Evolutionary adaptations to the hormonal regulation of vascular tissue development

Vascular tissues connect shoot and root organs in the vascular plant lineage, providing significant adaptive advantages. Although the crosstalk between auxin and cytokinin in promoting both vascular cell proliferation and differentiation has been well studied in angiosperms such as Arabidopsis thaliana, little is known about this regulation in other vascular plant lineages. Here, we found that unlike the hormonal cross talk found in all other species under study, the lycophyte Selaginella moellendorffii shows clear task separation with auxin driving vascular cell proliferation only, and cytokinin specifically triggering cell differentiation. Using a cross-species transcriptomics approach, we found members of the AUXINs/INDOLE-ACETIC ACIDs (Auxs/IAAs) and CYTOKININ OXIDASEs (CKXs) gene families exhibited divergent expression patterns in response to auxin and cytokinin. Despite these regulatory differences, we show that AtIAA/SmIAA and AtCKX/SmCKX proteins are functionally conserved. Taken together, our findings suggest an evolutionary adaptation to the hormonal regulation of vascular tissue development in which core protein functions are conserved, but regulatory circuits diverged in Lycophytes. Overall design: For Arabidopsis thaliana, the plants (Col-0) were grown on 1/2 Ms plates for 4 days and transferred to mock, 10µM NAA or 10µM BAP for 2h. Next, the 0,2 cm root tips were harvested for RNA-seq. For Selaginella moellendorffii, the experimental setup 1: the plants (WT) were grown on 1/2 Ms plates for 14 days and transferred to mock, 10µM NAA or 10µM BAP for 2h. Next, the 0.2 cm root tips were harvested for RNA-seq. The experimental setup 2: the plants (WT) were grown on 1/2 Ms plates for 14 days and transferred to 1µM NAA or Mock (directly harvest after the treatment) for 7d. Next, the plants were transferred to Mock for 0h, 3h, 6h, 12h, and 24h. The plants were harvest 0.2 cm root tips for RNA-seq in each indipendent time point.