Marcelo J. Yanovsky

Molecular basis of seasonal time measurement in Arabidopsis

Several organisms have evolved the ability to measure daylength, or photoperiod, allowing them to adjust their development in anticipation of annual seasonal changes. Daylength measurement requires the integration of temporal information, provided by the circadian system, with light/dark discrimination, initiated by specific photoreceptors. Here we demonstrate that in Arabidopsis this integration takes place at the level of CONSTANS (CO) function. CO is a transcriptional activator that accelerates flowering time in long days, at least in part by inducing the expression of FLOWERING LOCUS T (FT). First, we show that precise clock control of the timing of CO expression, such that it is high during daytime only in long days, is critical for daylength discrimination. We then provide evidence that CO activation of FT expression requires the presence of light perceived through cryptochrome 2 (cry2) or phytochrome A (phyA). We conclude that an external coincidence mechanism, based on the endogenous circadian control of CO messenger RNA levels, and the modulation of CO function by light, constitutes the molecular basis for the regulation of flowering time by daylength in Arabidopsis.

Network Discovery Pipeline Elucidates Conserved Time-of-Day–Specific cis-Regulatory Modules

Correct daily phasing of transcription confers an adaptive advantage to almost all organisms, including higher plants. In this study, we describe a hypothesis-driven network discovery pipeline that identifies biologically relevant patterns in genome-scale data. To demonstrate its utility, we analyzed a comprehensive matrix of time courses interrogating the nuclear transcriptome of Arabidopsis thaliana plants grown under different thermocycles, photocycles, and circadian conditions. We show that 89% of Arabidopsis transcripts cycle in at least one condition and that most genes have peak expression at a particular time of day, which shifts depending on the environment. Thermocycles alone can drive at least half of all transcripts critical for synchronizing internal processes such as cell cycle and protein synthesis. We identified at least three distinct transcription modules controlling phase-specific expression, including a new midnight specific module, PBX/TBX/SBX. We validated the network discovery pipeline, as well as the midnight specific module, by demonstrating that the PBX element was sufficient to drive diurnal and circadian condition-dependent expression. Moreover, we show that the three transcription modules are conserved across Arabidopsis, poplar, and rice. These results confirm the complex interplay between thermocycles, photocycles, and the circadian clock on the daily transcription program, and provide a comprehensive view of the conserved genomic targets for a transcriptional network key to successful adaptation.

Overlapping and Distinct Roles of PRR7 and PRR9 in the Arabidopsis Circadian Clock

The core mechanism of the circadian oscillators described to date rely on transcriptional negative feedback loops with a delay between the negative and the positive components . In plants, the first suggested regulatory loop involves the transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CAB EXPRESSION 1 (TOC1/PRR1). TOC1 is a member of the Arabidopsis circadian-regulated PRR gene family . Analysis of single and double mutants in PRR7 and PRR9 indicates that these morning-expressed genes play a dual role in the circadian clock, being involved in the transmission of light signals to the clock and in the regulation of the central oscillator. Furthermore, CCA1 and LHY had a positive effect on PRR7 and PRR9 expression levels, indicating that they might form part of an additional regulatory feedback loop. We propose that the Arabidopsis circadian oscillator is composed of several interlocking positive and negative feedback loops, a feature of clock regulation that appears broadly conserved between plants, fungi, and animals.

Living by the calendar: how plants know when to flower

Reproductive processes in plants and animals are usually synchronized with favourable seasons of the year. It has been known for 80 years that organisms anticipate seasonal changes by adjusting developmental programmes in response to daylength. Recent studies indicate that plants perceive daylength through the degree of coincidence of light with the expression of CONSTANS, which encodes a clock-regulated transcription factor that controls the expression of floral-inductive genes in a light-dependent manner.

Genome-Wide Expression Analysis in DrosophilaReveals Genes Controlling Circadian Behavior

In Drosophila, a number of key processes such as emergence from the pupal case, locomotor activity, feeding, olfaction, and aspects of mating behavior are under circadian regulation. Although we have a basic understanding of how the molecular oscillations take place, a clear link between gene regulation and downstream biological processes is still missing. To identify clock-controlled output genes, we have used an oligonucleotide-based high-density array that interrogates gene expression changes on a whole genome level. We found genes regulating various physiological processes to be under circadian transcriptional regulation, ranging from protein stability and degradation, signal transduction, heme metabolism, detoxification, and immunity. By comparing rhythmically expressed genes in the fly head and body, we found that the clock has adapted its output functions to the needs of each particular tissue, implying that tissue-specific regulation is superimposed on clock control of gene expression. Finally, taking full advantage of the fly as a model system, we have identified and characterized a cycling potassium channel protein as a key step in linking the transcriptional feedback loop to rhythmic locomotor behavior.

