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Sweet basil is a popular culinary herb used in many cuisines around the world and is widely grown commercially for retail as a live potted plant. However, basil is easily damaged by temperatures below 12 °C meaning plants must be transported from the grower to the retailer in a warm transport chain, adding considerable commercial cost in temperate countries. Improvement of chilling tolerance has been demonstrated in post-harvest crops such as tomato fruits and, indeed, fresh cut basil, by manipulation of the red:far red ratio of light provided to plants throughout the photoperiod and for a significant duration of the growing process in controlled environment chambers. We tested the effectiveness of periodic short-duration end-of-production supplementary far red light treatments designed for use with basil plants grown in a large scale commercial glasshouse for the live potted basil market. Four days of periodic, midday supplementary far red light given at end of production induced robust tolerance to 24 h of 4 °C cold treatment, resulting in greatly reduced visual damage, and reduced physiological markers of chilling injury including electrolyte leakage and reactive oxygen species accumulation. Antioxidant levels were also maintained at higher levels in live potted basil following this cold treatment. RNAseq-based analysis of gene expression changes associated with this response pointed to increased conversion of starch to soluble raffinose family oligosaccharide sugars; increased biosynthesis of anthocyanins and selected amino acids; inactivation of gibberellin signaling; and reduced expression of fatty acid desaturases, all previously associated with increased chilling tolerance in plants. Our findings offer an efficient, non-invasive approach to induce chilling tolerance in potted basil which is suitable for application in a large-scale commercial glasshouse., Competing Interests: Author SB was employed by the company Vitacress Herbs. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from the company Vitacress Herbs. The funder had the following involvement in the study: SB contributed to experimental design and supervision of the project., (Copyright © 2023 Begum, Skinner, Smieszek, Budge, Stead and Devlin.)

Culinary herbs are commercially cultivated for their wide range of volatile compounds that give characteristic aromas and tastes. Rosemary ( Salvia rosmarinus Spenn.) is an excellent model for assessment of methods improvement of volatile production as cultivars offer a wide variety of aromatic profiles due to the large family of terpene synthase genes. Arbuscular mycorrhizal fungi (AMF) associations have been shown to improve essential oil production in aromatic plants and offer one approach to enhance aroma in commercial herb production. Changes in the expression of seven different terpene synthases were compared in six rosemary cultivars in response to addition of AMF to a peat substrate. Addition of AMF profoundly influenced terpene synthase expression in all cultivars and did so without impacting the optimised plant size and uniformity achieved in these conditions. In addition, two methods for AMF application, developed with the horticultural industry in mind, were tested in this study. Uniform incorporation of AMF mixed into the growing substrate prior to planting of a root plug produced the most consistent root colonisation. Overall, our findings demonstrate the potential for the use of AMF in the improvement of aroma in culinary herbs within a commercial setting but show that outcomes are likely to greatly vary depending on variety.

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle's envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100cue8 identified a second mutation within the same gene, tic100soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling., (� The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The red-light regulated transcription factors FHY3 and FAR1 form a key point of light input to the plant circadian clock in positively regulating expression of genes within the central clock. However, the fhy3 mutant shows an additional red light-specific disruption of rhythmicity which is inconsistent with this role. Here we demonstrate that only fhy3 and not far1 mutants show this red specific disruption of rhythmicity. We examined the differences in rhythmic transcriptome in red versus white light and reveal differences in patterns of rhythmicity among the central clock proteins suggestive of a change in emphasis within the central mechanism of the clock, changes which underlie the red specificity of the fhy3 mutant. In particular, changes in enrichment of promoter elements were consistent with a key role for the HY5 transcription factor, a known integrator of the ratio of red to blue light in regulation of the clock. Examination of differences in the rhythmic transcriptome in the fhy3 mutant in red light identified specific disruption of the CCA1-regulated ELF3 and LUX central clock genes, while the CCA1 target TBS element, TGGGCC, was enriched among genes that became arrhythmic. Coupled with the known interaction of FHY3 but not FAR1 with CCA1 we propose that the red-specific circadian phenotype of fhy3 may involve disruption of the previously demonstrated moderation of CCA1 activity by FHY3 rather than a disruption of its own transcriptional regulatory activity. Together, this evidence suggests a conditional redundancy between FHY3 and HY5 in the integration of red and blue light input to the clock in order to enable a plasticity in response to light and optimise plant adaptation. Furthermore, our evidence also suggests changes in CCA1 activity between red and white light transcriptomes. This, together with the documented interaction of HY5 with CCA1, leads us to propose a model whereby this integration of red and blue signals may at least partly occur via direct FHY3 and HY5 interaction with CCA1 leading to moderation of CCA1 activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rhodes, Siddiqui, Khan and Devlin.)

