The SBP-Box Gene SPL8 Affects Reproductive Development and Gibberellin Response in Arabidopsis

Plant development requires a precise and dynamic regulatory network, in which transcription factors play major roles by temporally and spatially regulating gene expression. The SBP-box genes encode a group of plant-specific transcription factors, highly conserved in their DNA-binding domain, but quite heterogeneous outside. Gain- or loss-of-function of several SBP-box genes, including Maize LIGULELESS1 and Teosinte glume architecture1, Arabidopsis SPL3, SPL8, and SPL14, display diverse developmental defects, indicating that they function in different developmental processes. The work presented in this thesis aimed to uncover the role of the Arabidopsis SBP-box gene SPL8 in plant development and to integrate its function in an existing regulatory network. This has been achieved through the application of molecular genetic, biochemical and microscopic techniques. As deduced from its semi-sterile loss-of-function phenotype, previous studies have shown that SPL8 affects reproductive development. Remarkably, and extensively described in this thesis, SPL8 gain-of-function also caused sterility. However, unlike the loss-of-function mutant in which anther development is defective at early stage of sporogenesis, gain-of-function affects fertility through the non-dehiscence of anthers. Furthermore, constitutive SPL8 expression resulted in a pleiotropic phenotype partly resembling gibberellin-deficient mutants. Although some phenotypical changes could be rescued by exogenous GAs, seed germination, root elongation and fertility could not. Molecular data indicated that genes involved in GA biosynthesis/response are affected in SPL8 overexpressing lines, suggesting altered endogenous GA levels. Because SPL8 is assumed to encode a transcription factor, its nuclear targeting has also been briefly explored using GFP fusion constructs. Within the SBP-domain, a highly conserved serine residue has been predicted to be a target for phosphorylation. Mutation of this serine residue had a great impact on the subcellular localization of SPL8, suggesting the involvement of post-transcriptional regulation in its function. To obtain additional information on the regulatory network, SPL8 might be integrated in, a yeast two-hybrid screen was performed. Many putative SPL8 interacting proteins could be identified. The functions of most of these are unknown or predicted to be involved in general cellular activities and therefore not very helpful to clear the function of SPL8. For two putative interactors, selected on the basis of their role in gene regulation and expression pattern, respective mutant plants could be obtained. However, the absence of obvious mutant phenotypes allowed no further conclusions concerning the relevance of their interaction with SPL8. The experiments in this thesis investigated the role of SPL8 in plant development. Taken the results together, it could be proposed that SPL8 acts as a local regulator in a subset of GA-mediated developmental processes, positively in anther development, but negatively in seed germination and root elongation. Further studies, aiming at the identification of target genes and genetic interactions with other GA-signaling components, are needed to deepen our understanding of the role SPL8 plays in these more defined aspects of plant development