SERK1 links somatic embryogenesis to brassinosteroid signalling

Somatic embryogenesis, the phenomenon where a somatic cell supplied with the right cues can re-initiate embryogenesis, reveals the underlying totipotent character of plant cells. The acquisition of this totipotent state coincides with the expression of the Somatic Embryogenesis Receptor Kinase (SERK). Beyond the observation that SERK1 marks competent cells and increases embryogenic competence when overexpressed, the actual signaling cascade that SERK1 is involved in during the acquisition of embryogenic competence is unknown. The major goal of this study is to get a better insight into the function of SERK1 in somatic embryogenesis and in plant development.In Chapter 1 an introduction to plant stem cell niches, somatic embryogenesis and recent knowledge about the molecular basis of somatic embryogenesis and SERK1 signalling is provided.In Chapter 2, the localization of the SERK1 - Yellow Fluorescent Protein (YFP) fusion protein is described, which coincides with the gene expression pattern as described by Hecht et al (2001). The protein is present in the male and female gametophyte, during sporo- and gametogenesis, in developing embryos and in roots. As observed in protoplasts, also in plants the protein localizes to the plasma membrane.Application of the inhibitor of vesicle trafficking Brefeldin-A revealed that the protein undergoes endocytosis in planta , which is similar to what is seen in protoplasts.In Chapter 3, the application of the single molecule technique fluorescence correlation spectroscopy (FCS) on plants expressing the SERK-eYFP protein is described.The diffusion coefficients of SERK1-eYFP and of the membrane marker eYFP-CAAX were determined with FCS. In addition, the mobile fractions of SERK1-eYFP and BRASSINOSTEROID INSENSITIVE 1 (BRI1)-eGFP or BRI1-eYFP were determined in planta and in protoplasts with fluorescence recovery after photobleaching (FRAP).The FRAP measurements show that in contrast to protoplasts, in plants the major part of the SERK1 and BRI1 receptors is immobile.It was found to be possible to apply FCS in planta to determine the diffusion coefficient of plasma membrane localized SERK1-eYFP. When compared with SERK1-eYFP in protoplasts the diffusion coefficient of SERK1-eYFP in planta was significantly lower.In Chapter 4, the analysis of SERK1 localization in the vascular tissue is described. It is shown that SERK1 localizes to immature vascular cells, suggested to represent the vascular transit amplifying cells, and including the procambium. As a response to 2,4-D, SERK1 protein levels increase in the vascular cells. Prolonged 2,4-D exposure induces the proliferation of cells from the vascular bundle and SERK1 remains associated with these proliferating cells. In Arabidopsis, the procambium is reported to be the main origin of embryogenic cells, so SERK1 expression in this tissue may mark a plant cell population close to a totipotent state.In Chapter 5, experiments are described by which the response of serk1 mutants towards brassinosteroids (BRs) is investigated. It is shown that serk1 mutants have a shift in their responsiveness to BRs. In serk1 serk3 double mutants the sensitivity to BRs is reduced to the level of BRI1 mutants. None of the other SERK family members influences BR sensitivity, suggesting that SERK1 and SERK3 are the only family members redundantly involved in BR signaling.In Chapter 6, the cytological analysis of the vascular tissue of the strong serk1-3 mutant allele is described. In the inflorescence stem of adult plants, the serk1-3 mutant shows various vascular defects pointing to an altered procambium activity such as increased vascular differentiation. The serk1-3 vascular phenotype is not clearly influenced by weak bri1 or serk2 mutant alleles. In serk1 serk3 double mutants the vascular defects are partially reversed, suggesting that SERK1 and SERK3 have opposite roles in vascular tissue development.In Chapter 7, thestudy of the embryogenic competence of strong and weak serk and bri1 mutants is described. The serk1-3 allele, the bri1-201 and the det2 BR biosynthetic mutant have a marked reduction in embryogenic competence compared to wildtype. So, SERK1 not only marks and enhances embryogenic competence when overexpressed, but is also essential for the acquisition of embryogenic competence. These results suggest that BR signaling is required for the acquisition of embryogenic competence in Arabidopsis.In Chapter 8, the results described in the thesis are summarized and discussed and a model is given for the function of SERK1 and BR signaling in regulating cellular pluri- or totipotency.