ABA-dependent inhibition of the ubiquitin proteasome system during germination at high temperature in Arabidopsis.

During germination, endogenous and environmental factors trigger changes in the transcriptome, translatome and proteome to break dormancy. In Arabidopsis thaliana, the ubiquitin proteasome system (UPS) degrades proteins that promote dormancy to allow germination. While research on the UPS has focused on the identification of proteasomal substrates, little information is known about the regulation of its activity. Here we characterized the activity of the UPS during dormancy release and maintenance by monitoring protein ubiquitination and degradation of two proteasomal substrates: Suc-LLVY-AMC, a well characterized synthetic substrate, and FUSCA3 (FUS3), a dormancy-promoting transcription factor degraded by the 26S proteasome. Our data indicate that proteasome activity and protein ubiquitination increase during imbibition at optimal temperature (21°C), and are required for seed germination. However, abscisic acid (ABA) and supraoptimal temperature (32°C) inhibit germination by dampening both protein ubiquitination and proteasome activity. Inhibition of UPS function by high temperature is reduced by the ABA biosynthesis inhibitor, fluridone, and in ABA biosynthetic mutants, suggesting that it is ABA dependent. Accordingly, inhibition of FUS3 degradation at 32°C is also dependent on ABA. Native gels show that inhibition of proteasome activity is caused by interference with the 26S/30S ratio as well as free 19S and 20S levels, impacting the proteasome degradation cycle. Transfer experiments show that ABA-mediated inhibition of proteasome activity at 21°C is restricted to the first 2 days of germination, a time window corresponding to seed sensitivity to environmental and ABA-mediated growth inhibition. Our data show that ABA and high temperature inhibit germination under unfavourable growth conditions by repressing the UPS.

SnRK1 phosphorylation of FUSCA3 positively regulates embryogenesis, seed yield, and plant growth at high temperature in Arabidopsis.

The transcription factor FUSCA3 (FUS3) acts as a major regulator of seed maturation in Arabidopsis. FUS3 is phosphorylated by the SnRK1 catalytic subunit AKIN10/SnRK1α1, which belongs to a conserved eukaryotic kinase complex involved in energy homeostasis. Here we show that AKIN10 and FUS3 share overlapping expression patterns during embryogenesis, and that FUS3 is phosphorylated by AKIN10 in embryo cell extracts. To understand the role of FUS3 phosphorylation, we generated fus3-3 plants carrying FUS3 phosphorylation-null (FUS3S>A) and phosphorylation-mimic (FUS3S>D) variants. While FUS3S>A and FUS3S>D rescued all the fus3-3 seed maturation defects, FUS3S>A showed reduced transcriptional activity and enhanced fus3-3 previously uncharacterized phenotypes. FUS3S>A embryos displayed increased seed abortion due to maternal FUS3S>A and delayed embryo development, which correlated with a strong decrease in seed yield (~50%). Accordingly, the akin10 and akin11 mutants displayed a frequency of seed abortion similar to fus3-3. When plants were grown at elevated temperature, most phenotypes were exaggerated in FUS3S>A plants, and progeny seedlings overall grew poorly, suggesting that phosphorylation of FUS3 plays an important role during early embryogenesis and under heat stress. Collectively, these results suggest that FUS3 phosphorylation and SnRK1 are required for embryogenesis and integration of environmental cues to ensure the survival of the progeny.

The C-terminal domain of FUSCA3 negatively regulates mRNA and protein levels, and mediates sensitivity to the hormones abscisic acid and gibberellic acid in Arabidopsis.

The transcription factor FUSCA3 (FUS3) controls the transition from the embryonic to the vegetative phase of development by regulating abscisic acid (ABA) and gibberellic acid (GA) levels in Arabidopsis thaliana. In a feedback loop, FUS3 accumulation is negatively and positively regulated by GA and ABA, respectively, by an uncharacterized mechanism. Here, we use a FUS3-GFP construct to show that the level of the FUS3 protein decreases dramatically during mid to late embryogenesis, whereas its mRNA is present at a high level. Deletion studies identify a C-terminal domain (CTD) that negatively regulates mRNA and protein levels, and mediates sensitivity to ABA and GA. Indeed, a CTD-truncated FUS3 variant accumulates at high level, and is insensitive to the destabilizing and stabilizing effects of GA and ABA, respectively. In contrast, fusion of various fragments of the CTD with GFP is sufficient to greatly reduce GFP fluorescence. The GFP-CTD fluorescence can be increased by ABA and paclobutrazol, an inhibitor of GA biosynthesis. Cell-free degradation assays show that FUS3 is a short-lived protein. FUS3 degradation follows the 26S proteasome in vitro and in vivo, and the CTD affects its degradation rate. In contrast to the native form, the CTD-truncated FUS3 is unable to fully rescue the fus3-3 mutant, and is thus required for FUS3 function. In conclusion, this study identifies a CTD that maintains low levels of FUS3 during embryogenesis and early germination, and is required for normal FUS3 function and sensitivity to ABA and GA.

Inhibition of FUSCA3 degradation at high temperature is dependent on ABA signaling and is regulated by the ABA/GA ratio.

During seed imbibition at supra-optimal temperature, an increase in the abscisic acid (ABA)/gibberellin (GA) ratio imposes secondary dormancy to prevent germination (thermoinhibition). FUSCA3 (FUS3), a positive regulator of seed dormancy, accumulates in seeds imbibed at high temperature and increases ABA levels to inhibit germination. Recently, we showed that ABA inhibits FUS3 degradation at high temperature, and that ABA and high temperature also inhibit the ubiquitin-proteasome system, by dampening both proteasome activity and protein polyubiquitination. Here, we investigated the role of ABA signaling components and the ABA antagonizing hormone, GA, in the regulation of FUS3 levels. We show that the ABA receptor mutant, pyl1-1, is less sensitive to ABA and thermoinhibition. In this mutant background, FUS3 degradation in vitro is faster. Similarly, GA alleviates thermoinhibition and also increases FUS3 degradation. These results indicate that inhibition of FUS3 degradation at high temperature is dependent on a high ABA/GA ratio and a functional ABA signaling pathway. Thus, FUS3 constitutes an important node in ABA-GA crosstalk during germination at supra-optimal temperature.