C. Robertson McClung

Plant Circadian Rhythms

The earth rotates on its axis every 24 h, with the result that any position on the earths surface alternately faces toward or away from the sun—day and night. That the metabolism, physiology, and behavior of most organisms changes profoundly between day and night is obvious to even the most

Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

PSEUDO-RESPONSE REGULATOR 7 and 9 Are Partially Redundant Genes Essential for the Temperature Responsiveness of the Arabidopsis Circadian Clock

Environmental time cues, such as photocycles (light/dark) and thermocycles (warm/cold), synchronize (entrain) endogenous biological clocks to local time. Although much is known about entrainment of the Arabidopsis thaliana clock to photocycles, the determinants of thermoperception and entrainment to thermocycles are not known. The Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) genes, including the clock component TIMING OF CAB EXPRESSION 1/PRR1, are related to bacterial, fungal, and plant response regulators but lack the conserved Asp that is normally phosphorylated by an upstream sensory kinase. Here, we show that two PRR family members, PRR7 and PRR9, are partially redundant; single prr7-3 or prr9-1 mutants exhibit modest period lengthening, but the prr7-3 prr9-1 double mutant shows dramatic and more than additive period lengthening in the light and becomes arrhythmic in constant darkness. The prr7-3 prr9-1 mutant fails both to maintain an oscillation after entrainment to thermocycles and to reset its clock in response to cold pulses and thus represents an important mutant strongly affected in temperature entrainment in higher plants. We conclude that PRR7 and PRR9 are critical components of a temperature-sensitive circadian system. PRR7 and PRR9 could function in temperature and light input pathways or they could represent elements of an oscillator necessary for the clock to respond to temperature signals.

Enhancer Trapping Reveals Widespread Circadian Clock Transcriptional Control in Arabidopsis

The circadian clock synchronizes the internal biology of an organism with the environment and has been shown to be widespread among organisms. Microarray experiments have shown that the circadian clock regulates mRNA abundance of about 10% of the transcriptome in plants, invertebrates, and mammals. In contrast, the circadian clock regulates the transcription of the virtually all cyanobacterial genes. To determine the extent to which the circadian clock controls transcription in Arabidopsis, we used in vivo enhancer trapping. We found that 36% of our enhancer trap lines display circadian-regulated transcription, which is much higher than estimates of circadian regulation based on analysis of steady-state mRNA abundance. Individual lines identified by enhancer trapping exhibit peak transcription rates at circadian phases spanning the complete circadian cycle. Flanking genomic sequence was identified for 23 enhancer trap lines to identify clock-controlled genes (CCG-ETs). Promoter analysis of CCG-ETs failed to predict new circadian clock response elements (CCREs), although previously defined CCREs, the CCA1-binding site, and the evening element were identified. However, many CCGs lack either the CCA1-binding site or the evening element; therefore, the presence of these CCREs is insufficient to confer circadian regulation, and it is clear that additional elements play critical roles.

SPINDLY and GIGANTEA interact and act in Arabidopsis thaliana pathways involved in light responses, flowering, and rhythms in cotyledon movements.

SPINDLY (SPY) is a negative regulator of gibberellin signaling in Arabidopsis thaliana that also functions in previously undefined pathways. The N terminus of SPY contains a protein–protein interaction domain consisting of 10 tetratricopeptide repeats (TPRs). GIGANTEA (GI) was recovered from a yeast two-hybrid screen for proteins that interact with the TPR domain. GI and SPY also interacted in Escherichia coli and in vitro pull-down assays. The phenotypes of spy and spy-4 gi-2 plants support the hypothesis that SPY functions with GI in pathways controlling flowering, circadian cotyledon movements, and hypocotyl elongation. GI acts in the long-day flowering pathway upstream of CONSTANS (CO) and FLOWERING LOCUS T (FT). Loss of GI function causes late flowering and reduces CO and FT RNA levels. Consistent with SPY functioning in the long-day flowering pathway upstream of CO, spy-4 partially suppressed the reduced abundance of CO and FT RNA and the late flowering of gi-2 plants. Like gi, spy affects the free-running period of cotyledon movements. The free-running period was lengthened in spy-4 mutants and shortened in plants that overexpress SPY under the control of the 35S promoter of Cauliflower mosaic virus. When grown under red light, gi-2 plants have a long hypocotyl. This hypocotyl phenotype was suppressed in spy-4 gi-2 double mutants. Additionally, dark-grown and far-red-light–grown spy-4 seedlings were found to have short and long hypocotyls, respectively. The different hypocotyl length phenotypes of spy-4 seedlings grown under different light conditions are consistent with SPY acting in the GA pathway to inhibit hypocotyl elongation and also acting as a light-regulated promoter of elongation.

