Adamchek, C. | bioRxiv Science

bioRxiv2023

The circadian clock regulates PIF3 protein stability in parallel to red light

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.

plant biology↗

bioRxiv2017

Decoys reveal the genetic and biochemical roles of redundant plant E3 ubiquitin ligases

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.

plant biology↗