Isabelle A. Carré

The late elongated hypocotyl Mutation of Arabidopsis Disrupts Circadian Rhythms and the Photoperiodic Control of Flowering

The dominant late elongated hypocotyl (lhy) mutation of Arabidopsis disrupted circadian clock regulation of gene expression and leaf movements and caused flowering to occur independently of photoperiod. LHY was shown to encode a MYB DNA-binding protein. In wild-type plants, the LHY mRNA showed a circadian pattern of expression with a peak around dawn but in the mutant was expressed constantly at high levels. Increased LHY expression from a transgene caused the endogenous gene to be expressed at a constant level, suggesting that LHY was part of a feedback circuit that regulated its own expression. Thus, constant expression of LHY disrupts several distinct circadian rhythms in Arabidopsis, and LHY may be closely associated with the central oscillator of the circadian clock.

LHY and CCA1 are partially redundant genes required to maintain circadian rhythms in Arabidopsis

Several genes are known to regulate circadian rhythms in Arabidopsis, but the identity of the central oscillator has not been established. LHY and CCA1 are related MYB-like transcription factors proposed to be closely involved. Here we demonstrate that, as shown previously for CCA1, inactivation of LHY shortens the period of circadian rhythms in gene expression and leaf movements. By constructing lhy cca1-1 double mutants, we show that LHY and CCA1 are partially redundant and essential for the maintenance of circadian rhythms in constant light. Under light/dark cycles the lhy cca1-1 plants show dramatically earlier phases of expression of GI and TOC1, genes associated with the generation of circadian rhythms and the promotion of LHY and CCA1 expression. We conclude that LHY and CCA1 appear to be negative regulatory elements required for central oscillator function.

Light-regulated translation mediates gated induction of the Arabidopsis clock protein LHY.

The transcription factor LHY and the related protein CCA1 perform overlapping functions in a regulatory feedback loop that is closely associated with the circadian oscillator of Arabidopsis. Overexpression of LHY abolished function of the circadian clock in constant light, but rhythmic expression of several circadian clock‐regulated transcripts was observed under light–dark cycles. These oscillations correlated with high amplitude changes in LHY protein levels, caused by light‐induced translation of the LHY transcript. Increases in LHY protein levels were also observed in light‐grown wild‐type plants, when light signals coincided with the circadian‐regulated peak of LHY transcription at dawn. Unexpectedly, translational induction coincided with acute downregulation of LHY transcript levels. We suggest that the simultaneous translational induction and transcriptional repression of LHY expression play a role to narrow the peak of LHY protein synthesis at dawn and increase the robustness and accuracy of circadian oscillations. Strong phase shifting responses to light signals were observed in plants lacking function of LHY, CCA1 or both, suggesting that light‐regulated expression of these proteins does not mediate entrainment of the clock to light–dark cycles.

Floral responses to photoperiod are correlated with the timing of rhythmic expression relative to dawn and dusk in Arabidopsis

Daylength, or photoperiod, is perceived as a seasonal signal for the control of flowering of many plants. The measurement of daylength is thought to be mediated through the interaction of phototransduction pathways with a circadian rhythm, so that flowering is induced (in long-day plants) or repressed (in short-day plants) when light coincides with a sensitive phase of the circadian cycle. To test this hypothesis in the facultative long-day plant, Arabidopsis thaliana, we used varying, non-24-hr light/dark cycles to alter the timing of circadian rhythms of gene expression relative to dawn and dusk. Effects on circadian rhythms were correlated with those on flowering times. We show that conditions that displaced subjective night events, such as expression of the flowering time regulator CONSTANS into the light portion of the cycle, were perceived as longer days. This work demonstrates that the perception of daylength in Arabidopsis relies on adjustments of the phase angle of circadian rhythms relative to the light/dark cycle, rather than on the measurement of the absolute duration of light and darkness.

Prediction of Photoperiodic Regulators from Quantitative Gene Circuit Models

Photoperiod sensors allow physiological adaptation to the changing seasons. The prevalent hypothesis is that day length perception is mediated through coupling of an endogenous rhythm with an external light signal. Sufficient molecular data are available to test this quantitatively in plants, though not yet in mammals. In Arabidopsis, the clock-regulated genes CONSTANS (CO) and FLAVIN, KELCH, F-BOX (FKF1) and their light-sensitive proteins are thought to form an external coincidence sensor. Here, we model the integration of light and timing information by CO, its target gene FLOWERING LOCUS T (FT), and the circadian clock. Among other predictions, our models show that FKF1 activates FT. We demonstrate experimentally that this effect is independent of the known activation of CO by FKF1, thus we locate a major, novel controller of photoperiodism. External coincidence is part of a complex photoperiod sensor: modeling makes this complexity explicit and may thus contribute to crop improvement.

