Repressing a repressor: E3 ligase COP1/SPA promotes seed germination by targeting the DELLA protein RGL2

Timely germination ensures proper seedling establishment and therefore has vital importance in the dispersal, adaptation, and survival of plant species. After ripening, seeds enter into dormancy, a physiological state that suppresses germination, caused partly by the interplay of internal hormone signaling with external factors such as light, temperature, and the availability of moisture. Under favorable environmental conditions, the spatio-temporal dynamics of the antagonistic plant hormones gibberellins (GA) and abscisic acid (ABA) determine the timing of dormancy-breaking and seed germination. While a high GA-to-ABA ratio favors germination of imbibed seeds, a low ratio promotes dormancy (Carrera-Castaño et al., 2020). In line with the major role of GA in seed germination, Arabidopsis (Arabidopsis thaliana) mutants with impaired GA-biosynthesis fail to germinate without exogenous GA (Holdsworth et al., 2008). Central to GA signaling is the DELLA domain-containing transcriptional regulators (DELLAs) that repress GA-responsive genes. The GA-receptor GA INSENSITIVE DWARF1 (GID1) perceives bioactive GA and undergoes conformational changes that enable the interaction between GID1 and DELLAs. The F-box proteins SLEEPY1 and SNEEZY, as components of the Skp1–Cul1–F-box-protein (SCF) ubiquitin ligase, target the GA-GID1-DELLA complex for ubiquitin-mediated proteolysis, thereby alleviating the repression of GA-responsive genes and initiating the GA signaling cascade (Bao et al., 2020). Among the five Arabidopsis DELLA proteins (GA INSENSITIVE [GAI], REPRESSOR OF ga1-3 [RGA], RGA-LIKE 1 [RGL1], RGL2, and RGL3), RGL2 functions as the major repressor of GA-mediated seed germination (Carrera-Castaño et al., 2020). Recent work demonstrates that the DELLA proteins GAI and RGA can also be degraded (Blanco-Touriñán et al., 2020) via the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)/SUPPRESSOR OF PHYA-105 (SPA) complex, an evolutionarily conserved E3 ligase that acts as a master regulator of photomorphogenesis (Ponnu and Hoecker, 2021). The COP1/SPA complex directly interacts with and facilitates the degradation of GAI and RGA to regulate plant growth under warm temperature and shade conditions (Blanco-Touriñán et al., 2020). In this issue of Plant Physiology, Lee and co-workers (Lee et al., 2022) show that the COP1/SPA complex promotes seed germination by targeting yet another DELLA protein, RGL2, for ubiquitination and subsequent degradation. The authors observed that exogenous GA restored the slight but significant delay in the germination of cop1-4 (a weak cop1 allele), suggesting a positive role of COP1 in seed germination. However, the presence of the GA-biosynthesis inhibitor paclobutrazol (PAC) impaired the germination of cop1-4 more severely than the wild-type Col-0, implying an enhanced effect of COP1 under GA-deficient conditions. In contrast, the Arabidopsis rgl2 mutant showed PAC insensitivity and the rgl2 mutation completely rescued the PAC-hypersensitive phenotype of cop1-4, indicating the epistatic effect of RGL2 over COP1. Furthermore, the authors showed that RGL2 and COP1 physically interact (Lee et al., 2022). Similar to COP1-RGA/GAI interactions (Blanco-Touriñán et al., 2020), the presence of SPA1 as a bridge protein is essential for the in vivo association of COP1 with RGL2 (Lee et al., 2022), reinforcing the important role of SPA proteins (Hoecker, 2017) in the COP1/SPA E3 ligase complex. Consistent with the idea that COP1 destabilizes RGL2, the recombinant MBP-RGL2 degraded faster when incubated with soluble protein extracts of a COP1-overexpressing line, but slower with extracts of cop1-4 mutant, compared to Col-0 in a cell-free degradation assay. In addition, COP1 could directly ubiquitinate RGL2 in vitro, confirming RGL2 as a direct target of COP1. Furthermore, germination-promoting genes suppressed by RGL2, such as GA-STIMULATED ARABIDOPSIS 6, expansins, and xyloglucan endotransglucosylases/endohydrolases, were downregulated in the cop1-4 mutant. Collectively, Lee and co-workers (Lee et al., 2022) demonstrate that Arabidopsis COP1 destabilizes the downstream ubiquitination target RGL2 and thereby relieves the expression of germination-promoting genes (Figure 1). Whether COP1 targets the other RGLs (RGL1 and RGL3) is presently unknown. COP1 destabilizes RGL2 to promote seed germination. The arrows and dashed arrows denote direct and indirect effects, respectively. The lines with blunt ends show negative regulation. The question mark (?) represents unknown mechanisms. Modified from Lee et al. (2022). The components that regulate seed germination via determining the spatiotemporal balance of GA and ABA are often interconnected. For example, PHYTOCHROME INTERACTING FACTOR 1 (PIF1), which enhances COP1 activity during photomorphogenesis, negatively regulates seed germination by promoting the expression of DELLA proteins. Similarly, ELONGATED HYPOCOTYL 5 (HY5), an important target of COP1, may suppress seed germination by promoting ABA signaling (Carrera-Castaño et al., 2020). However, germination assays using the respective mutants suggested a PIF1- and HY5-independent role of COP1 in GA-mediated seed germination (Lee et al., 2022). Also, the purple coloration phenotype (fusca) observed in many light signaling mutants, including cop1-4, cannot generally be associated with GA-related germination defects (Lee et al., 2022). The effect of COP1 on GA and ABA signaling seems to be antagonistic in seeds, but not in seedlings. COP1 enhances GA-mediated seed germination via destabilizing RGL2 (Lee et al., 2022), but suppresses post-germination seedling establishment by participating in the ABA signaling pathway (Yadukrishnan et al., 2020). Reciprocally, both GA and ABA may influence COP1 activity. In line with this, GA application enhanced COP1 protein accumulation in the imbibed seeds via unknown mechanisms (Lee et al., 2022; Figure 1). Similarly, a recent preprint reported the ABA-induced translocation of COP1 into the cytoplasm in seeds (Chen et al., 2021). However, the ABA-induced nucleo-cytoplasmic repartitioning of COP1 negatively regulates ABA-mediated seed germination inhibition. Hence, COP1 may promote seed germination via enhancing downstream signaling of GA and repressing the activities of ABA. Elucidation of the complex interplay between COP1 and the two antagonistic plant hormones GA and ABA requires further research.