Background: Jasmonates (JAs) mediate trade-off between responses to both biotic and abiotic stress and growth in plants. The Arabidopsis thaliana HISTONE DEACETYLASE 6 is part of the CORONATINE INSENSITIVE1 receptor complex, co-repressing the HDA6/COI1-dependent acetic acid-JA pathway that confers plant drought tolerance. The decrease in HDA6 binding to target DNA mirrors histone H4 acetylation (H4Ac) changes during JA-mediated drought response, and mutations in HDA6 also cause depletion in the constitutive repressive marker H3 lysine 27 trimethylation (H3K27me3). However, the genome-wide effect of HDA6 on H4Ac and much of the impact of JAs on histone modifications and chromatin remodelling remain elusive., Results: We performed high-throughput ChIP-Seq on the HDA6 mutant, axe1-5, and wild-type plants with or without methyl jasmonate (MeJA) treatment to assess changes in active H4ac and repressive H3K27me3 histone markers. Transcriptional regulation was investigated in parallel by microarray analysis in the same conditions. MeJA- and HDA6-dependent histone modifications on genes for specialized metabolism; linolenic acid and phenylpropanoid pathways; and abiotic and biotic stress responses were identified. H4ac and H3K27me3 enrichment also differentially affects JAs and HDA6-mediated genome integrity and gene regulatory networks, substantiating the role of HDA6 interacting with specific families of transposable elements in planta and highlighting further specificity of action as well as novel targets of HDA6 in the context of JA signalling for abiotic and biotic stress responses., Conclusions: The findings demonstrate functional overlap for MeJA and HDA6 in tuning plant developmental plasticity and response to stress at the histone modification level. MeJA and HDA6, nonetheless, maintain distinct activities on histone modifications to modulate genetic variability and to allow adaptation to environmental challenges., (© 2022. The Author(s).)

Plant microbiomes and immune responses have coevolved through history, and this applies just as much to the phyllosphere microbiome and defense phytohormone signaling. When in homeostasis, the phyllosphere microbiome confers benefits to its host. However, the phyllosphere is also dynamic and subject to stochastic events that can modulate community assembly. Investigations into the impact of defense phytohormone signaling on the microbiome have so far been limited to culture-dependent studies; or focused on the rhizosphere. In this study, the impact of the foliar phytohormone salicylic acid (SA) on the structure and composition of the phyllosphere microbiome was investigated. 16S rRNA amplicons were sequenced from aerial tissues of two Arabidopsis mutants that exhibit elevated SA signaling through different mechanisms. SA signaling was shown to increase community diversity and to result in the colonization of rare, satellite taxa in the phyllosphere. However, a stable core community remained in high abundance. Therefore, we propose that SA signaling acts as a source of intermediate disturbance in the phyllosphere. Predictive metagenomics revealed that the SA-mediated microbiome was enriched for antibiotic biosynthesis and the degradation of a diverse range of xenobiotics. Core taxa were predicted to be more motile, biofilm-forming and were enriched for traits associated with microbe-microbe communication; offering potential mechanistic explanation of their success despite SA-mediated phyllospheric disturbance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vincent, Ebertz, Spanu and Devlin.)

The circadian clock provides a time-keeping mechanism that synchronizes various biological activities with the surrounding environment. Arabidopsis ( Arabidopsis thaliana ) CIRCADIAN CLOCK ASSOCIATED1 ( CCA1 ), encoding a MYB-related transcription factor, is a key component of the core oscillator of the circadian clock, with peak expression in the morning. The molecular mechanisms regulating the light induction and rhythmic expression of CCA1 remain elusive. In this study, we show that two phytochrome signaling proteins, FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its paralog FAR-RED IMPAIRED RESPONSE1 (FAR1), are essential for the light-induced expression of CCA1 FHY3 and FAR1 directly bind to the CCA1 promoter and activate its expression, whereas PHYTOCHROME INTERACTING FACTOR5 (PIF5) directly binds to its promoter and represses its expression. Furthermore, PIF5 and TIMING OF CAB EXPRESSION1 physically interact with FHY3 and FAR1 to repress their transcriptional activation activity on CCA1 expression. These findings demonstrate that the photosensory-signaling pathway integrates with circadian oscillators to orchestrate clock gene expression. This mechanism might form the molecular basis of the regulation of the clock system by light in response to daily changes in the light environment, thus increasing plant fitness., (© 2020 American Society of Plant Biologists. All rights reserved.)

