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Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we first employed mathematical modeling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrhythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modeling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod-insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship among plant circadian genes. [ABSTRACT FROM AUTHOR]

The core plant circadian oscillator is composed of multiple interlocked transcriptional–translational feedback loops, which synchronize endogenous diel physiological rhythms to the cyclic changes of environmental cues. PSEUDO-RESPONSE REGULATORS (PRRs) have been identified as negative components in the circadian clock, though their underlying molecular mechanisms remain largely unknown. Here, we found that a subfamily of zinc finger transcription factors, B-box (BBX)-containing proteins, have a critical role in fine-tuning circadian rhythm. We demonstrated that overexpressing Arabidopsis thaliana BBX19 and BBX18 significantly lengthened the circadian period, while the null mutation of BBX19 accelerated the circadian speed. Moreover, BBX19 and BBX18, which are expressed during the day, physically interacted with PRR9, PRR7, and PRR5 in the nucleus in precise temporal ordering from dawn to dusk, consistent with the respective protein accumulation pattern of PRRs. Our transcriptomic and genetic analysis indicated that BBX19 and PRR9, PRR7, and PRR5 cooperatively inhibited the expression of morning-phased clock genes. PRR proteins affected BBX19 recruitment to the CCA1 , LHY , and RVE8 promoters. Collectively, our findings show that BBX19 interacts with PRRs to orchestrate circadian rhythms, and suggest the indispensable role of transcriptional regulators in fine-tuning the circadian clock. [ABSTRACT FROM AUTHOR]

The circadian clock provides a time-keeping mechanism that synchronizes various biological activities with the surrounding environment. Arabidopsis (Arabidopsis thaliana) CIRCADIAN CLOCK ASSOCIATED1 (CCA1), encoding a MYB-related transcription factor, is a key component of the core oscillator of the circadian clock, with peak expression in the morning. The molecular mechanisms regulating the light induction and rhythmic expression of CCA1 remain elusive. In this study, we show that two phytochrome signaling proteins, FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its paralog FAR-RED IMPAIRED RESPONSE1 (FAR1), are essential for the light-induced expression of CCA1. FHY3 and FAR1 directly bind to the CCA1 promoter and activate its expression, whereas PHYTOCHROME INTERACTING FACTOR5 (PIF5) directly binds to its promoter and represses its expression. Furthermore, PIF5 and TIMING OF CAB EXPRESSION1 physically interact with FHY3 and FAR1 to repress their transcriptional activation activity on CCA1 expression. These findings demonstrate that the photosensory-signaling pathway integrates with circadian oscillators to orchestrate clock gene expression. This mechanism might form the molecular basis of the regulation of the clock system by light in response to daily changes in the light environment, thus increasing plant fitness. [ABSTRACT FROM AUTHOR]

Transcriptional feedback loops are central to the architecture of eukaryotic circadian clocks. Models of the Arabidopsis thaliana circadian clock have emphasized transcriptional repressors, but recently, Myb-like REVEILLE (RVE) transcription factors have been established as transcriptional activators of central clock components, including PSEUDO-RESPONSE REGULATOR5 (PRR5) and TIMING OF CAB EXPRESSION1 (TOC1). We show here that NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED1 (LNK1) and LNK2, members of a small family of four LNK proteins, dynamically interact with morning-expressed oscillator components, including RVE4 and RVE8. Mutational disruption of LNK1 and LNK2 function prevents transcriptional activation of PRR5 by RVE8. The LNKs lack known DNA binding domains, yet LNK1 acts as a transcriptional activator in yeast and in planta. Chromatin immunoprecipitation shows that LNK1 is recruited to the PRR5 and TOC1 promoters in planta. We conclude that LNK1 is a transcriptional coactivator necessary for expression of the clock genes PRR5 and TOC1 through recruitment to their promoters via interaction with bona fide DNA binding proteins such as RVE4 and RVE8. [ABSTRACT FROM AUTHOR]

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9 , and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. [ABSTRACT FROM AUTHOR]