We show that activation of WUS function in the root inhibits the responses to exogenous auxin and suppresses the initiation and growth of lateral roots. Using time lapse movies to follow the cell-cycle marker CYCB1;1::GFP, we also show that activation of WUS function suppresses cell division and cell elongation. In addition, activation of WUS represses the auxin-induced expression of the PLETHORA1 root identity gene and promotes shoot fate. Shoot apical meristem formation requires a high cytokinin-to-auxin ratio. Our findings provide evidence for the manner by which WUS specifies stem-cell identity: by affecting auxin responses, by reducing the cell mitotic activity and by repressing other developmental pathways. At the meristem, the stem-cells which are characterized by low division rate are surrounded by the highly proliferative meristematic cells. Our results also provide a model for WUS establishing the differential mitotic rates between two cell populations at the minute structure of the meristem.

Competency for shoot regeneration from Arabidopsis root explants is regulated by DNA methylation.

Plants exhibit high capacity to regenerate in three alternative pathways: tissue repair, somatic embryogenesis and de novo organogenesis. For most plants, de novo organ initiation can be easily achieved in tissue culture by exposing explants to auxin and/or cytokinin, yet the competence to regenerate varies among species and within tissues from the same plant. In Arabidopsis, root explants incubated directly on cytokinin-rich shoot inducing medium (SIM-direct), are incapable of regenerating shoots, and a pre-incubation step on auxin-rich callus inducing medium (CIM) is required to acquire competency to regenerate on the SIM. However the mechanism underlying competency acquisition still remains elusive. Here we show that the chromomethylase 3 (cmt3) mutant which exhibits significant reduction in CHG methylation, shows high capacity to regenerate on SIM-direct and that regeneration occurs via direct organogenesis. In WT, WUSCHEL (WUS) promoter, an essential gene for shoot formation, is highly methylated, and its expression on SIM requires pre-incubation on CIM. However, in cmt3, WUS expression induced by SIM-direct. We propose that pre-incubation on CIM is required for the re-activation of cell division. Following the transfer of roots to SIM, the intensive cell division activity continues, and in the presence of cytokinin leads to a dilution in DNA methylation that allows certain genes required for shoot regeneration to respond to SIM, thereby advancing shoot formation.