Many plants exhibit circadian clock-driven leaf movements whereby the leaves are raised during the day to achieve a relatively high angle during the evening, before lowering late in the night. Such leaf movements were first recorded over 2000 years ago but there is still much debate as to their purpose. We investigated whether such leaf movements within Arabidopsis, a ruderal rosette plant, can aid in overtopping leaves of neighboring plants. Wild type and circadian clock mutant plants were grown in an alternating grid system so that their leaves would meet as the plants grew. Experiments were performed using day lengths that matched the endogenous rhythm of either wild type or mutant. Plants grown in a day length shorter than their endogenous rhythm were consistently overtopped by plants which were in synchrony with the day night cycle, demonstrating a clear overtopping advantage resulting from circadian leaf movement rhythms. Furthermore, we found that this leaf overtopping as a result of correctly synchronized circadian leaf movements is additive to leaf overtopping due to shade avoidance. Curiously, this did not apply to plants grown in a day length longer than their endogenous period. Plants grown in a day length longer than their endogenous period were able to adapt their leaf rhythms and suffered no overtopping disadvantage. Crucially, our results show that, in a context-dependent manner, circadian clock-driven leaf movements in resonance with the external light/dark cycle can facilitate overtopping of the leaves of neighboring plants., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The use of microbial inoculants containing plant growth-promoting rhizobacteria as a promoter of plant fitness and health is becoming increasingly popular in agriculture. However, whether and how these bacteria affect indigenous bacterial communities in field conditions is sparsely explored. We studied the effects of seed inoculation and field soil application of ubiquitous soil bacteria, B. cereus, B. subtilis, and B. amyloliquefaciens, on the diversity, evenness, and richness of endophytic bacterial communities in sprouting broccoli roots using high-throughput metagenome sequencing. The multiple operational taxonomic units (OTUs) assigned to different bacterial taxa clearly showed changes in ecological measures and relative abundances of certain taxa between control and treatment groups. The Bacillus inocula, themselves, failed to flourish as endophytes; however, the effects they extended on the endophytic bacterial community were both generic as well as species specific. In each case, Pseudomonadales, Rhizobiales, Xanthomonadales, and Burkholderiales were the most abundant orders in the endosphere. B. amyloliquefaciens drastically reduced the most abundant genus, Pseudomonas, while increasing the relative abundance of a range of minor taxa. The Shannon-Weiner diversity and Buzas and Gibson's evenness indices showed that the diversity and evenness were increased in both B. amyloliquefaciens and mixed treated plants. The UniFrac measurement of beta diversity showed that all treatments affected the specific composition of the endophytic bacterial community, with an apparent interspecies competition in the mixed treatment. Taken together, Bacillus species influenced the diversity, evenness, and composition of the endophytic bacterial community. However, these effects varied between different Bacillus spp. in a context-specific manner.

When plants grow in close proximity basic resources such as light can become limiting. Under such conditions plants respond to anticipate and/or adapt to the light shortage, a process known as the shade avoidance syndrome (SAS). Following genetic screening using a shade-responsive luciferase reporter line (PHYB:LUC), we identified DRACULA2 (DRA2), which encodes an Arabidopsis homolog of mammalian nucleoporin 98, a component of the nuclear pore complex (NPC). DRA2, together with other nucleoporins, participates positively in the control of the hypocotyl elongation response to plant proximity, a role that can be considered dependent on the nucleocytoplasmic transport of macromolecules (i.e. is transport dependent). In addition, our results reveal a specific role for DRA2 in controlling shade-induced gene expression. We suggest that this novel regulatory role of DRA2 is transport independent and that it might rely on its dynamic localization within and outside of the NPC. These results provide mechanistic insights in to how SAS responses are rapidly established by light conditions. They also indicate that nucleoporins have an active role in plant signaling., (© 2016. Published by The Company of Biologists Ltd.)

