The key clock component ZEITLUPE (ZTL) negatively regulates ABA signaling by degradation of CHLH in Arabidopsis.

Ubiquitination-mediated protein degradation plays important roles in ABA signal transduction and delivering responses to chloroplast stress signals in plants, but additional E3 ligases of protein ubiquitination remain to be identified to understand the complex signaling network. Here we reported that ZEITLUPE (ZTL), an F-box protein, negatively regulates abscisic acid (ABA) signaling during ABA-inhibited early seedling growth and ABA-induced stomatal closure in Arabidopsis thaliana. Using molecular biology and biochemistry approaches, we demonstrated that ZTL interacts with and ubiquitinates its substrate, CHLH/ABAR (Mg-chelatase H subunit/putative ABA receptor), to modulate CHLH stability via the 26S proteasome pathway. CHLH acts genetically downstream of ZTL in ABA and drought stress signaling. Interestingly, ABA conversely induces ZTL phosphorylation, and high levels of ABA also induce CHLH proteasomal degradation, implying that phosphorylated ZTL protein may enhance the affinity to CHLH, leading to the increased degradation of CHLH after ABA treatment. Taken together, our results revealed a possible mechanism of reciprocal regulation between ABA signaling and the circadian clock, which is thought to be essential for plant fitness and survival.

The functional interplay between protein kinase CK2 and CCA1 transcriptional activity is essential for clock temperature compensation in Arabidopsis.

Circadian rhythms are daily biological oscillations driven by an endogenous mechanism known as circadian clock. The protein kinase CK2 is one of the few clock components that is evolutionary conserved among different taxonomic groups. CK2 regulates the stability and nuclear localization of essential clock proteins in mammals, fungi, and insects. Two CK2 regulatory subunits, CKB3 and CKB4, have been also linked with the Arabidopsis thaliana circadian system. However, the biological relevance and the precise mechanisms of CK2 function within the plant clockwork are not known. By using ChIP and Double-ChIP experiments together with in vivo luminescence assays at different temperatures, we were able to identify a temperature-dependent function for CK2 modulating circadian period length. Our study uncovers a previously unpredicted mechanism for CK2 antagonizing the key clock regulator CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). CK2 activity does not alter protein accumulation or subcellular localization but interferes with CCA1 binding affinity to the promoters of the oscillator genes. High temperatures enhance the CCA1 binding activity, which is precisely counterbalanced by the CK2 opposing function. Altering this balance by over-expression, mutation, or pharmacological inhibition affects the temperature compensation profile, providing a mechanism by which plants regulate circadian period at changing temperatures. Therefore, our study establishes a new model demonstrating that two opposing and temperature-dependent activities (CCA1-CK2) are essential for clock temperature compensation in Arabidopsis.

A functional connection between the clock component TOC1 and abscisic acid signaling pathways.

The hormone abscisic acid (ABA) regulates the stress signals crucial for plant tolerance to adverse environmental conditions. The circadian clock also uses environmental cues for appropriate timing of plant physiology and metabolism. Despite previous studies showing the connections between ABA and clock signalling pathways, the molecular nodes underlying these connections remained unknown. In a recent study, we have shown that the essential clock component TOC1 (Timing of CAB expression 1) regulates the diurnal expression of the ABA-related gene ABAR/CHLH/GUN5 by direct binding to its promoter. Treatment with ABA specifically induces TOC1 at midday and this induction controls both the phase of TOC1 binding and the expression of ABAR. TOC1 induction by ABA is abolished in ABAR RNAi plants revealing a new feedback loop that reciprocally links ABAR and TOC1 expression. This regulation is essential for ABA function as TOC1 and ABAR overexpressing and mutant plants display altered ABA-mediated tolerance to drought conditions. Notably, TOC1 is also implicated in ABA-mediated inhibition of seed germination but in an opposite direction to that observed for dehydration responses. These opposing functions open interesting questions about the spatial and temporal networks connecting ABA and clock signaling pathways.

Interaction of shade avoidance and auxin responses: a role for two novel atypical bHLH proteins.