Critical role for CCA1 and LHY in maintaining circadian rhythmicity in Arabidopsis

Circadian clocks are autoregulatory, endogenous mechanisms that allow organisms, from bacteria to humans, to advantageously time a wide range of activities within 24-hr environmental cycles. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are thought to be important components of the circadian clock in the model plant Arabidopsis. The similar circadian phenotypes of lines overexpressing either CCA1 or LHY have suggested that the functions of these two transcription factors are largely overlapping. cca1-1 plants, which lack CCA1 protein, show a short-period phenotype for the expression of several genes when assayed under constant light conditions. This suggests that LHY function is able to only partially compensate for the lack of CCA1 protein, resulting in a clock with a faster pace in cca1-1 plants. We have obtained plants lacking CCA1 and with LHY function strongly reduced, cca1-1 lhy-R, and show that these plants are unable to maintain sustained oscillations in both constant light and constant darkness. However, these plants exhibit some circadian function in light/dark cycles, showing that the Arabidopsis circadian clock is not entirely dependent on CCA1 and LHY activities.

Dual Role of TOC1 in the Control of Circadian and Photomorphogenic Responses in Arabidopsis

To examine the role of the TOC1 (TIMING OF CAB EXPRESSION1) gene in the Arabidopsis circadian system, we generated a series of transgenic plants expressing a gradation in TOC1 levels. Silencing of the TOC1 gene causes arrhythmia in constant darkness and in various intensities of red light, whereas in blue light, the clock runs faster in silenced plants than in wild-type plants. Increments in TOC1 gene dosage delayed the pace of the clock, whereas TOC1 overexpression abolished rhythmicity in all light conditions tested. Our results show that TOC1 RNA interference and toc1-2 mutant plants displayed an important reduction in sensitivity to red and far-red light in the control of hypocotyl elongation, whereas increments in TOC1 gene dosage clearly enhanced light sensitivity. Furthermore, the red light–mediated induction of CCA1/LHY expression was decreased in TOC1 RNA interference and toc1-2 mutant plants, indicating a role for TOC1 in the phytochrome regulation of circadian gene expression. We conclude that TOC1 is an important component of the circadian clock in Arabidopsis with a crucial function in the integration of light signals to control circadian and morphogenic responses.

A Genomic Analysis of the Shade Avoidance Response in Arabidopsis

Plants respond to the proximity of neighboring vegetation by elongating to prevent shading. Red-depleted light reflected from neighboring vegetation triggers a shade avoidance response leading to a dramatic change in plant architecture. These changes in light quality are detected by the phytochrome family of photoreceptors. We analyzed global changes in gene expression over time in wild-type, phyB mutant, and phyA phyB double mutant seedlings of Arabidopsis in response to simulated shade. Using pattern fitting software, we identified 301 genes as shade responsive with patterns of expression corresponding to one of various physiological response modes. A requirement for a consistent pattern of expression across 12 chips in this way allowed more subtle changes in gene expression to be considered meaningful. A number of previously characterized genes involved in light and hormone signaling were identified as shade responsive, as well as several putative, novel shade-specific signal transduction factors. In addition, changes in expression of genes in a range of pathways associated with elongation growth and stress responses were observed. The majority of shade-responsive genes demonstrated antagonistic regulation by phyA and phyB in response to shade following the pattern of many physiological responses. An analysis of promoter elements of genes regulated in this way identified conserved promoter motifs potentially important in shade regulation.

A methyl transferase links the circadian clock to the regulation of alternative splicing

Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day–night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5′-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.

Phytochrome B enhances photosynthesis at the expense of water-use efficiency in Arabidopsis.

In open places, plants are exposed to higher fluence rates of photosynthetically active radiation and to higher red to far-red ratios than under the shade of neighbor plants. High fluence rates are known to increase stomata density. Here we show that high, compared to low, red to far-red ratios also increase stomata density in Arabidopsis (Arabidopsis thaliana). High red to far-red ratios increase the proportion of phytochrome B (phyB) in its active form and the phyB mutant exhibited a constitutively low stomata density. phyB increased the stomata index (the ratio between stomata and epidermal cells number) and the level of anphistomy (by increasing stomata density more intensively in the adaxial than in the abaxial face). phyB promoted the expression of FAMA and TOO MANY MOUTHS genes involved in the regulation of stomata development in young leaves. Increased stomata density resulted in increased transpiration per unit leaf area. However, phyB promoted photosynthesis rates only at high fluence rates of photosynthetically active radiation. In accordance to these observations, phyB reduced long-term water-use efficiency estimated by the analysis of isotopic discrimination against 13CO2. We propose a model where active phyB promotes stomata differentiation in open places, allowing plants to take advantage of the higher irradiances at the expense of a reduction of water-use efficiency, which is compensated by a reduced leaf area.