Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In Arabidopsis thaliana, ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 278C), whereas they decrease for LHY. However, as temperatures decrease (from 17 to 128C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 278C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in lhy and gi mutant plants. However, at 128C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than lhy at the lower temperature. At 178C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperaturespecific phenotypes of gi mutants.

TERMINAL FLOWER1 (TFL1) is a key regulator of flowering time and the development of the inflorescence meristem in Arabidopsis thaliana. TFL1 and FLOWERING LOCUS T (FT) have highly conserved amino acid sequences but opposite functions. For example, FT promotes flowering and TFL1 represses it; FT-overexpressing plants and TFL1 loss-of-function mutants have a similar phenotype production of terminal flowers in the shoot apex. FT is believed to function in a transcriptional activator complex by interacting with FD. Here, we demonstrate that TFL1 is involved in the transcriptional repression of genes that are activated by FT. We analyzed transgenic plants overexpressing TFL1 fused to a transcriptional repressor domain (TFL1-SRDX) or an activator domain (TFL1-VP16). Plants carrying 35S:TFL1-SRDX showed delayed flowering similar to 35S:TFL1 plants, and plants carrying 35S:TFL1-VP16 showed an early flowering phenotype and produced terminal flowers. Furthermore, the tfl1 and 35S:TFL1-VP16 plant phenotypes were strongly suppressed by the fd mutation, and TFL1 interacted with FD in the cell nucleus, as shown by bimolecular fluorescence complementation experiments. We conclude that TFL1 negatively modulates the FD-dependent transcription of target genes to fine-tune flowering time and the development of the inflorescence meristem.

Plants synchronize developmental and metabolic processes with the earth's 24-h rotation through the integration of circadian rhythms and responses to light. We characterize the time for coffee ( tic ) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b -binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 ( ELF3 ) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify TIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. Even the robust circadian clock of plants cannot function with defects at two different phases.

Circadian systems coordinate endogenous events with external signals. In mammals, hormone-clock feedbacks are a well-known integration system. Here, we investigated phytohormone effects on plant-circadian rhythms via the promoter:luciferase system. We report that many hormones control specific features of the plant-circadian system, and do so in distinct ways. In particular, cytokinins delay circadian phase, auxins regulate circadian amplitude and clock precision, and brassinosteroid and abscisic acid modulate circadian periodicity. We confirmed the pharmacology in hormone synthesis and perception mutants, as rhythmic expression is predictably altered in an array of hormone-related mutants. We genetically dissected one mechanism that integrates hormone signals into the clock, and showed that the hormone-activated ARABIDOPSIS RESPONSE REGULATOR 4 and the photoreceptor phytochrome B are elements in the input of the cytokinin signal to circadian phase. Furthermore, molecular-expression targets of this signal were found. Collectively, we found that plants have multiple input/output feedbacks, implying that many hormones can function on the circadian system to adjust the clock to external signals to properly maintain the clock system.

Lys-63-linked multiubiquitin chains play important roles in signal transduction in yeast and in mammals, but the functions for this type of chain in plants remain to be defined. The RING domain protein RGLG2 (for RING domain Ligase2) from Arabidopsis thaliana can be N-terminally myristoylated and localizes to the plasma membrane. It can form Lys-63-linked multiubiquitin chains in an in vitro reaction. RGLG2 has overlapping functions with its closest sequelog, RGLG1, and single mutants in either gene are inconspicuous. rglg1 rglg2 double mutant plants exhibit loss of apical dominance and altered phyllotaxy, two traits critically influenced by the plant hormone auxin. Auxin and cytokinin levels are changed, and the plants show a decreased response to exogenously added auxin. Changes in the abundance of PIN family auxin transport proteins and synthetic lethality with a mutation in the auxin transport regulator BIG suggest that the directional flow of auxin is modulated by RGLG activity. Modification of proteins by Lys-63-linked multiubiquitin chains is thus important for hormoneregulated, basic plant architecture.

The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in darkgrown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci.

TIMING OF CAB EXPRESSION 1 ( TOC1) functions with CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) in a transcriptional feedback loop that is important for the circadian clock in Arabidopsis thaliana (L.) Heynh. TOC1 and its four paralogues, the Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) genes, are expressed in an intriguing daily sequence. This was proposed to form a second feedback loop, similar to the interlocking clock gene circuits in other taxa. We show that prr9 and prr5 null mutants have reciprocal period defects for multiple circadian rhythms, consistent with subtly altered expression patterns of CCA1 and TOC1. The period defects are conditional on light quality and combine additively in double-mutant plants. Thus PRR9 and PRR5 modulate light input to the circadian clock but are neither uniquely required for rhythm generation nor form a linear series of mutual PRR gene regulation.

BackgroundPlant circadian systems regulate various biological processes in harmony with daily environmental changes. In Arabidopsis thaliana, the underlying clock mechanism is comprised of multiple integrated transcriptional feedbacks, which collectively lead to global patterns of rhythmic gene expression. The transcriptional networks are essential within the clock itself and in its output pathway.ResultsHere, to expand understanding of transcriptional networks within and associated to the clock, we performed both an in silico analysis of transcript rhythmicity of transcription factor genes, and a pilot assessment of functional phenomics on the MYB, bHLH, and bZIP families. In our in silico analysis, we defined which members of these families express a circadian waveform of transcript abundance. Up to 20% of these families were over-represented as clock-controlled genes. To detect members that contribute to proper oscillator function, we systematically measured rhythmic growth via an imaging system in hundreds of misexpression lines targeting members of the transcription-factor families. Three transcription factors were found that conferred aberrant circadian rhythms when misexpressed: MYB3R2, bHLH69, and bHLH92.ConclusionTranscript abundance of many transcription factors in Arabidopsis oscillates in a circadian manner. Further, a developed pipeline assessed phenotypic contribution of a panel of transcriptional regulators in the circadian system.