FHY3 and FAR1 are positively acting transcription factors that directly regulate expression of a number of target genes in Arabidopsis thaliana. Here, we looked at the regulation of one specific target gene, ELF4. We demonstrate that the action of FHY3 and FAR1 in upregulation of ELF4 is light dependent. Furthermore, although FHY3 and FAR1 have been exclusively characterized as components of the phytochrome A signaling pathway because of their importance in regulating expression of phyA nuclear importers, we show that, as transcription factors in their own right, FHY3 and FAR1 act downstream of light stable phytochromes, phyB, phyD, and phyE. We demonstrate that light stable phytochrome acts in a red/far-red reversible manner to regulate the level of FHY3 protein. We also observed that ELF4 shows specific FHY3 and FAR1-mediated light induction in the evening and we show that regulation by light stable phytochromes at this time is important as it allows the plant to maintain normal ELF4 expression beyond dusk when the day length shortens, something which would not be possible through light labile phytochrome action. Without FHY3 and FAR1, ELF4 expression falls rapidly at dusk and in short days this results in an early drop in ELF4 expression, accompanied by a de-repression of an ELF4 target gene later in the night. Our results, therefore, demonstrate an important role for FHY3 and FAR1 as mediators of light stable phytochrome signaling.

We aimed to test the proposal that progressive combinations of multiple promoter elements acting in concert may be responsible for the full range of phases observed in plant circadian output genes. In order to allow reliable selection of informative phase groupings of genes for our purpose, intrinsic cyclic patterns of expression were identified using a novel, non-biased method for the identification of circadian genes. Our non-biased approach identified two dominant, inherent orthogonal circadian trends underlying publicly available microarray data from plants maintained under constant conditions. Furthermore, these trends were highly conserved across several plant species. Four phase-specific modules of circadian genes were generated by projection onto these trends and, in order to identify potential combinatorial promoter elements that might classify genes into these groups, we used a Random Forest pipeline which merged data from multiple decision trees to look for the presence of element combinations. We identified a number of regulatory motifs which aggregated into coherent clusters capable of predicting the inclusion of genes within each phase module with very high fidelity and these motif combinations changed in a consistent, progressive manner from one phase module group to the next, providing strong support for our hypothesis.

The circadian clock regulates many aspects of plant physiology, growth and development. It produces daily rhythms of growth and metabolism, and interacts with signalling pathways controlling environmental responses over the course of a day or a year. Over the last decade, a combination of empirical research in molecular genetics and mathematical modelling, mostly utilising Arabidopsis thaliana, has led to the identification of many plant clock components and an understanding of their interlocking roles within the biochemical mechanism. The plant clock shares many characteristics of circadian clocks in other taxa, being temperature-compensated, capable of generating endogenous rhythms, of entraining to environmental cycles and regulated by means of transcription-translation feedback loops; however, few, if any, components of the plant clock appear to be shared with other organisms, indicating an independent evolutionary origin. In this review, we describe our current understanding of the central clockwork and how it receives input and regulates outputs. We also discuss the interaction between the clock and the environment, identifying areas, such as the integration of non-photic stimuli, where future work will lead to a fuller understanding of how the circadian system is embedded in plant physiology., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The shade avoidance syndrome (SAS) allows plants to anticipate and avoid shading by neighbouring plants by initiating an elongation growth response. The phytochrome photoreceptors are able to detect a reduction in the red:far red ratio in incident light, the result of selective absorption of red and blue wavelengths by proximal vegetation. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 1 (dra1) mutant, that showed no avoidance of shade for the PHYB::LUC response, was the result of a mutation in the PHYA gene. Like previously characterized phyA mutants, dra1 showed a long hypocotyl in far red light and an enhanced hypocotyl elongation response to shade. However, dra1 additionally showed a long hypocotyl in red light. Since phyB levels are relatively unaffected in dra1, this gain-of-function red light phenotype strongly suggests a disruption of phyB signalling. The dra1 mutation, G773E within the phyA PAS2 domain, occurs at a residue absolutely conserved among phyA sequences. The equivalent residue in phyB is absolutely conserved as a threonine. PAS domains are structurally conserved domains involved in molecular interaction. Structural modelling of the dra1 mutation within the phyA PAS2 domain shows some similarity with the structure of the phyB PAS2 domain, suggesting that the interference with phyB signalling may be the result of non-functional mimicry. Hence, it was hypothesized that this PAS2 residue forms a key distinction between the phyA and phyB phytochrome species.

