Redox and hormone profiling of a Nicotiana tabacum dedifferentiated protoplast culture suggests a role for a cytokinin and gibberellin in plant totipotency

The ability of plant tissues to retain totipotency despite being fully differentiated has been documented for decades. The transition from mature plant tissue to rejuvenated tissue first requires dedifferentiation of mature tissue, followed by rejuvenation (re-entry into the cell cycle) and somatic embryogenesis. We used a Nicotiana tabacum protoplast-based culture system to elucidate the role played by redox and phytohormone networks during the process of dedifferentiation and rejuvenation. Classical markers of redox homeostasis were measured during the rejuvenation process and lipid peroxidation is proposed as the best marker for indicating recovery of cells from oxidative stress sustained during the process of protoplast preparation and culture, prior to rejuvenation. A transient increase at 24 h after culture (HAC) in levels of a cytokinin riboside, iPA, suggests a putative novel function in initiating a stem-cell niche in an auxin dependent manner. A sharp rise at 72 HAC of gibberellin GA₄, furthermore suggests a function for this hormone during the process of rejuvenation. These two key findings are consistent with previously described plant models for lateral root developmental. Therein, iPA could be involved in ‘stem-cell-niche’ initiation. Subsequently, GA₄ could be involved in rapidly suppressing this initiation step following the earliest cell divisions, thereby enabling the establishment of this ‘niche’ into a callus-like tissue.