The plant specific transcription factor LEAFY (LFY) is a master regulator of the vital switch from vegetative to reproductive development. It has a second role in patterning of the reproductive structures, the flowers. Despite its central role in the organism, the direct events set in motion by this transcription factor are as yet poorly understood. Two general approaches will be taken to address this problem. One is a genetic analysis of the biological role of direct LFY targets the investigators previously identified using expression arrays. This study will reveal the regulatory logic connecting known components of the network downstream of LFY. The second approach will use sensitive genomic LFY binding studies to identify missing targets of LFY during the switch to reproductive development and in flower patterning. In addition to advancing our understanding of the events set in motion by LFY in these two processes, this approach will define the binding motif of this master transcription factor with great precision and thus may reveal new roles of LFY. Finally, by comparing LFY binding and activity at two developmental stages (during the switch to reproduction and during flower patterning), this study will provide first evidence as to how LFY activity is regulated during development. Conclusions derived from these experiments will provide much needed insight into a fundamental developmental switch and key patterning events in Arabidopsis. Based on the conserved role of LFY, these findings will likely also contribute to understanding of the onset of reproduction in other flowering plants. This knowledge is of high potential importance for agriculture, plant breeding, and biofuel production. In addition, an important aspect of this project is mentoring and training of students as well as outreach to local schools.
Optimal timing of the onset of flower formation and flower patterning are key for plant survival and for human sustenance, which depends on the seeds formed by flowers. This grant proposal is aimed at characterization of the regulatory network that controls the timing of the formation of the first flower - the meristem identity switch – downstream of LEAFY (LFY) as LFY is a master regulator of this transition. It is a continuation of IOB-0130804 and IOB-0516622, in which we performed microarray-based screens to identify new targets of the plant specific transcription factor LEAFY (LFY) during the meristem identity switch (Wagner et al., 2003; Wagner et al., 2004; William et al., 2004). Our subsequent efforts have focused on (1) characterizing the biological role of the direct LFY we identified in the initial study (William et al., 2004), (2) further elucidating the role of LFY in the onset of flower formation and flower patterning by performing genome-wide binding studies and (3) beginning to unravel the regulatory inputs that control LFY expression. Intellectual Merit 1. Mutants in each of the top five LFY targets we identified (William et al., 2004) enhance the timing defects in the onset of flower formation of a weak lfy allele. Genetic placement of several of these in the regulatory pathway that leads to flower formation has revealed presence of two prominent types of network motifs: coherent feed-forward loops and positive feedback that explain key features of the switch to flower formation in Arabidopsis: its abruptness and irreversibility (Saddic et al., 2006) (Pastore et al., 2011). 2 . We conducted a genome-wide LFY binding study (Winter et al., 2011), which was highly successful, more than 40% of the genes bound by LFY were significantly differentially expressed after LFY activation. We devised stringent criteria for identification of high confidence LFY targets: regulatory regions of such targets should be bound by LFY, the expression of these genes should be LFY dependent and the genes should be co-expressed with LFY. Further examination of the newly identified LFY targets revealed novel insight into how LFY directs flower patterning. In addition, this study revealed a hitherto unknown role for LFY in repression of biotic stimulus response just prior to formation of the first flower. It is likely that this role of LFY serves to optimize resource allocation for optimal fitness during reproductive development. Finally, we identified -using de novo motif analyses- predictive stage specific LFY binding motifs. The de novo motif analysis also identified LFY cofactor motifs, including one frequently present in regions that recruit Polycomb repressors to silence gene expression. This led us to propose that LFY may play a role in overcoming Polycomb repression of genes important in flower development. Indeed LFY recruits another types of chromatin regulator to overcome polycomb repression (Wu et al., 2012). 3. Given that presence of LFY in a new primordium directs it to adopt floral fate, it is critical to understand what pathways control spatiotemporal expression of LFY. We recently identified the age-sensing pathway as a new input into LFY upregulation (Yamaguchi et al., 2009). Additional insight was derived from analysis of evolutionary conservation of the LFY promoter in closely related species. Two highly conserved and functionally important regions were identified. One of these conatined 4 highly conserved Auxin Response Elements, suggesting a possible link between auxin and LFY. Indeed, LFY is induced by auxin and plays a role in flower primordium initiation downstream of auxin (Yamaguchi et al., 2013). Broader Impact We have published 11 research manuscripts and 2 reviews on our studies, with one paper expected to be submitted shortly. I have given a large number of talks on the research funded by this grant at various symposia and meetings. My lab has trained 12 undergraduates over 3 semesters each, 10 are authors on publications resulting from this grant with 2 being first authors. The majority of the undergraduates were female, three were underrepresented minorities. I have trained an additional 20 undergraduates/semester for two semesters in a ‘hands on’ laboratory class, which engages undergraduates in experimental research by addressing an unanswered biological question (supplied by me). I have actively participated in two outreach programs aimed at (1) providing professional training to Philadelphia Inner City high school Biology teachers (last in the fall of 2012) and a (2) offering laboratory research to underprivileged inner city high school students (last in the summer of 2012). We have trained many visiting researchers, including an African American PI on a ROA supplement. Throughout the years, I have focused on helping women and underrepresented minorities overcome obstacles that may impact their research careers. For example, I went to Japan in the summer of 2010 on an NSF funded visit aimed at overcoming the glass ceiling that prevents female STEM scientists from obtaining high level positions in academia.
