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

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.

Functional characterization of PETIOLULE-LIKE PULVINUS (PLP) gene in abscission zone development in Medicago truncatula and its application to genetic improvement of alfalfa.

Alfalfa (Medicago sativa L.) is one of the most important forage crops throughout the world. Maximizing leaf retention during the haymaking process is critical for achieving superior hay quality and maintaining biomass yield. Leaf abscission process affects leaf retention. Previous studies have largely focused on the molecular mechanisms of floral organ, pedicel and seed abscission but scarcely touched on leaf and petiole abscission. This study focuses on leaf and petiole abscission in the model legume Medicago truncatula and its closely related commercial species alfalfa. By analysing the petiolule-like pulvinus (plp) mutant in M. truncatula at phenotypic level (breakstrength and shaking assays), microscopic level (scanning electron microscopy and cross-sectional analyses) and molecular level (expression level and expression pattern analyses), we discovered that the loss of function of PLP leads to an absence of abscission zone (AZ) formation and PLP plays an important role in leaflet and petiole AZ differentiation. Microarray analysis indicated that PLP affects abscission process through modulating genes involved in hormonal homeostasis, cell wall remodelling and degradation. Detailed analyses led us to propose a functional model of PLP in regulating leaflet and petiole abscission. Furthermore, we cloned the PLP gene (MsPLP) from alfalfa and produced RNAi transgenic alfalfa plants to down-regulate the endogenous MsPLP. Down-regulation of MsPLP results in altered pulvinus structure with increased leaflet breakstrength, thus offering a new approach to decrease leaf loss during alfalfa haymaking process.