Post-translational Regulation of the Arabidopsis Circadian Clock through Selective Proteolysis and Phosphorylation of Pseudo-response Regulator Proteins

The circadian clock controls the period, phasing, and amplitude of processes that oscillate with a near 24-h rhythm. One core group of clock components in Arabidopsis that controls the pace of the central oscillator is comprised of five PRR (pseudo-response regulator) proteins whose biochemical function in the clock remains unclear. Peak expression of TOC1 (timing of cab expression 1)/PRR1, PRR3, PRR5, PRR7, and PRR9 are each phased differently over the course of the day and loss of any PRR protein alters period. Here we show that, together with TOC1, PRR5 is the only other likely proteolytic substrate of the E3 ubiquitin ligase SCFZTL within this PRR family. We further demonstrate a functional significance for the phosphorylated forms of PRR5, TOC1, and PRR3. Each PRR protein examined is nuclear-localized and is differentially phosphorylated over the circadian cycle. The more highly phosphorylated forms of PRR5 and TOC1 interact best with the F-box protein ZTL (ZEITLUPE), suggesting a mechanism to modulate their proteolysis. In vivo degradation of both PRR5 and ZTL is inhibited by blue light, likely the result of blue light photoperception by ZTL. TOC1 and PRR3 interact in vivo and phosphorylation of both is necessary for their optimal binding in vitro. Additionally, because PRR3 and ZTL both interact with TOC1 in vivo via the TOC1 N terminus, taken together these data suggest that the TOC1/PRR3 phosphorylation-dependent interaction may protect TOC1 from ZTL-mediated degradation, resulting in an enhanced amplitude of TOC1 cycling.

Phase-Specific Circadian Clock Regulatory Elements in Arabidopsis

We have defined a minimal Arabidopsis CATALASE 3(CAT3) promoter sufficient to drive evening-specific circadian transcription of a LUCIFERASE reporter gene. Deletion analysis and site-directed mutagenesis reveal a circadian response element, the evening element (EE: AAAATATCT), that is necessary for evening-specific transcription. The EE differs only by a single base pair from the CIRCADIAN CLOCK ASSOCIATED 1-binding site (CBS: AAAAAATCT), which is important for morning-specific transcription. We tested the hypothesis that the EE and the CBS specify circadian phase by site-directed mutagenesis to convert theCAT3 EE into a CBS. Changing the CAT3 EE to a CBS changes the phase of peak transcription from the evening to the morning in continuous dark and in light-dark cycles, consistent with the specification of phase by the single base pair that distinguishes these elements. However, rhythmicity of the CBS-containing CAT3 promoter is dramatically compromised in continuous light. Thus, we conclude that additional information normally provided in the context of a morning-specific promoter is necessary for full circadian activity of the CBS.

Ambient Thermometers in Plants: From Physiological Outputs towards Mechanisms of Thermal Sensing

Plants respond to ambient temperature changes over a series of timescales. Genetic and physiological studies over the last decades have revealed myriad thermally sensitive pathways in plants. A recent study provides a genetic and biochemical mechanistic description of how thermal changes can be transduced to influence gene expression. What remains to be revealed in this, and other thermally controlled responses, is a description of the primary temperature-sensing event. Cooling and warming alter membrane fluidity and elicit intracellular free-calcium elevations, a process that has been considered the primary event controlling plant responses to temperature. Such direct thermal sensors appear to process temperature information. Future efforts will be required to identify the effector proteins linking perception to response. This review considers the evidence for plant thermometers to date, provides a description of several notable physiological and developmental processes under ambient temperature control, and outlines major questions that remain to be addressed in the understanding of thermometers in plants.

Regulation of Catalases in Arabidopsis

The catalase multi-gene family in Arabidopsis includes three genes encoding individual subunits which associate to form at least six isozymes that are readily resolved by non-denaturing gel electrophoresis. CAT1 and CAT3 map to chromosome 1, and CAT2 maps to chromosome 4. The nucleotide and deduced amino acids sequences of the three coding regions are highly related to each other and to other catalases. Both the individual isozymes and the individual subunit mRNAs show distinct patterns of spatial (organ-specific) expression. Six isozymes are detected in flowers and leaves and two are seen in roots. All three mRNAs are highly expressed in inflorescences, and CAT2 and CAT3 are highly expressed in leaves. All three mRNAs are detectable in freshly imbibed seeds, although the pattern of mRNA relative abundance varies among the three genes during early germination. CAT1 and CAT2 mRNA abundance is induced by light. In contrast, CAT3 is negatively light-responsive. CAT2 and CAT3 mRNA abundance is controlled by the circadian clock. Interestingly, the peak in CAT3 mRNA abundance occurs in the subjective evening, which is out of phase with expression of the Arabidopsis CAT2 catalase gene that shows clock-regulated expression gated to the subjective early morning. CAT1 mRNA abundance is not clock-regulated.

The Arabidopsis thaliana Clock

A combination of forward and reverse genetic approaches together with transcriptome-scale gene expression analyses have allowed the elaboration of a model for the Arabidopsis thaliana circadian clock. The working model largely conforms to the expected negative feedback loop model that has emerged from studies in other model systems. Although a core loop has emerged, it is clear that additional components remain to be identified and that the workings of the Arabidopsis clock have been established only in outline. Similarly, the details of resetting by light and temperature are only incompletely known. In contrast, the mechanism of photoperiodic induction of flowering is known in considerable detail and is consistent with the external coincidence model.