Research Summary

The researchers identified a key negative regulator of stem regeneration, WOX13, which promotes the fate of non-meristematic cells through a transcriptional repressor, thereby affecting regeneration efficiency. This finding provides new insights into cell fate specification pathways and suggests that knockdown of WOX13 can improve stem regeneration efficiency, which could be a valuable tool in agriculture and horticulture.

Japanese researchers have discovered that the WOX13 gene negatively controls the fate of plant cell regeneration and affects the efficiency of plant stem regeneration.

Plants possess the unique ability to regenerate completely from a somatic cell, that is, a normal cell that normally does not participate in reproduction. This process involves the de novo (or new) formation of a shoot apical meristem (SAM), which generates lateral organs, which are key to plant reconstruction.

At the cellular scale, SAM formation is tightly controlled by positive or negative regulators (genes/protein molecules) that induce or limit stem regeneration, respectively. But which molecules are involved? Are there other regulatory layers that have yet to be discovered? To find answers to the above questions, a research team led by the Nara Institute of Science and Technology (NAIST) in Japan investigated this process in Arabidopsis thaliana, a plant commonly used in genetic research.

Their study, published in Science Advances, discovered and characterized a key negative regulator of shoot regeneration. They show how the USCHEL-associated HOMEOBOX 13 (WOX13) gene and its protein affect regeneration efficiency by acting as transcriptional (RNA level) repressors that promote the non-meristematic (non-dividing) function of callus cells.

"The search for strategies to improve the efficiency of plant stem regeneration has been ongoing for a long time. However, progress has been hampered because the relevant regulatory mechanisms are unclear. Our study fills this gap by defining a new cell fate specification pathway blank," explains Momoko Ikeuchi, lead researcher on the study. Previous research by her team had identified a role for WOX13 in tissue repair and organ adhesion after transplantation. Therefore, they first tested this gene's potential role in controlling stem regeneration in WOX13 Arabidopsis mutants (plants with dysfunctional WOX13) using a two-step tissue culture system.

Phenotypic and imaging analyzes showed that shoot regeneration was accelerated (3 days faster) in plants lacking WOX13, whereas it was slower in plants induced to express WOX13. Furthermore, in normal plants, WOX13 was locally expressed at reduced levels in SAMs. The above results indicated that WOX13 has a negative regulatory effect on stem regeneration.

To validate their findings, the researchers compared WOX13 mutants to wild-type (normal) plants using RNA sequencing at multiple time points. Under callus-inducing conditions, deletion of WOX13 did not significantly change Arabidopsis gene expression. However, shoot-inducing conditions significantly enhanced the changes induced by the WOX13 mutation, resulting in the upregulation of shoot meristem regulatory genes.

Interestingly, in mutant plants, these genes were repressed within 24 hours of WOX13 overexpression. Collectively, they found that WOX13 represses a subset of stem meristem regulators while directly activating cell wall modifying genes involved in cell expansion and cell differentiation. Subsequent Quartz-Seq2-based single-cell RNA sequencing (scRNA-seq) confirmed that WOX13 plays a key role in pluripotent callus cell fate.

This study highlights that, unlike other known negative regulators of shoot regeneration, which only prevent callus transition to SAM, WOX13 inhibits SAM specification by promoting the acquisition of alternative fates. It achieves this suppression through a regulatory circuit that reciprocally inhibits the regulatory factor WUS, which promotes the fate of non-meristematic cells by inducing cell wall modification factors through transcriptional repression of WUS and other SAM regulators.

Thus, WOX13 is a master regulator of regeneration efficiency. “Our results show that knocking out WOX13 can promote the acquisition of shoot fate and improve the efficiency of shoot regulation. This means that WOX13 knockout can be used as a tool in agriculture and horticulture to promote tissue culture-mediated regeneration of new crop shoots,” concluded Ikeuchi.