Arabidopsis PSEUDORESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome-dependent degradation to discretely phase maximal expression in early evening. While its repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 abundance is unknown. We used mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein-protein interactions at low temperatures. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP-qPCR at C-REPEAT BINDING FACTOR1 (CBF1) and COLD-REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. HOS15 mediates PRR7 turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoters of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15-mediated degradation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways that lead to increased freezing tolerance.

Arabidopsis PSEUDO RESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome-dependent degradation to discretely phase maximal expression in early evening. While its transcriptional repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 protein activity is unknown. We used double mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein-protein interactions. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP-qPCR at C-REPEAT BINDING FACTOR (CBF) and COLD REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. We establish that HOS15 mediates PRR7 protein turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoter regions of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15-mediated regulation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways leading to freezing tolerance through upregulation of CBF1 and COR15A.

Circadian clocks are biological timekeeping systems that coordinate genetic, metabolic and physiological behaviors with the external day-night cycle. The clock in plants relies on the transcriptional-translational feedback loops transcription-translation feedback loop (TTFL), consisting of transcription factors including PSUEDO-RESPONSE REGULATOR (PRR) proteins, plant lineage-specific transcriptional repressors. Here, we report that a novel synthetic small-molecule modulator, 5-(3,4-dichlorophenyl)-1-phenyl-1,7-dihydro-4H-pyrazolo[3,4-d] pyrimidine-4,6(5H)-dione (TU-892), affects the PRR7 protein amount. A clock reporter line of Arabidopsis was screened against the 10,000 small molecules in the Maybridge Hitfinder 10K chemical library. This screening identified TU-892 as a period-lengthening molecule. Gene expression analyses showed that TU-892 treatment upregulates CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) mRNA expression. TU-892 treatment reduced the amount of PRR7 protein, a transcriptional repressor of CCA1. Other PRR proteins including TIMING OF CAB EXPRESSION 1 were altered less by TU-892 treatment. TU-892-dependent CCA1 upregulation was attenuated in mutants impaired in PRR7. Collectively, TU-892 is a novel type of clock modulator that reduces the levels of PRR7 protein.

Phytochrome B (phyB) expressed in the epidermis is sufficient to promote red light responses, including the inhibition of hypocotyl elongation and hypocotyl negative gravitropism. Nonetheless, the downstream mechanism of epidermal phyB in promoting light responses had been elusive. Here, we mutagenized the epidermis-specific phyB-expressing line (MLB) using ethyl methanesulfonate (EMS) and characterized a novel mutant allele of RRC1 (rrc1-689), which causes reduced epidermal phyB-mediated red light responses. The rrc1-689 mutation increases the alternative splicing of major clock gene transcripts, including PRR7 and TOC1, disrupting the rhythmic expression of the entire clock and clock-controlled genes. Combined with the result that MLB/prr7 exhibits the same red-hyposensitive phenotypes as MLB/rrc1-689, our data support that the circadian clock is required for the ability of epidermal phyB to promote light responses. We also found that, unlike phyB, RRC1 preferentially acts in the endodermis to maintain the circadian rhythm by suppressing the alternative splicing of core clock genes. Together, our results suggest that epidermal phyB requires RRC1 to promote light responses by activating the circadian rhythm in Arabidopsis thaliana.

The core of the plant circadian clock involves multiple interlocking gene expression loops and post-translational controls along with inputs from light and metabolism. The complexity of the interactions is such that few specific functions can be ascribed to single components. In previous work, we reported differences in the operation of the clocks in Arabidopsis shoots and roots, including the effects of mutations of key clock components. Here, we have used luciferase imaging to study prr7 mutants expressing CCA1::LUC and GI::LUC markers. In mature shoots expressing CCA1::LUC, loss of PRR7 radically altered behaviour in light:dark cycles and caused loss of rhythmicity in constant light but had little effect on roots. In contrast, in mature plants expressing GI::LUC, loss of PRR7 had little effect in light:dark cycles but in constant light increased the circadian period in shoots and reduced it in roots. We conclude that most or all of the circadian input to the CCA1 promoter in shoots is mediated by PRR7 and that loss of PRR7 has organ-specific effects. The results emphasise the differences in operation of the shoot and root clocks, and the importance of studying clock mutants in both light:dark cycles and constant light.

[This corrects the article DOI: 10.3389/fpls.2019.00667.].

Control of protein turnover is a key post-translational control point in the oscillatory network of the circadian clock. Some elements, such as TOC1 and PRR5 are engaged by a well-described F-box protein, ZEITLUPE, dedicated to their proteolytic turnover to shape their expression profile to a specific time of night. For most other clock components the regulation of their protein abundance is unknown, though turnover is often rapid and often lags the cycling of the respective mRNA. Here we report the design and results of an unbiased genetic screen in Arabidopsis to uncover proteolytic regulatory factors of PSEUDO-RESPONSE REGULATOR 7 (PRR7), a transcriptional repressor that peaks in the late afternoon. We performed EMS mutagenesis on a transgenic line expressing a PRR7::PRR7-luciferase (PRR7-LUC) translational fusion that accurately recapitulates the diurnal and circadian oscillations of the endogenous PRR7 protein. Using continuous luciferase imaging under constant light, we recovered mutants that alter the PRR7-LUC waveform and some that change period. We have identified novel alleles of ELF3 and ELF4, core components of the ELF3-ELF4-LUX Evening Complex (EC), that dampen the oscillation of PRR7-LUC. We report the characterization of two new hypomorphic alleles of ELF3 that help to understand the relationship between molecular potency and phenotype.

Transmembrane adaptor proteins (TRAPs) are structurally related proteins that have no enzymatic function, but enable inducible recruitment of effector molecules to the plasma membrane, usually in a phosphorylation dependent manner. Numerous surface receptors employ TRAPs for either propagation or negative regulation of the signal transduction. Several TRAPs (LAT, NTAL, PAG, LIME, PRR7, SCIMP, LST1/A, and putatively GAPT) are known to be palmitoylated that could facilitate their localization in lipid rafts or tetraspanin enriched microdomains. This review summarizes expression patterns, binding partners, signaling pathways, and biological functions of particular palmitoylated TRAPs with an emphasis on the three most recently discovered members, PRR7, SCIMP, and LST1/A. Moreover, we discuss in silico methodology used for discovery of new family members, nature of their binding partners, and microdomain localization.