WOX9 functions antagonistic to STF and LAM1 to regulate leaf blade expansion in Medicago truncatula and Nicotiana sylvestris

Plant specific WOX family transcription factors are known to regulate embryogenesis, meristem maintenance and lateral organ development. Modern clade WOX genes function through a transcriptional repression mechanism, and the intermediate clade transcriptional activator WOX9 functions with the repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. WOX1 homologues STF and LAM1 are required for leaf blade outgrowth in Medicago truncatula and Nicotiana Sylvestris , respectively. Here we show that WOX9 negatively regulates leaf blade outgrowth and functions antagonistically to STF and LAM1 . While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression of NsWOX9 and knockout by CRISPR/Cas9 genome editing in N. sylvestris resulted in a range of severe leaf blade distortions, indicating that controlled negative regulation by NsWOX9 is required for proper blade development. Our results indicate that direct repression of WOX9 transcriptional activation activity by the transcriptional repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris . These findings suggest that a balance between transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in higher plants. One sentence summary WOX9 negatively regulates blade outgrowth antagonizing STF function but directly repressed by STF indicating WOX-mediated homeostasis in cell proliferation and differentiation during leaf morphogenesis.