The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

Animals that inhabit mid- to high-latitude regions exhibit various adaptive behaviors, such as migration, reproduction, molting and hibernation in response to seasonal cues. These adaptive behaviors are tightly regulated by seasonal changes in photoperiod, the relative day length vs night length. Recently, the regulatory pathway of seasonal reproduction has been elucidated using quail. In birds, deep brain photoreceptors receive and transmit light information to the pars tuberalis in the pituitary gland, which induces the secretion of thyroid-stimulating hormone. Thyroid-stimulating hormone locally activates thyroid hormone via induction of type 2 deiodinase in the mediobasal hypothalamus. Thyroid hormone then induces morphological changes in the terminals of neurons that express gonadotropin-releasing hormone and facilitates gonadotropin secretion from the pituitary gland. In mammals, light information is received by photoreceptors in the retina and neurally transmitted to the pineal gland, where it inhibits the synthesis and secretion of melatonin, which is crucial for seasonal reproduction. Importantly, the signaling pathway downstream of light detection and signaling is fully conserved between mammals and birds. In fish, the regulatory components of seasonal reproduction are integrated, from light detection to neuroendocrine output, in a fish-specific organ called the saccus vasculosus. Various physiological processes in humans are also influenced by seasonal environmental changes. The findings discussed herein may provide clues to addressing human diseases, such as seasonal affective disorder.

SUMMARY Thyroid-stimulating hormone (TSH: thyrotropin) is a glycoprotein secreted from the pituitary gland. Pars distalis-derived TSH (PD-TSH) stimulates the thyroid gland to produce thyroid hormones (THs), whereas pars tuberalis-derived TSH (PT-TSH) acts on the hypothalamus to regulate seasonal physiology and behavior. However, it had not been clear how these two TSHs avoid functional crosstalk. Here, we show that this regulation is mediated by tissue-specific glycosylation. Although PT-TSH is released into the circulation, it does not stimulate the thyroid gland. PD-TSH is known to have sulfated bi-antennary N-glycans, and sulfated TSH is rapidly metabolized in the liver. By contrast, PT-TSH has sialylated multi-branched N-glycans; in the circulation, it forms the macro-TSH complex with immunoglobulin or albumin, resulting in the loss of its bioactivity. Glycosylation is fundamental to a wide range of biological processes. This is the first report demonstrating its involvement in preventing functional crosstalk of signaling molecules in the body.

The cyanobacterial circadian oscillator can be reconstituted in vitro. In the presence of KaiA and KaiB, the phosphorylation state of KaiC oscillates with a periodicity of ∼24 h. KaiC is a hexameric P-loop ATPase with autophosphorylation and autodephosphorylation activities. Recently, we found that dephosphorylation of KaiC occurs via reversal of the phosphorylation reaction: a phosphate group attached to Ser431/Thr432 is transferred to KaiC-bound ADP to generate ATP, which is subsequently hydrolyzed. This unusual reaction mechanism suggests that the KaiC phosphorylation rhythm is sustained by periodic shifts in the equilibrium of the reversible autophosphorylation reaction, the molecular basis of which has never been elucidated. Because KaiC-bound ATP and ADP serve as substrates for the forward and reverse reactions, respectively, we investigated the regulation of the nucleotide-bound state of KaiC. In the absence of KaiA, the condition in which the reverse reaction proceeds, KaiC favored the ADP-bound state. KaiA increased the ratio of ATP to total KaiC-bound nucleotides by facilitating the release of bound ADP and the incorporation of exogenous ATP, allowing the forward reaction to proceed. When both KaiA and KaiB were present, the ratio of ATP to total bound nucleotides exhibited a circadian rhythm, whose phase was advanced by several hours relative to that of the phosphorylation rhythm. Based on these findings, we propose that the direction of the reversible autophosphorylation reaction is regulated by KaiA and KaiB at the level of substrate availability and that this regulation sustains the oscillation of the phosphorylation state of KaiC.in vitro reconstitution | self-sustained oscillation | nucleotide exchange

Organisms living outside the tropics measure the changes in the length of the day to adapt to seasonal changes in the environment. Animals that breed during spring and summer are called long-day breeders, while those that breed during fall are called short-day breeders. Although the influence of thyroid hormone in the regulation of seasonal reproduction has been known for several decades, its precise mechanism remained unknown. Recent studies revealed that the activation of thyroid hormone within the mediobasal hypothalamus plays a key role in this phenomenon. This localized activation of the thyroid hormone is controlled by thyrotropin (thyroid-stimulating hormone) secreted from the pars tuberalis of the pituitary gland. Although seasonal reproduction is a rate-limiting factor in animal production, genes involved in photoperiodic signal transduction pathway could emerge as potential targets to facilitate domestication.