Blue Light- and Low Temperature-Regulated COR27 and COR28 Play Roles in the Arabidopsis Circadian Clock.

Light and temperature are two key environmental signals that profoundly affect plant growth and development, but underlying molecular mechanisms of how light and temperature signals affect the circadian clock are largely unknown. Here, we report that COR27 and COR28 are regulated not only by low temperatures but also by light signals. COR27 and COR28 are negative regulators of freezing tolerance but positive regulators of flowering, possibly representing a trade-off between freezing tolerance and flowering. Furthermore, loss-of-function mutations in COR27 and COR28 result in period lengthening of various circadian output rhythms and affect central clock gene expression. Also, the cor27 cor28 double mutation affects the pace of the circadian clock. Additionally, COR27 and COR28 are direct targets of CCA1, which represses their transcription via chromatin binding. Finally, we report that COR27 and COR28 bind to the chromatin of TOC1 and PRR5 to repress their transcription, suggesting that their effects on rhythms are in part due to their regulation of TOC1 and PRR5 These data demonstrate that blue light and low temperature-regulated COR27 and COR28 regulate the circadian clock as well as freezing tolerance and flowering time.

Pulsed induction of circadian clock genes in Arabidopsis seedlings.

The Alc-inducible system is a simple, yet effective, "gene switch" that can be used to transiently induce gene expression in Arabidopsis. Here we provide a protocol for using the Alc-inducible system to give a pulse in expression of a circadian clock gene in transgenic seedlings. The line we use as an example harbors an Alc-inducible copy of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene (Alc∷CCA1). Alc∷CCA1 seedlings are grown on solid MS medium and subsequently treated with ethanol vapor. Because the ethanol is quickly absorbed into the medium upon exposure, the seedlings are moved to fresh plates following treatment to avoid continuous induction. After the induction, the seedlings are harvested over a time-course for future total RNA and/or protein extraction that can be used for subsequent gene expression analyses.

The myristoylated amino-terminus of an Arabidopsis calcium-dependent protein kinase mediates plasma membrane localization.

Calcium-dependent protein kinases (CDPK) are a major group of calcium-stimulated kinases found in plants and some protists. Many CDPKs are membrane-associated, presumably because of lipid modifications at their amino termini. We investigated the subcellular location and myristoylation of AtCPK5, a member of the Arabidopsis CDPK family. Most AtCPK5 was associated with the plasma membrane as demonstrated by two-phase fractionation of plant microsomes and by in vivo detection of AtCPK5-GFP fusion proteins. AtCPK5 was a substrate for plant N-myristoyltransferase and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In transgenic plants, a G2A mutation completely abolished AtCPK5 membrane association, indicating that myristoylation was essential for membrane binding. The first sixteen amino acids of AtCPK5 were sufficient to direct plasma membrane localization. In addition, differentially phosphorylated forms of AtCPK5 were detected both in planta and after expression of AtCPK5 in a cell-free plant extract. Our results demonstrate that AtCPK5 is myristoylated at its amino terminus and that myristoylation is required for membrane binding.

CCA1 and ELF3 Interact in the control of hypocotyl length and flowering time in Arabidopsis.

The circadian clock is an endogenous oscillator with a period of approximately 24 h that allows organisms to anticipate, and respond to, changes in the environment. In Arabidopsis (Arabidopsis thaliana), the circadian clock regulates a wide variety of physiological processes, including hypocotyl elongation and flowering time. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a central clock component, and CCA1 overexpression causes circadian dysfunction, elongated hypocotyls, and late flowering. EARLY FLOWERING3 (ELF3) modulates light input to the clock and is also postulated to be part of the clock mechanism. elf3 mutations cause light-dependent arrhythmicity, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is not clear. Here, we show that CCA1 represses ELF3 by associating with its promoter, completing a CCA1-ELF3 negative feedback loop that places ELF3 within the oscillator. We also show that ELF3 acts downstream of CCA1, mediating the repression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5 in the control of hypocotyl elongation. In the regulation of flowering, our findings show that ELF3 and CCA1 either cooperate or act in parallel through the CONSTANS/FLOWERING LOCUS T pathway. In addition, we show that CCA1 represses GIGANTEA and SUPPRESSOR OF CONSTANS1 by direct interaction with their promoters, revealing additional connections between the circadian clock and the flowering pathways.

