Carlos T. Hotta

The Arabidopsis circadian clock incorporates a cADPR-based feedback loop

Transcriptional feedback loops are a feature of circadian clocks in both animals and plants. We show that the plant circadian clock also incorporates the cytosolic signaling molecule cyclic adenosine diphosphate ribose (cADPR). cADPR modulates the circadian oscillators transcriptional feedback loops and drives circadian oscillations of Ca2+ release. The effects of antagonists of cADPR signaling, manipulation of cADPR synthesis, and mathematical simulation of the interaction of cADPR with the circadian clock indicate that cADPR forms a feedback loop within the plant circadian clock.

How plants tell the time

Plants, like all eukaryotes and most prokaryotes, have evolved sophisticated mechanisms for anticipating predictable environmental changes that arise due to the rotation of the Earth on its axis. These mechanisms are collectively termed the circadian clock. Many aspects of plant physiology, metabolism and development are under circadian control and a large proportion of the transcriptome exhibits circadian regulation. In the present review, we describe the advances in determining the molecular nature of the circadian oscillator and propose an architecture of several interlocking negative-feedback loops. The adaptive advantages of circadian control, with particular reference to the regulation of metabolism, are also considered. We review the evidence for the presence of multiple circadian oscillator types located in within individual cells and in different tissues.

Distinct Light and Clock Modulation of Cytosolic Free Ca2+ Oscillations and Rhythmic CHLOROPHYLL A/B BINDING PROTEIN2 Promoter Activity in Arabidopsis

Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca2+]cyt). To dissect the circadian Ca2+-signaling network, we monitored circadian [Ca2+]cyt oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca2+]cyt. Red light signaling is mediated by PHYTOCHROME B (PHYB). Blue light signaling occurs through the redundant action of CRYPTOCHROME1 (CRY1) and CRY2. Blue light also increases the basal level of [Ca2+]cyt, and this response requires PHYB, CRY1, and CRY2. Light input into the oscillator controlling [Ca2+]cyt rhythms is gated by EARLY FLOWERING3. Signals generated in the dark also regulate the circadian behavior of [Ca2+]cyt. Oscillations of [Ca2+]cyt and CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are dependent on the rhythmic expression of LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK-ASSOCIATED1, but [Ca2+]cyt and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca2+]cyt and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.

Correct biological timing in Arabidopsis requires multiple light-signaling pathways

Circadian oscillators provide rhythmic temporal cues for a range of biological processes in plants and animals, enabling anticipation of the day/night cycle and enhancing fitness-associated traits. We have used engineering models to understand the control principles of a plant’s response to seasonal variation. We show that the seasonal changes in the timing of circadian outputs require light regulation via feed-forward loops, combining rapid light-signaling pathways with entrained circadian oscillators. Linear time-invariant models of circadian rhythms were computed for 3,503 circadian-regulated genes and for the concentration of cytosolic-free calcium to quantify the magnitude and timing of regulation by circadian oscillators and light-signaling pathways. Bioinformatic and experimental analysis show that rapid light-induced regulation of circadian outputs is associated with seasonal rephasing of the output rhythm. We identify that external coincidence is required for rephasing of multiple output rhythms, and is therefore important in general phase control in addition to specific photoperiod-dependent processes such as flowering and hypocotyl elongation. Our findings uncover a fundamental design principle of circadian regulation, and identify the importance of rapid light-signaling pathways in temporal control.

Are there multiple circadian clocks in plants

We have reported that Arabidopsis might have genetically distinct circadian oscillators in multiple cell-types. Rhythms of CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are 2.5 h longer in phytochromeB mutants in constant red light and in cryptocrome1 cry2 double mutant (hy4-1 fha-1) in constant blue light than the wild-type. However, we found that cytosolic free Ca2+ ([Ca2+]cyt) oscillations were undetectable in these mutants in the same light conditions1. Furthermore, mutants of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) have short period rhythms of leaf movement but have arrhythmic [Ca2+]cyt oscillations. More important, the timing of cab1-1 (toc1-1) mutant has short period rhythms of CAB2 promoter activity (~21 h) but, surprisingly, has a wild-type period for circadian [Ca2+]cyt oscillations (~24 h). In contrast, toc1-2, a TOC1 loss-of-function mutant, has a short period of both CAB2 and [Ca2+]cyt rhythms (~21h). Here we discuss the difference between the phenotypes of toc1-1 and toc1-2 and how rhythms of CAB2 promoter activity and circadian [Ca2+]cyt oscillations might be regulated differently.

Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock

In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterized. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).The circadian clock involves daily variations in transcription of a set of core genes. Here, the authors show that oscillations in free calcium concentration, read by calmodulin-like proteins, regulate the clock and are part of this complex mechanism.