Analysis of COP1/SPA signalling events in plant developmental responses

Plants are able to perceive the light cues and adjust its development according to the prevalent conditions. An essential core component of the light signaling is the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex, that operates downstream of the photoreceptors to inhibit the light responses. Thus, the COP1/SPA complex is involved in the regulation of a variety of plant developmental processes, such as the tPlants are able to perceive the light cues and adjust its development according to the prevalent conditions. An essential core component of the light signaling is the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex, that operates downstream of the photoreceptors to inhibit the light responses. Thus, the COP1/SPA complex is involved in the regulation of a variety of plant developmental processes, such as the timing of flowering and seedling growth. At the molecular level, COP1/SPA acts as an E3 ubiquitin ligase that promotes the selective degradation of its substrates, which mainly consist of transcription factors, i.e. CONSTANS (CO/BBX1). Since the spanQ and cop1 mutants exhibit severe growth and developmental compromises that cannot be explained by the targets described so far, this study aims to identify and characterize a novel COP1/SPA substrate. In addition, COP1 and SPAs have been shown to act in concert to regulate post-embryonic development. However, the degree at which COP1 and SPAs act independently of each other was not addressed so far. Furthermore, it was unknown whether COP1/SPA plays a role in the regulation of embryogenesis. These novel aspects of the COP1/SPAs co-action are addressed in part II of this thesis. Previously, a screening for interactors of COP1/SPA in vitro suggested that several B-Box transcription factors (BBX), that share similarity with CO, interact with COP1 and SPA1. This study confirms that several BBX proteins bind to COP1/SPA in planta, and focuses on the phenotypical and molecular characterization of CONSTANS-LIKE 12 (COL12/ BBX10). A domain mapping analysis indicated that the CO, CO-Like, TOC1 (CCT) domain of COL12 is important for binding to COP1 whereas the B-boxes are important for binding to SPA1, indicating that COP1 and SPAs might reinforce substrate binding. In agreement with the protein interactions, the COL12 protein undergoes proteolysis at the 26S proteasome in the darkness in a COP1-dependent manner. Both monochromatic red and blue light enhance COL12 protein stability, which contrasts with the destabilizing effects of red light on CO. Therefore, we conclude that COL12 is a novel COP1/SPA substrate. The phenotypical analysis reveals that overexpression of COL12 delays the plant flowering time and enhances branching. Importantly, COL12 repression of flowering is photoperiod-dependent indicating that the protein takes part in the photoperiodic pathway that controls flowering. At the center of this pathway, the CO-FT accumulation is restricted to the LD. The visible light has a prominent role as a positive regulator of CO protein levels. Here, biochemical analyses reveal that COL12 physically interacts with the master regulator of flowering CO, possibly antagonizing its function. In addition, FT mRNA accumulation is reduced in COL12 ox plants compared to WT. Altogether our experiments suggest that COL12 might constitute a fine-tuning mechanism thereby the visible light can enhance CO function by stabilization of the COL12 repressor. In the second part of this thesis, we isolated the quintuple cop1-5 spaQn mutant seedlings, demonstrating that COP1/SPA do not play a role in embryogenesis. We also isolated the cop1-4 spaQn mutant that is completely devoid in the conserved WD-repeats. These quintuple mutant plants resemble the null cop1-5 mutant. This result indicates that the WD- repeat domains provided by the SPA proteins can partially substitute for the lack of the COP1 WD-repeat domain in the COP1-4 protein. Therefore, the WD-repeats are essential for the signaling activity of COP1/SPA. Timing of flowering and seedling growth. At the molecular level, COP1/SPA acts as an E3 ubiquitin ligase that promotes the selective degradation of its substrates, which mainly consist of transcription factors, i.e. CONSTANS (CO/BBX1). Since the spanQ and cop1 mutants exhibit severe growth and developmental compromises that cannot be explained by the targets described so far, this study aims to identify and characterize a novel COP1/SPA substrate. In addition, COP1 and SPAs have been shown to act in concert to regulate post-embryonic development. However, the degree at which COP1 and SPAs act independently of each other was not addressed so far. Furthermore, it was unknown whether COP1/SPA plays a role in the regulation of embryogenesis. These novel aspects of the COP1/SPAs co-action are addressed in part II of this thesis. Previously, a screening for interactors of COP1/SPA in vitro suggested that several B-Box transcription factors (BBX), that share similarity with CO, interact with COP1 and SPA1. This study confirms that several BBX proteins bind to COP1/SPA in planta, and focuses on the phenotypical and molecular characterization of CONSTANS-LIKE 12 (COL12/ BBX10). A domain mapping analysis indicated that the CO, CO-Like, TOC1 (CCT) domain of COL12 is important for binding to COP1 whereas the B-boxes are important for binding to SPA1, indicating that COP1 and SPAs might reinforce substrate binding. In agreement with the protein interactions, the COL12 protein undergoes proteolysis at the 26S proteasome in the darkness in a COP1-dependent manner. Both monochromatic red and blue light enhance COL12 protein stability, which contrasts with the destabilizing effects of red light on CO. Therefore, we conclude that COL12 is a novel COP1/SPA substrate. The phenotypical analysis reveals that overexpression of COL12 delays the plant flowering time and enhances branching. Importantly, COL12 repression of flowering is photoperiod-dependent indicating that the protein takes part in the photoperiodic pathway that controls flowering. At the center of this pathway, the CO-FT accumulation is restricted to the LD. The visible light has a prominent role as a positive regulator of CO protein levels. Here, biochemical analyses reveal that COL12 physically interacts with the master regulator of flowering CO, possibly antagonizing its function. In addition, FT mRNA accumulation is reduced in COL12 ox plants compared to WT. Altogether our experiments suggest that COL12 might constitute a fine-tuning mechanism thereby the visible light can enhance CO function by stabilization of the COL12 repressor. In the second part of this thesis, we isolated the quintuple cop1-5 spaQn mutant seedlings, demonstrating that COP1/SPA do not play a role in embryogenesis. We also isolated the cop1-4 spaQn mutant that is completely devoid in the conserved WD-repeats. These quintuple mutant plants resemble the null cop1-5 mutant. This result indicates that the WD- repeat domains provided by the SPA proteins can partially substitute for the lack of the COP1 WD-repeat domain in the COP1-4 protein. Therefore, the WD-repeats are essential for the signaling activity of COP1/SPA.