A role for protein kinase casein kinase2 α-subunits in the Arabidopsis circadian clock.

Circadian rhythms are autoregulatory, endogenous rhythms with a period of approximately 24 h. A wide variety of physiological and molecular processes are regulated by the circadian clock in organisms ranging from bacteria to humans. Phosphorylation of clock proteins plays a critical role in generating proper circadian rhythms. Casein Kinase2 (CK2) is an evolutionarily conserved serine/threonine protein kinase composed of two catalytic α-subunits and two regulatory ß-subunits. Although most of the molecular components responsible for circadian function are not conserved between kingdoms, CK2 is a well-conserved clock component modulating the stability and subcellular localization of essential clock proteins. Here, we examined the effects of a cka1a2a3 triple mutant on the Arabidopsis (Arabidopsis thaliana) circadian clock. Loss-of-function mutations in three nuclear-localized CK2α subunits result in period lengthening of various circadian output rhythms and central clock gene expression, demonstrating that the cka1a2a3 triple mutant affects the pace of the circadian clock. Additionally, the cka1a2a3 triple mutant has reduced levels of CK2 kinase activity and CIRCADIAN CLOCK ASSOCIATED1 phosphorylation in vitro. Finally, we found that the photoperiodic flowering response, which is regulated by circadian rhythms, was reduced in the cka1a2a3 triple mutant and that the plants flowered later under long-day conditions. These data demonstrate that CK2α subunits are important components of the Arabidopsis circadian system and their effects on rhythms are in part due to their phosphorylation of CIRCADIAN CLOCK ASSOCIATED1.

Chromatin remodeling and the circadian clock: Jumonji C-domain containing proteins.

Circadian rhythms are a universal way for organisms, ranging from prokaryotes to humans, to maintain coordination with the daily changes of light and temperature. It is known that a functional circadian clock confers enhanced fitness. In both animals and plants, diverse physiological processes are affected by the clock and more than 10% of transcripts show a circadian rhythm. Recent advances in the field have revealed a link between circadian regulated gene expression and dynamic changes in chromatin. Jumonji C (JmjC) domain-containing proteins have been shown to be involved in chromatin remodeling, acting as histone demethylases. The recent discovery that a JmjC-domain containing protein functions as a novel clock component suggests that histone modification has evolved as an important mechanism at the core of the circadian machinery. 

Phosphorylation by CK2 enhances the rapid light-induced degradation of phytochrome interacting factor 1 in Arabidopsis.

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four ß subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.

The Jumonji C domain-containing protein JMJ30 regulates period length in the Arabidopsis circadian clock.

Histone methylation plays an essential role in regulating chromatin structure and gene expression. Jumonji C (JmjC) domain-containing proteins are generally known as histone demethylases. Circadian clocks regulate a large number of biological processes, and recent studies suggest that chromatin remodeling has evolved as an important mechanism for regulating both plant and mammalian circadian systems. Here, we analyzed a subgroup of JmjC domain-containing proteins and identified Arabidopsis (Arabidopsis thaliana) JMJ30 as a novel clock component involved in controlling the circadian period. Analysis of loss- and gain-of-function mutants of JMJ30 indicates that this evening-expressed gene is a genetic regulator of period length in the Arabidopsis circadian clock. Furthermore, two key components of the central oscillator of plants, transcription factors CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL, bind directly to the JMJ30 promoter to repress its expression, suggesting that JMJ30 regulates the pace of the circadian clock in close association with the central oscillator. JMJ30 represents, to our knowledge, the first JmjC domain-containing protein involved in circadian function, and we envision that this provides a possible molecular connection between chromatin remodeling and the circadian clock.

