Article by: Harper Mason, on 08 July 2023, at 08:27 am PDT

Plants possess a remarkable ability to regenerate completely from a regular cell, known as a somatic cell, which doesn't typically partake in reproduction. This process involves the formation of a new shoot apical meristem (SAM), which gives rise to lateral organs crucial for the plant's reconstruction.

At a cellular level, the formation of SAM is tightly controlled by specific genes and protein molecules that can either encourage or restrict shoot regeneration. However, the identity of these molecules and the existence of additional regulatory mechanisms have remained unknown.

To shed light on these questions, a team of researchers led by the Nara Institute of Science and Technology (NAIST) in Japan conducted a study on Arabidopsis, a plant commonly used in genetic research. Their findings, published in Science Advances, identified and characterized a key negative regulator of shoot regeneration.

The study demonstrated how the WUSCHEL-RELATED HOMEOBOX 13 (WOX13) gene and its protein play a role in promoting the non-dividing function of callus cells, which are non-meristematic. WOX13 acts as a transcriptional repressor at the RNA level, thereby influencing the efficiency of regeneration.

"The search for methods to enhance shoot regeneration efficiency in plants has been a long-standing endeavor. However, progress has been impeded due to unclear regulatory mechanisms. Our study addresses this gap by uncovering a novel pathway for cell-fate specification," explains Momoko Ikeuchi, the principal investigator of the study.

Previous research from the same group had already established the involvement of WOX13 in tissue repair and organ adhesion following grafting. Hence, they initially investigated the potential role of this gene in controlling shoot regeneration by using a two-step tissue culture system and examining a wox13 mutant of Arabidopsis (a plant with dysfunctional WOX13).

Through phenotypic and imaging analysis, they discovered that shoot regeneration occurred at an accelerated rate (three days faster) in plants lacking WOX13, while it was slower when WOX13 expression was induced. Additionally, in normal plants, WOX13 showed reduced expression levels in SAM. These findings suggest that WOX13 negatively regulates shoot regeneration.

To confirm their findings, the researchers compared the wox13 mutants with wild-type plants using RNA sequencing at multiple time points. They observed that the absence of WOX13 did not significantly alter the gene expression of Arabidopsis under callus-inducing conditions. However, under shoot-inducing conditions, the alterations induced by the wox13 mutation were significantly enhanced, leading to the upregulation of genes responsible for regulating shoot meristem.

Interestingly, these genes were suppressed within 24 hours of WOX13 overexpression in mutant plants. Overall, the study revealed that WOX13 inhibits a subset of shoot meristem regulators while directly activating genes that modify cell walls and contribute to cell expansion and differentiation. Further analysis using single-cell RNA sequencing confirmed the critical role of WOX13 in determining the fate of pluripotent callus cells.

This research highlights that unlike other known negative regulators of shoot regeneration, which only prevent the transition from callus to SAM, WOX13 inhibits SAM specification by promoting the acquisition of alternative fates. It accomplishes this inhibition through a mutually repressive regulatory circuit with the WUS regulator. By transcriptionally inhibiting WUS and other SAM regulators while inducing cell wall modifiers, WOX13 acts as a major regulator of regeneration efficiency.

"Our findings demonstrate that knocking out WOX13 can promote the acquisition of shoot fate and enhance shoot regeneration efficiency. This means that WOX13 knockout can serve as a valuable tool in agriculture and horticulture, enabling the tissue culture-mediated de novo shoot regeneration of crops," concludes Ikeuchi.