Expression of the Arabidopsis gai gene under its own promoter causes a reduction in plant height in chrysanthemum by attenuation of the gibberellin response

Several transgenic chrysanthemum (Chrysanthemum morifolium Ramat.) lines have been produced that express the Arabidopsis thaliana gai (gibberellic acid insensitive) gene under its own promoter. These transformants exhibit a range of dwarf phenotypes, the extent of dwarfing being related to the reduction in the response to gibberellin in each transgenic line. Physiological measurements of growth, chlorophyll content and flowering time demonstrate that the extent of the transgene effects correlate with the level of transgene expression. Production of many ornamental crops relies heavily on the use of expensive and harmful agrochemicals in order to produce a blemish free and uniform product. We demonstrate the feasibility of producing a dwarf (pot) chrysanthemum without the need for growth retardant chemicals through heterologous expression of the mutant Arabidopsis gai gene driven from its own promoter.

Emerging design principles in the Arabidopsis circadian clock

Recent experimental advances have enabled the identification of direct regulatory targets for transcription factors. Application of these techniques to the circadian regulatory network in Arabidopsis has uncovered a number of discrepancies within established models as well as novel regulatory interactions. This review integrates these new findings and discusses the functional implications of the revised transcriptional network for the oscillatory mechanism of the clock.

Isoform switching facilitates period control in the Neurospora crassa circadian clock

A striking and defining feature of circadian clocks is the small variation in period over a physiological range of temperatures. This is referred to as temperature compensation, although recent work has suggested that the variation observed is a specific, adaptive control of period. Moreover, given that many biological rate constants have a Q10 of around 2, it is remarkable that such clocks remain rhythmic under significant temperature changes. We introduce a new mathematical model for the Neurospora crassa circadian network incorporating experimental work showing that temperature alters the balance of translation between a short and long form of the FREQUENCY (FRQ) protein. This is used to discuss period control and functionality for the Neurospora system. The model reproduces a broad range of key experimental data on temperature dependence and rhythmicity, both in wild‐type and mutant strains. We present a simple mechanism utilising the presence of the FRQ isoforms (isoform switching) by which period control could have evolved, and argue that this regulatory structure may also increase the temperature range where the clock is robustly rhythmic.

Evolutionarily Conserved Regulatory Motifs in the Promoter of the Arabidopsis Clock Gene LATE ELONGATED HYPOCOTYL

The transcriptional regulation of the LATE ELONGATED HYPOCOTYL (LHY) gene is key to the structure of the circadian oscillator, integrating information from multiple regulatory pathways. We identified a minimal region of the LHY promoter that was sufficient for rhythmic expression. Another upstream sequence was also required for appropriate waveform of transcription and for maximum amplitude of oscillations under both diurnal and free-running conditions. We showed that two classes of protein complexes interact with a G-box and with novel 5A motifs; mutation of these sites reduced the amplitude of oscillation and broadened the peak of expression. A genome-wide bioinformatic analysis showed that these sites were enriched in phase-specific clusters of rhythmically expressed genes. Comparative genomic analyses showed that these motifs were conserved in orthologous promoters from several species. A position-specific scoring matrix for the 5A sites suggested similarity to CArG boxes, which are recognized by MADS box transcription factors. In support of this, the FLOWERING LOCUS C (FLC) protein was shown to interact with the LHY promoter in planta. This suggests a mechanism by which FLC might affect circadian period.

Circadian regulation of abiotic stress tolerance in plants.

Extremes of temperatures, drought and salinity cause widespread crop losses throughout the world and impose severe limitations on the amount of land that can be used for agricultural purposes. Hence, there is an urgent need to develop crops that perform better under such abiotic stress conditions. Here, we discuss intriguing, recent evidence that circadian clock contributes to plants’ ability to tolerate different types of environmental stress, and to acclimate to them. The clock controls expression of a large fraction of abiotic stress-responsive genes, as well as biosynthesis and signaling downstream of stress response hormones. Conversely, abiotic stress results in altered expression and differential splicing of the clock genes, leading to altered oscillations of downstream stress-response pathways. We propose a range of mechanisms by which this intimate coupling between the circadian clock and environmental stress-response pathways may contribute to plant growth and survival under abiotic stress.