Plants sense the presence of potentially competing nearby individuals as a reduction in the red to far-red ratio of the incoming light. In anticipation of eventual shading, a set of plant responses known as the shade avoidance syndrome (SAS) is initiated soon after detection of this signal by the phytochrome photoreceptors. Here we analyze the function of PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2, two Arabidopsis thaliana genes rapidly upregulated after simulated shade perception. These genes encode two closely related atypical basic helix-loop-helix proteins with no previously assigned function in plant development. Using reverse genetic approaches, we show that PAR1 and PAR2 act in the nucleus to broadly control plant development, acting as negative regulators of a variety of SAS responses, including seedling elongation and photosynthetic pigment accumulation. Molecularly, PAR1 and PAR2 act as direct transcriptional repressors of two auxin-responsive genes, SMALL AUXIN UPREGULATED15 (SAUR15) and SAUR68. Additional results support that PAR1 and PAR2 function in integrating shade and hormone transcriptional networks, rapidly connecting phytochrome-sensed light changes with auxin responsiveness.

Altered oscillator function affects clock resonance and is responsible for the reduced day-length sensitivity of CKB4 overexpressing plants.

Most organisms have evolved a timing mechanism or circadian clock that is able to generate 24 h rhythmic oscillations in multiple biological events. The environmental fluctuations in light and temperature synchronize the expression and activity of key oscillator components that ultimately define the period, phase and amplitude of output rhythms. In Arabidopsis, overexpression of the casein kinase 2 (CK2) regulatory subunits, CKB3 or CKB4, alters the function of the clock under free-running conditions, and results in period-shortening of genes peaking at different phase angles. Here, we examine the effects of CKB4 overexpression (CKB4-ox) on a number of clock outputs that are modulated by day length or photoperiod. We have found a phase shift in gene expression, shortening of hypocotyl elongation and earlier than wild-type initiation of flowering under short-day conditions. Our study shows that the earlier expression phases of the floral induction genes GIGANTEA, FLAVIN-BINDING KELCH REPEAT F-BOX1 and CONSTANS correlate with higher abundance of the FLOWERING LOCUS T transcript under short-day conditions. Matching the period of the external light/dark cycles relative to the endogenous short period of the CKB4-ox oscillator restores the phase of gene expression and the flowering sensitivity to day length, indicating that a clock defect is responsible for the CKB4-ox phenotypes. Our studies suggest a function for CKB4 very close to the oscillator, as expression of the core components TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 is also altered in CKB4-ox plants. Based on our results, we propose that oscillator dysfunction is responsible for the period defect of CKB4-ox plants that leads to clock dissonance with the environment and reduced sensitivity to day length.

The proteasome-dependent degradation of CKB4 is regulated by the Arabidopsis biological clock.

Most organisms have evolved an internal timing mechanism, the circadian clock, that is able to generate and maintain 24 h rhythmic oscillation in molecular, biochemical and metabolic activities. In Arabidopsis, the clock-dependent synchronization of physiology with the environment is essential for successful growth and development. The mechanisms of the Arabidopsis clockwork have been described as transcriptional feedback loops at the core of the oscillator. However, an increasing body of evidence points towards a key role of post-translational regulation of clock components as an essential mechanism of circadian function. Here, we identify CKB4, a CK2 regulatory subunit, as a component of the Arabidopsis circadian system. We demonstrate that the nuclear-localized CKB4 protein exists in vivo as different isoforms, resulting from phosphorylation on serine residues. Our findings show that the phosphorylated isoforms are the preferred substrate for ubiquitination and degradation by the proteasome pathway. We provide evidence of the involvement of the biological clock in the circadian regulation of CKB4 protein abundance, which itself is important for an accurate control of circadian period by the clock. Overexpression of CKB4 results in elevated CK2 overall activity and period-shortening of clock-controlled genes peaking at different phase angles. Restriction of CKB4 protein phosphorylation and/or degradation to specific phases within the circadian cycle might provide the cell with a fine-tuning mechanism to selectively regulate the CK2 phosphorylation activity on specific substrates.