The circadian clock controls many metabolic, developmental and physiological processes in a time-of-day-specific manner in both plants and animals. The photoreceptors involved in the perception of light and entrainment of the circadian clock have been well characterized in plants. However, how light signals are transduced from the photoreceptors to the central circadian oscillator, and how the rhythmic expression pattern of a clock gene is generated and maintained by diurnal light signals remain unclear. Here, we show that in Arabidopsis thaliana, FHY3, FAR1 and HY5, three positive regulators of the phytochrome A signalling pathway, directly bind to the promoter of ELF4, a proposed component of the central oscillator, and activate its expression during the day, whereas the circadian-controlled CCA1 and LHY proteins directly suppress ELF4 expression periodically at dawn through physical interactions with these transcription-promoting factors. Our findings provide evidence that a set of light- and circadian-regulated transcription factors act directly and coordinately at the ELF4 promoter to regulate its cyclic expression, and establish a potential molecular link connecting the environmental light-dark cycle to the central oscillator.

In vitro analyses of plant GATA transcription factors have implicated some proteins in light-mediated and circadian-regulated gene expression, and, more recently, the analysis of mutants has uncovered further diverse roles for plant GATA factors. To facilitate function discovery for the 29 GATA genes in Arabidopsis (Arabidopsis thaliana), we have experimentally verified gene structures and determined expression patterns of all family members across adult tissues and suspension cell cultures, as well as in response to light and signals from the circadian clock. These analyses have identified two genes that are strongly developmentally light regulated, expressed predominantly in photosynthetic tissue, and with transcript abundance peaking before dawn. In contrast, several GATA factor genes are light down-regulated. The products of these light-regulated genes are candidates for those proteins previously implicated in light-regulated transcription. Coexpression of these genes with well-characterized light-responsive transcripts across a large microarray data set supports these predictions. Other genes show additional tissue-specific expression patterns suggesting novel and unpredicted roles. Genome-wide analysis using coexpression scatter plots for paralogous gene pairs reveals unexpected differences in cocorrelated gene expression profiles. Clustering the Arabidopsis GATA factor gene family by similarity of expression patterns reveals that genes of recent descent do not uniformly show conserved current expression profiles, yet some genes showing more distant evolutionary origins have acquired common expression patterns. In addition to defining developmental and environmental dynamics of GATA transcript abundance, these analyses offer new insights into the evolution of gene expression profiles following gene duplication events.

Circadian gating of light signaling limits the timing of maximum responsiveness to light to specific times of day. The fhy3 (for far-red elongated hypocotyl3) mutant of Arabidopsis thaliana is involved in independently gating signaling from a group of photoreceptors to an individual response. fhy3 shows an enhanced response to red light during seedling deetiolation. Analysis of two independent fhy3 alleles links enhanced inhibition of hypocotyl elongation in response to red light with an arrhythmic pattern of hypocotyl elongation. Both alleles also show disrupted rhythmicity of central-clock and clock-output gene expression in constant red light. fhy3 exhibits aberrant phase advances under red light pulses during the subjective day. Release-from-light experiments demonstrate clock disruption in fhy3 during the early part of the subjective day in constant red light, suggesting that FHY3 is important in gating red light signaling for clock resetting. The FHY3 gating function appears crucial in the early part of the day for the maintenance of rhythmicity under these conditions. However, unlike previously described Arabidopsis gating mutants that gate all light signaling, gating of direct red light-induced gene expression in fhy3 is unaffected. FHY3 appears to be a novel gating factor, specifically in gating red light signaling to the clock during daytime.

