To seek answers to the above questions, a research group led by Nara Institute of Science and Technology (NAIST), Japan studied the process inArabidopsis, a plant commonly used in genetic research. Their research -- which was published inScience Advances-- identified and characterized a key negative regulator of shoot regeneration. They demonstrated how theWUSCHEL-RELATED HOMEOBOX 13(WOX13) gene and its protein can promote the non-meristematic (non-dividing) function of callus cells by acting as a transcriptional (RNA-level) repressor, thereby impacting regeneration efficiency.

"The search for strategies to enhance shoot regeneration efficiency in plants has been a long one. But progress has been hindered because the related regulatory mechanisms have been unclear. Our study fills this gap by defining a new cell-fate specification pathway," explains Momoko Ikeuchi, the principal investigator of this study.

Previous studies from her team had already established the role ofWOX13in tissue repair and organ adhesion after grafting. Hence, they first tested the potential role of this gene in the control of shoot regeneration in awox13 Arabidopsismutant (plant with dysfunctionalWOX13) using a two-step tissue culture system. Phenotypic and imaging analysis revealed that shoot regeneration was accelerated (3 days faster) in plants lackingWOX13, and slower whenWOX13expression was induced. Moreover, in normal plants,WOX13showed locally reduced expression levels in SAM. These findings suggest thatWOX13can negatively regulate shoot regeneration.

To validate their findings, the researchers compared thewox13mutants and wild-type (normal) plants using RNA-sequencing at multiple time points. The absence ofWOX13did not considerably alterArabidopsisgene expression under callus-inducing conditions. However, shoot-inducing conditions significantly enhanced the alterations induced by thewox13的突变,导致upregulation梅里开枪stem regulator genes. Interestingly, these genes were suppressed within 24 hours ofWOX13overexpression in mutant plants. Overall, they found thatWOX13inhibits a subset of shoot meristem regulators while directly activating cell wall modifier genes involved in cell expansion and cellular differentiation. Subsequent Quartz-Seq2-based single cell RNA sequencing (scRNA-seq) confirmed the key role ofWOX13in specifying the fate of pluripotent callus cells.

This study highlights that unlike other known negative regulators of shoot regeneration, which only prevent the shift from callus toward SAM,WOX13inhibits SAM specification by promoting the acquisition of alternative fates. It achieves this inhibition through a mutually repressive regulatory circuit with the regulatorWUS, promoting the non-meristematic cell fate by transcriptionally inhibitingWUSand other SAM regulators and inducing cell wall modifiers. In this way,WOX13acts as a major regulator of regeneration efficiency. "Our findings show that knocking outWOX13can promote the acquisition of shoot fate and enhance shoot regulation efficiency. This means thatWOX13knockout can serve as a tool in agriculture and horticulture and boost the tissue culture-mediated de novo shoot regeneration of crops," concludes Ikeuchi.