F-box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control Arabidopsis clock progression.

Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.

CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL function synergistically in the circadian clock of Arabidopsis.

The circadian clock is an endogenous mechanism that coordinates biological processes with daily and seasonal changes in the environment. Heterodimerization of central clock components is an important way of controlling clock function in several different circadian systems. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are Myb-related proteins that function in or close to the central oscillator in Arabidopsis (Arabidopsis thaliana). Single mutants of cca1 and lhy have a phenotype of short-period rhythms. cca1 lhy double mutants show an even shorter period phenotype than the cca1 single mutant, suggesting that CCA1 and LHY are only partially functionally redundant. To determine whether CCA1 and LHY act in parallel or synergistically in the circadian clock, we examined their expression in both light-grown and etiolated seedlings. We have shown that LHY and CCA1 bind to the same region of the promoter of a Light-harvesting chlorophyll a/b protein (Lhcb, also known as CAB). CCA1 and LHY can form homodimers, and they also colocalize in the nucleus and heterodimerize in vitro and in vivo. In Arabidopsis, CCA1 and LHY physically interact in a manner independent of photoperiod. Moreover, results from gel filtration chromatography indicate that CCA1 and LHY are present in the same large complex in plants. Taken together, these results imply that CCA1 and LHY function synergistically in regulating circadian rhythms of Arabidopsis.

Testing time: can ethanol-induced pulses of proposed oscillator components phase shift rhythms in Arabidopsis?

Circadian rhythms are generated by endogenous central oscillators that respond to input from the environment and regulate rhythmic outputs. In Arabidopsis, more than a dozen components that affect rhythms have been identified and used to propose models of the central oscillator. However, none has been shown to fulfill one of the expected characteristics of an oscillator component: that a pulse of its expression shifts the phase of circadian rhythms. Here we show that a pulse of the proposed oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) causes dramatic phase shifts in rhythms of expression of the circadian reporter CAB2::LUC, as well as of the clock-associated genes TIMING OF CAB EXPRESSION 1 (TOC1) and GIGANTEA (GI). These results demonstrate that pulses of either CCA1 or LHY are capable of resetting the circadian clock. In contrast, a pulse of TOC1 expression did not elicit phase shifts. Control of TOC1 protein level is in part posttranscriptional; thus a pulse of TOC1 protein could be induced only at times when it is already high. Our work also shows that the ethanol-inducible system can be useful for achieving relatively short (<8 h) pulses of gene expression in seedlings.

An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum.

Arabidopsis contains 34 genes that are predicted to encode calcium-dependent protein kinases (CDPKs). CDPK enzymatic activity previously has been detected in many locations in plant cells, including the cytosol, the cytoskeleton, and the membrane fraction. However, little is known about the subcellular locations of individual CDPKs or the mechanisms involved in targeting them to those locations. We investigated the subcellular location of one Arabidopsis CDPK, AtCPK2, in detail. Membrane-associated AtCPK2 did not partition with the plasma membrane in a two-phase system. Sucrose gradient fractionation of microsomes demonstrated that AtCPK2 was associated with the endoplasmic reticulum (ER). AtCPK2 does not contain transmembrane domains or known ER-targeting signals, but does have predicted amino-terminal acylation sites. AtCPK2 was myristoylated in a cell-free extract and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In plants, the G2A mutation decreased AtCPK2 membrane association by approximately 50%. A recombinant protein, consisting of the first 10 amino acids of AtCPK2 fused to the amino-terminus of beta-glucuronidase, was also targeted to the ER, indicating that the amino terminus of AtCPK2 can specify ER localization of a soluble protein. These results indicate that AtCPK2 is localized to the ER, that myristoylation is likely to be involved in the membrane association of AtCPK2, and that the amino terminal region of AtCPK2 is sufficient for correct membrane targeting.