The phytochrome (phy) photoreceptors modulate plant development after perception of light. Upon illumination of etiolated seedlings, phys initiate a transcriptional cascade by directly transducing light signals to the promoters of genes encoding regulators of morphogenesis. In light-grown plants, however, little is known about the transcriptional cascade modulated by phys in response to changes in light. The phy entry points in this cascade are completely unknown. We are particularly interested in the shade avoidance syndrome (SAS). Here we describe a subset of six genes whose expression is rapidly modulated by phys during both deetiolation and SAS in Arabidopsis (Arabidopsis thaliana). Using cycloheximide, we provide evidence that four of these phy rapidly regulated (PAR) genes are direct targets of phy signaling during SAS, revealing these genes as upstream components of the transcriptional cascade. Promoter-beta-glucuronidase fusions confirmed that PAR genes are photoregulated at the transcriptional level. Analysis of gene expression in light signal transduction mutants showed that COP1 and DET1 (but not DET2 or HY5) play a role in modulating PAR expression in response to shade in light-grown seedlings. Moreover, genetic analyses showed that one of the genes identified as a direct target of phy signaling was phy-interacting factor 3-like-1 (PIL1). PIL1 has previously been implicated in SAS in response to transient shade, but we show here that it also plays a key role in response to long-term shade. The action of PIL1 was particularly apparent in a phyB background, suggesting an important negative role for PIL1 under dense vegetation canopies.

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.

The circadian clock forms one of the most fascinating adaptations to life on earth. Organisms can not only anticipate the day/night cycle but can make use of an internal clock to measure daylength as an indicator of the changing of the seasons. The innate period of the clock is not exactly equal to 24 h, but is reset each day by environmental signals at dawn and dusk, most notably by changes in light and temperature. This ability to re-entrain also ensures that the clock is synchronized with the day/night cycle which in turn is crucial for anticipation of dawn and dusk. Recent advances in the field have identified the photoreceptors involved in resetting the clock in several systems. This has revealed surprising similarities, but also key differences in the circadian systems of plants, fungi, insects, and mammals. One recurring feature emerging from this research is that the photoreceptors themselves are under the control of the clock with transcript abundance being tightly regulated. Furthermore, elements of a feedback pathway whereby the clock modulates the activity of the light input pathway are now being identified.

The circadian clock is intrinsically linked to the daily cycle of day and night. A capacity for entrainment to light-dark cycles has proven to be a universal feature of the clock in all organisms examined. Here we review a wealth of recent advances that reveal more about the light input mechanisms by which the circadian clock is set to the correct time in a range of different systems. Now that we are identifying more of the molecular components of both the light input pathway and the clock mechanism itself, we are becoming increasingly less able to distinguish between the two.

The circadian clock is entrained to the daily cycle of day and night by light signals at dawn and dusk. Plants make use of both the phytochrome (phy) and cryptochrome (cry) families of photoreceptors in gathering information about the light environment for setting the clock. We demonstrate that the phytochromes phyA, phyB, phyD, and phyE act as photoreceptors in red light input to the clock and that phyA and the cryptochromes cry1 and cry2 act as photoreceptors in blue light input. phyA and phyB act additively in red light input to the clock, whereas cry1 and cry2 act redundantly in blue light input. In addition to the action of cry1 as a photoreceptor that mediates blue light input into the clock, we demonstrate a requirement of cry1 for phyA signaling to the clock in both red and blue light. Importantly, Arabidopsis cry1 cry2 double mutants still show robust rhythmicity, indicating that cryptochromes do not form a part of the central circadian oscillator in plants as they do in mammals.

Light is a crucial environmental signal that controls many photomorphogenic and circadian responses in plants. Perception and transduction of light is achieved by at least two principal groups of photoreceptors, phytochromes and cryptochromes. Phytochromes are red/far-red light-absorbing receptors encoded by a gene family of five members (phyA to phyE) in Arabidopsis. Cryptochrome 1 (cry1), cryptochrome 2 (cry2) and phototropin are the blue/ultraviolet-A light receptors that have been characterized in Arabidopsis. Previous studies showed that modulation of many physiological responses in plants is achieved by genetic interactions between different photoreceptors; however, little is known about the nature of these interactions and their roles in the signal transduction pathway. Here we show the genetic interaction that occurs between the Arabidopsis photoreceptors phyB and cry2 in the control of flowering time, hypocotyl elongation and circadian period by the clock. PhyB interacts directly with cry2 as observed in co-immunoprecipitation experiments with transgenic Arabidopsis plants overexpressing cry2. Using fluorescent resonance energy transfer microscopy, we show that phyB and cry2 interact in nuclear speckles that are formed in a light-dependent fashion.

