Ectopic cambia in wisteria vines are associated with the expression of conserved KNOX genes.

Secondary vascular growth is a conserved mechanism that gives rise to vascular tissues produced via a single vascular cambium. Molecular mechanisms underlying this process are characterized mainly in model species with typical vascular architectures, while the genetics underlying ecologically-important atypical vascular architectures remain unexplored. We use developmental anatomy, comparative transcriptomics, molecular evolutionary analyses, and heterologous gene expression to address this knowledge gap, investigating how multiple ectopic cambia (EC) form in the woody vine Japanese wisteria. Anatomical studies show EC in Japanese wisteria arise from cortical parenchyma, and cambium-specific transcriptome comparisons reveal that genes acting as regulators of typical cambium development in model species are likewise associated with EC development. Gene trees of KNOX genes suggest that duplication events may contribute to EC formation, including a Fabaceae-specific duplication of KNAT2/6, which is detected as being under positive selection. We also demonstrate that KNOX genes from Japanese wisteria show canonical KNOX-like activity in heterologous functional assays, although no vascular aberrations were observed. Overall, these findings provide the first insights into the genetics of EC formation in "natural variants", advancing our understanding of the molecular mechanisms regulating vascular variants in seed plants.