One sentence summary: 32 WOX9 negatively regulates blade outgrowth antagonizing STF function but directly 33 repressed by STF indicating WOX-mediated homeostasis in cell proliferation and 34 differentiation during leaf morphogenesis. 35 36 44 this article in accordance with the policy described in the Instructions for Authors 45 (www.plantcell.org) is: Million Tadege (million.tadege@okstate.edu). 46 47 48 Plant specific WOX family transcription factors are known to regulate embryogenesis, 49 meristem maintenance and lateral organ development. Modern clade WOX genes 50 function through a transcriptional repression mechanism, and the intermediate clade51 transcriptional activator WOX9 functions with the repressor WOX genes in 52 embryogenesis and meristems maintenance, but the mechanism of this interaction is 53 unclear. WOX1 homologues STF and LAM1 are required for leaf blade outgrowth in 54 Medicago truncatula and Nicotiana Sylvestris, respectively. Here we show that WOX9 55 negatively regulates leaf blade outgrowth and functions antagonistically to STF and 56 LAM1. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and 57 antisense expression partially rescues the lam1 mutant, both overexpression of NsWOX9 58 and knockout by CRISPR/Cas9 genome editing in N. sylvestris resulted in a range of 59 severe leaf blade distortions, indicating that controlled negative regulation by NsWOX9 60 is required for proper blade development. Our results indicate that direct repression of 61 WOX9 transcriptional activation activity by the transcriptional repressor STF/LAM1 is 62 required for correct blade architecture and patterning in M. truncatula and N. sylvestris. 63 These findings suggest that a balance between transcriptional activation and repression 64 mechanisms by direct interaction of activator and repressor WOX genes may be required 65 for cell proliferation and differentiation homeostasis, and could be an evolutionarily 66 conserved mechanism for the development of complex and diverse morphology in higher 67 plants. 68 69 Introduction 70 WUSCHEL-related homeobox (WOX) factors are plant-specific transcriptional regulator 71 proteins that contain a DNA binding homeodomain similar to WUSCHEL (WUS), the 72 founding member of the family from Arabidopsis. Several elegant studies demonstrated 73 that the WOX family is involved in the regulation of a wide range of key developmental 74 programs ranging from the modulation of zygotic development and embryogenesis by 75 WOX2, WOX8, and WOX9 ( Haecker et al., 2004; Breuninger et al., 2008; Ueda et al., 76 2011) to maintenance of shoot and root apical meristems orchestrated by WUS and 77 WOX5, respectively (Mayer et al., 1998; Sarkar, 2007), along with several other 78 4 developmental pathways (Matsumoto and Okada, 2001; Park et al., 2005; Deyhle et al.,