The molecular network underlying BABY BOOM‐mediated somatic embryogenesis

Somatic embryogenesis is a form of totipotency that can be induced by applying plant growth regulators or by ectopic overexpression of specific transcription factors. It has a great value in commercial plant breeding for clonal propagation of valuable germplasm. This thesis aims to extend the knowledge of the molecular components of transcription factor-induced somatic embryogenesis, in particular the different levels of BBM-mediated somatic embryogenesis.BBM is one of the eight AIL family transcription factors found in arabidopsis. Chapter 1 introduces the AIL transcription factor family and describes the functions of arabidopsis AIL genes during plant developmental processes, like embryo development, root and shoot development, flower development and regeneration. Some of the signaling pathways in which these proteins function are also presented.Chapter 2 identifies BBM target genes in somatic embryos and characterizes the function of these targets. We show that BBM binds to and regulates expression of the LAFL embryo identity transcription factors, which themselves are positive regulators of (somatic) embryo identity. We uncovered positive (LEC1, LEC2, FUS) and negative (ABI3) roles for these transcription factors in BBM-mediated somatic embryogenesis. We also show that the BBM-PLT clade of AIL proteins are functionally equivalent for somatic embryogenesis induction and that BBM and PLT2 induce somatic embryogenesis in a dose- and context-dependent manner, as was demonstrated previously for root development.We also identified BBM target genes in other signaling pathways, among which auxin pathways genes that were found in early and late stages of somatic embryogenesis. In Chapter 3 we identify BBM target genes during the initiation phase of BBM-mediated somatic embryogenesis in imbibed seeds. We show that genes involved in two tryptophan-dependent pathways, IAA biosynthesis and IAOx metabolism, are some of the earliest target genes during BBM-mediated somatic embryo induction, and that these genes are positive regulators of somatic embryogenesis. We show that auxin is not required for the initial reinstatement of totipotency in cotyledons, despite the early induction of YUCCA auxin biosynthesis genes and auxin accumulation. Rather, auxin is essential for maintaining embryonic identity and for embryo patterning. This chapter also shows that loss of function mutants in the IAOx pathway genes regulated by BBM, negatively affect BBM-induced somatic embryogenesis, even though some of these genes are repressed by BBM. This data identifies a new role for the IAOx pathway in cell totipotency and suggests that the extent and/or timing of these metabolic changes is tightly regulated.BBM and other AILs interact with HOMEODOMAIN GLABROUS (HDG) transcription factors. They act antagonistically to regulate plant cell proliferation and differentiation via protein-protein interaction and regulation of common target genes. Functional analysis of the HDG protein family has been limited by the functional redundancy in this gene family and by the use of T-DNA insertion alleles with different activities. Chapter 4 characterizes the functions of HDG transcription factors in arabidopsis seedling cotyledons. Using CRISPR-Cas9 induced null mutants, we confirmed previously reported seedling phenotypes observed in presumed dominant negative and partial T-DNA insertion lines and also uncovered new interactions and functions. This study highlights the utility of genome editing for targeted and simultaneous mutation of multigene families.A chromatin remodeling protein, PICKLE-RELATED1 (PKR1), was also identified as BBM protein interactor in the same in vitro screen that identified the HDG proteins. In Chapter 5, we use in vivo immunoprecipitation followed by MS/MS of a BBM:GFP protein to identify the components of BBM protein complexes in somatic embryos. We identified PKR1 as BBM protein complex partner, as well as a large number of additional chromatin remodeling proteins. We show that PKR1 regulates the timing of zygotic embryo development and promotes BBM-induced somatic embryogenesis.Finally, Chapter 6 summarizes and discusses the major results of this thesis. We discuss the remaining questions and provide a perspective on future research that can help to better understand BBM function.