Regulatory mechanisms of the LBD40 transcription factor in somatic embryogenesis.

Somatic embryogenesis (SE) is a process by which an embryo is derived from somatic tissue. Transcription factors (TFs) have been identified that control this process. One such TF that promotes SE is AGAMOUS-like 15 (AGL15). Prior work has shown that AGL15 can both induce and repress gene expression. One way this type of dual function TF works is via protein interactions, so a yeast 2-hybrid (Y2H) screen was undertaken. One intriguing protein with which AGL15 interacted in Y2H was LBD40. LBD40 encodes a LATERAL ORGAN BOUNDARIES (LOB)-domain TF that is unique to plants and is primarily expressed during seed development. Here, we confirm the AGL15-LBD40 interaction by quantitative assays and co-immunoprecipation. We also document a role for LBD40, and the closely related protein LBD41, in supporting SE. To determine downstream genes potentially controlled by LBD40, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) was used. More than 400 binding regions for LBD40 were consistently found genome-wide. To determine genes responsive to LBD40/41 accumulation, RNA-seq analysis of transcriptomes of wild-type control and loss-of-function was performed. Combining these datasets provides insight into genes directly and indirectly controlled by these LOB domain TFs. The gene ontology (GO) enrichment analysis of these regulated genes showed an overrepresentation of biological processes that are associated with SE, further indicating the importance of LBD40 in SE. This work provides insight into SE, a poorly understood, but essential process to generate transgenic plants to meet agricultural demands or test gene function. This manuscript reports on experiments to understand the role that LDB40, a TF, plays in support of SE by investigating genes directly and indirectly controlled by LBD40 and examining physical and genetic interactions with other TFs active in SE. We uncover targets of LBD40 and an interacting TF of the MADS family and investigate targets involvement in SE.