The plant-specific family of WUSCHEL (WUS)-related homeobox (WOX) transcription factors is key regulators of embryogenesis, meristem maintenance, and lateral organ development in flowering plants. The modern/WUS clade transcriptional repressor STENOFOLIA/LAMINA1(LAM1), and the intermediate/WOX9 clade transcriptional activator MtWOX9/NsWOX9 antagonistically regulate leaf blade expansion, but the molecular mechanism is unknown. Using transcriptome profiling and biochemical methods, we determined that NsCKX3 is the common target of LAM1 and NsWOX9 in Nicotiana sylvestris. LAM1 and NsWOX9 directly recognize and bind to the same cis-elements in the NsCKX3 promoter to repress and activate its expression, respectively, thus controlling the levels of active cytokinins in vivo. Disruption of NsCKX3 in the lam1 background yielded a phenotype similar to the knockdown of NsWOX9 in lam1, while overexpressing NsCKX3 resulted in narrower and shorter lam1 leaf blades reminiscent of NsWOX9 overexpression in the lam1 mutant. Moreover, we established that LAM1 physically interacts with NsWOX9, and this interaction is required to regulate NsCKX3 transcription. Taken together, our results indicate that repressor and activator WOX members oppositely regulate a common downstream target to function in leaf blade outgrowth, offering a novel insight into the role of local cytokinins in balancing cell proliferation and differentiation during lateral organ development.

Summary WOX family transcription factors regulate multiple developmental programs. The intermediate clade transcriptional activator WOX9 functions together with the modern clade transcriptional repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. STF and LAM1 are WOX1 orthologs required for leaf blade outgrowth in Medicago truncatula and Nicotiana sylvestris, respectively. Using biochemical methods and genome editing technology, here we show that WOX9 is an abaxial factor and functions antagonistically to STF and LAM1 to regulate leaf blade development. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression and knockout of NsWOX9 in N. sylvestris resulted in a range of severe leaf blade distortions, indicating important role in blade development. Our results indicate that direct repression of WOX9 by WUS clade repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. These findings suggest that controlling 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 flowering plants.

Transgenic switchgrass overexpressing Lolium perenne L. delta1-pyrroline 5-carboxylate synthase (LpP5CS) in group I (TG4 and TG6 line) and group II (TG1 and TG2 line) had significant P5CS and ProDH enzyme activities, with group I plants (TG4 and TG6) having higher P5CS and lower ProDH enzyme activity, while group II plants had higher ProDH and lower P5CS enzyme activity. We found group II transgenic plants showed stunted growth, and the changed proline content in overexpressing transgenic plants may influence the growth and development in switchgrass. RNA-seq analysis showed that KEGG enrichment included phenylpropanoid biosynthesis pathway among group I, group II and WT plants, and the expression levels of genes related to lignin biosynthesis were significantly up-regulated in group II. We also found that lignin content in group II transgenic plants was higher than that in group I and WT plants, suggesting that increased lignin content may suppress switchgrass growth and development. This study uncover that proline can appropriately reduce lignin biosynthesis to improve switchgrass growth and development. Therefore, appropriate reduction in lignin content and increase in biomass are important for bioenergy crop to lower processing costs for biomass fermentation-derived fuels.

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.,

Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield.