The Interaction Between Cytokinin And Auxin Controls Stem Cell Specification During In Vitro Shoot Induction In Arabidopsis

Plant organ regeneration plays an important role in plant reproduction and genetic engineering, and also provides an experimental system to study molecular mechanism of plant development. Shoot regeneration is an important way for plant propagation in vitro. So far, shoot regeneration of Arabidopsis was achieved successfully. Stem cells are critical for the formation of shoot apical meristem and must be induced within the callus during shoot induction. Little is understood however about the regulation of this process and how the stem-cell fate becomes determined during this process. In our current study, we showed that the fate of stem cells is determined by cytokinin and auxin within the callus after induction. Using stage-10 pistils as explants, calli were induced on the callus induced medium.Shoots were subsequently induced after each callus was transferred onto the shoot induced medium. Following induction treatment, WUSCHEL (WUS) was firstly expressed in a group of cells within callus, named it the organizing center and then, the expression of CLAVATA3(CLV3) was detected in the cells of layer 1-3 above the organization center. Finally, the transcripts of CLV3 and WUS were localized in the tissues that form the shoot meristem. By inhibition of WUS expression, the regeneration of shoot was significantly reduced. Thus, these results indicate that WUS is induced within the callus and its expression is essential for the in vitro shoot regeneration.We next visualized the behavior and interaction of cytokinin and auxin during stem-cell formation. In the non-induced callus, either cytokinin or auxin gradients were established in its surrounding edge region. However, the regional distribution of both hormones occurred following induction after which WUS was found to be induced in the region containing high levels of cytokinin and low levels of auxin. Furthermore, the polarized PIN-FORMED (PIN) proteins, which are auxin-efflux carriers, were also observed in the regions close to the edge of the callus after induction. Upon the inhibition of cytokinin biosynthesis, the auxin levels decreased and a normal distribution pattern for WUS induction was not observed. Disruption of cytokinin signaling also resulted in an abnormal distribution pattern for auxin. Following inhibition of auxin transport or inhibition of PIN1 expression, the regional distribution of auxin gradients was not detected within the callus and notably, the regional distribution of cytokinin gradients was also not observed. In addition, the pattern of cell proliferation supports the notion that cytokinin and auxin interact.Taken together, our current results suggest that a positive interaction between cytokinin and auxin controls the stem-cell fate, and we propose a model for stem-cell specification control during in vitro shoot induction in Arabidopsis by cytokinin and auxin.