Loss of developmental pluripotency occurs in two stages during leaf aging in Arabidopsis thaliana

Aging in plants and animals can result in the loss of cellular pluripotency and the inability to regenerate multiple organs from differentiated, somatic tissues. Detached cotyledons from Arabidopsis thaliana (L.) Heynh. seedlings can proliferate to form callus as well as root and shoot organs in the presence of the phytohormones auxin and cytokinin or their synthetic analogs. In this study, detached cotyledons from the ecotype Landsberg erecta (Ler-0) demonstrated two separate developmental intervals of pluripotency loss. During a 48- to 60-h interval, occurring approximately 4 to 6 d after germination (DAG), detached cotyledons from Ler-0 could proliferate new cells (callus) but lost shoot regeneration competency, which the ecotype Nossen-0 (No-0) retained. In older cotyledons, the pluripotency loss was more severe, as detached cotyledons failed to proliferate callus. In the first interval, Ler-0 cotyledons lost the ability to respond to treatment with a high concentration of synthetic auxin, a level previously hypothesized to promote dedifferentiation. However, both young and old Ler-0 cotyledons were found to induce WUSCHEL::NLS-GUS, a marker for undifferentiated shoot apical meristem cells. The late-stage interval positively correlated to the timing of slowed growth in cotyledons still attached to intact seedlings. Also, cytokinin overexpression could rescue (or delay) this late-stage callus proliferation failure, but not the early-stage decline in shoot regeneration ability. Combined, the results demonstrate that loss of pluripotency in vitro occurs in distinct temporal stages and involves auxin and/or cytokinin signaling. Furthermore, there is genetic variation for this loss of pluripotency within Arabidopsis.