The circadian clock is an endogenous oscillator, and its importance lies in its ability to impart rhythmicity on downstream biological processes, or outputs. Our knowledge of output regulation, however, is often limited to an understanding of transcriptional connections between the clock and outputs. For instance, the clock is linked to plant growth through the gating of photoreceptors via rhythmic transcription of the nodal growth regulators, PHYTOCHROME-INTERACTING FACTORs (PIFs), but the clock's role in PIF protein stability is less clear. Here, we identified a clock-regulated, F-box type E3 ubiquitin ligase, CLOCK-REGULATED F-BOX WITH A LONG HYPOCOTYL 1 (CFH1), that specifically interacts with and degrades PIF3 during the daytime. Additionally, genetic evidence indicates that CFH1 functions primarily in monochromatic red light, yet CFH1 confers PIF3 degradation independent of the prominent red-light photoreceptor phytochrome B (phyB). This work reveals a clock-mediated growth regulation mechanism in which circadian expression of CFH1 promotes sustained, daytime PIF3 degradation in parallel with phyB signaling.

The circadian clock is an endogenous oscillator, but its importance lies in its ability to impart rhythmicity on downstream biological processes or outputs. Focus has been placed on understanding the core transcription factors of the circadian clock and how they connect to outputs through regulated gene transcription. However, far less is known about posttranslational mechanisms that tether clocks to output processes through protein regulation. Here, we identify a protein degradation mechanism that tethers the clock to photomorphogenic growth. By performing a reverse genetic screen, we identify a clock-regulated F-box type E3 ubiquitin ligase, CLOCK-REGULATED F-BOX WITH A LONG HYPOCOTYL 1 ( CFH1 ), that controls hypocotyl length. We then show that CFH1 functions in parallel to red light signaling to target the transcription factor PIF3 for degradation. This work demonstrates that the circadian clock is tethered to photomorphogenesis through the ubiquitin proteasome system and that PIF3 protein stability acts as a hub to integrate information from multiple environmental signals.

The Tubby domain, named after the TUBBY protein in mice, binds to phosphatidylinositol 4,5-bisphosphate. Arabidopsis has 11 Tubby domain-containing proteins referred to as Tubby-Like Proteins (TLPs). Of the 11 TLPs, 10 possess the N-terminal F-box domain, which can interact with SKP-like proteins and form SKP1-Cullin-F-box E3 ligase complexes. Although mice TUBBY has been extensively studied, plant TLPs' functions are scarcely detailed. In this study, we show that the Arabidopsis Tubby-like protein 6 (TLP6) and its redundant homologs, TLP1, TLP2, TLP5, and TLP10, positively regulate Arabidopsis immune responses. Furthermore, in an immunoprecipitation mass spectrometry analysis to search for ubiquitination substrates of the TLPs, we identified two redundant phosphoinositide biosynthesis enzymes, phosphatidylinositol 4-kinase β proteins (PI4Kβs), PI4Kβ1 and PI4Kβ2, as TLP interactors. Importantly, TLP6 overexpression lines fully phenocopy the phenotypes of the pi4kβ1,2 mutant, while TLP6 overexpression also leads to increased PI4Kβ2 ubiquitination and reduction in its protein level in a proteasome-dependent manner. Most significantly, TLP6 overexpression does not further enhance the autoimmunity of the pi4kβ1,2 double mutant, supporting the hypothesis that TLP6 targets the PI4Kβs for ubiquitination and degradation. Thus, our study reveals a novel mechanism where TLPs promote plant immune responses by modulating the PI4Kβs protein levels.

Eukaryotic circadian clocks utilize the ubiquitin proteasome system to precisely degrade clock proteins. In plants, the F-box-type E3 ubiquitin ligases ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1), and LOV KELCH PROTEIN2 (LKP2) regulate clock period and couple the clock to photoperiodic flowering in response to end-of-day light conditions. To better understand their functions, we expressed decoy ZTL, FKF1, and LKP2 proteins that associate with target proteins but are unable to ubiquitylate their targets in Arabidopsis (Arabidopsis thaliana). These dominant-negative forms of the proteins inhibit the ubiquitylation of target proteins and allow for the study of ubiquitylation-independent and -dependent functions of ZTL, FKF1, and LKP2. We demonstrate the effects of expressing ZTL, FKF1, and LKP2 decoys on the circadian clock and flowering time. Furthermore, the decoy E3 ligases trap substrate interactions, and using immunoprecipitation-mass spectrometry, we identify interacting partners. We focus studies on the clock transcription factor CCA1 HIKING EXPEDITION (CHE) and show that ZTL interacts directly with CHE and can mediate CHE ubiquitylation. We also demonstrate that CHE protein is degraded in the dark and that degradation is reduced in a ztl mutant plant, showing that CHE is a bona fide ZTL target protein. This work increases our understanding of the genetic and biochemical roles for ZTL, FKF1, and LKP2 and also demonstrates an effective methodology for studying complicated genetic redundancy among E3 ubiquitin ligases.

The mapping of protein-protein interaction (PPI) networks and their dynamics are crucial steps to deciphering the function of a protein and its role in cellular pathways, making it critical to have comprehensive knowledge of a protein's interactome. Advances in affinity purification and mass spectrometry technology (AP-MS) have provided a powerful and unbiased method to capture higher-order protein complexes and decipher dynamic PPIs. However, the unbiased calling of nonspecific interactions and the ability to detect transient interactions remains challenging when using AP-MS, thereby hampering the detection of biologically meaningful complexes. Additionally, there are plant-specific challenges with AP-MS, such as a lack of protein-specific antibodies, which must be overcome to successfully identify PPIs. Here we discuss and describe a protocol designed to bypass the traditional challenges of AP-MS and provide a roadmap to identify bona fide PPIs in plants.

The ubiquitin proteasome system (UPS) is the main cellular route for protein degradation in plants and is important for a wide range of biological processes including daily and seasonal timing. The UPS relies on the action of E3 ubiquitin ligases to specifically recognize substrate proteins and facilitate their ubiquitylation. In plants, there are three major challenges that inhibit studies of E3 ligase function: 1) rampant genetic redundancy, 2) labile interactions between an E3 ligase and its cognate substrates, and 3) a lack of tools for rapid validation of bona fide substrates. To overcome these 3 challenges, we have developed a decoy method that allows for rapid genetic analysis of E3 ligases, in vivo identification of substrates using immunoprecipitation followed by mass spectrometry, and reconstitution of the ubiquitylation reaction in mammalian cells to rapidly validate potential substrates. We employ the strategy to study the plant F-box proteins, ZTL, LKP2, and FKF1 revealing differential genetic impacts on circadian clock period and seasonal flowering. We identify a group of circadian clock transcriptional regulators that interact with ZTL, LKP2, and FKF1 in vivo providing a host of potential substrates that have not been seen previously. We then validate one substrate of ZTL, the plant circadian clock transcription factor CHE, and show that ZTL mediates CHE ubiquitylation and that the levels of the CHE protein cycle in daily timecourses. This work further untangles the complicated genetic roles of this family of E3 ligases and suggests that ZTL is a master regulator of a diverse set of critical clock transcription factors. Furthermore, the method that is validated here can be tool employed widely to overcome traditional challenges in studying redundant plant E3 ubiquitin ligases.