Cryptochromes are blue/UV-A-absorbing photoreceptor proteins discovered originally in plants and so named because their nature proved elusive in over a century of research. Now we know that the photoreceptor essential for proper seedling establishment in blue light has homologues in the animal kingdom - in insects, in mice and in humans. In recent months, evidence has emerged pointing to a common role for cryptochromes in all of these organisms in entraining the circadian clock, a biochemical timing mechanism running within cells, synchronizing metabolism to the daily light-dark cycle and having consequences on a much larger scale in the regulation of behaviour such as the sleep-wake cycle.

Shade avoidance in higher plants is regulated by the action of multiple phytochrome (phy) species that detect changes in the red/far-red ratio (R/FR) of incident light and initiate a redirection of growth and an acceleration of flowering. The phyB mutant of Arabidopsis is constitutively elongated and early flowering and displays attenuated responses to both reduced R/FR and end-of-day far-red light, conditions that induce strong shade-avoidance reactions in wild-type plants. This indicates that phyB plays an important role in the control of shade avoidance. In Arabidopsis phyB and phyD are the products of a recently duplicated gene and share approximately 80% identity. We investigated the role played by phyD in shade avoidance by analyzing the responses of phyD-deficient mutants. Compared with the monogenic phyB mutant, the phyB-phyD double mutant flowers early and has a smaller leaf area, phenotypes that are characteristic of shade avoidance. Furthermore, compared with the monogenic phyB mutant, the phyB-phyD double mutant shows a more attenuated response to a reduced R/FR for these responses. Compared with the phyA-phyB double mutant, the phyA-phyB-phyD triple mutant has elongated petioles and displays an enhanced elongation of internodes in response to end-of-day far-red light. These characteristics indicate that phyD acts in the shade-avoidance syndrome by controlling flowering time and leaf area and that phyC and/or phyE also play a role.

Circadian clocks are synchronized by environmental cues such as light. Photoreceptor-deficient Arabidopsis thaliana mutants were used to measure the effect of light fluence rate on circadian period in plants. Phytochrome B is the primary high-intensity red light photoreceptor for circadian control, and phytochrome A acts under low-intensity red light. Cryptochrome 1 and phytochrome A both act to transmit low-fluence blue light to the clock. Cryptochrome 1 mediates high-intensity blue light signals for period length control. The presence of cryptochromes in both plants and animals suggests that circadian input pathways have been conserved throughout evolution.

Plants have evolved exquisite sensory systems for monitoring their light environment. The intensity, quality, direction and duration of light are continuously monitored by the plant and the information gained is used to modulate all aspects of plant development. Several classes of distinct photoreceptors, sensitive to different regions of the light spectrum, mediate the developmental responses of plants to light signals. The red-far-red light-absorbing, reversibly photochromic phytochromes are perhaps the best characterized of these. Higher plants possess a family of phytochromes, the apoproteins of which are encoded by a small, divergent gene family. Arabidopsis has five apophytochrome-encoding genes, PHYA-PHYE. Different phytochromes have discrete biochemical and physiological properties, are differentially expressed and are involved in the perception of different light signals. Photoreceptor and signal transduction mutants of Arabidopsis are proving to be valuable tools in the molecular dissection of photomorphogenesis. Mutants deficient in four of the five phytochromes have now been isolated. Their analysis indicates considerable overlap in the physiological functions of different phytochromes. In addition, mutants defining components acting downstream of the phytochromes have provided evidence that different members of the family use different signalling pathways.

From a screen of M2 seedlings derived from gamma-mutagenesis of seeds doubly null for phytochromes phyA and phyB, we isolated a mutant lacking phyE. The PHYE gene of the selected mutant, phyE-1, was found to contain a 1-bp deletion at a position equivalent to codon 726, which is predicted to result in a premature stop at codon 739. Immunoblot analysis showed that the phyE protein was undetectable in the phyE-1 mutant. In the phyA- and phyB-deficient background, phyE deficiency led to early flowering, elongation of internodes between adjacent rosette leaves, and reduced petiole elongation. This is a phenocopy of the response of phyA phyB seedlings to end-of-day far-red light treatments. Furthermore, a phyE deficiency attenuated the responses of phyA phyB seedlings to end-of-day far-red light treatments. Monogenic phyE mutants were indistinguishable from wild-type seedlings. However, phyB phyE double mutants flowered earlier and had longer petioles than did phyB mutants. The elongation and flowering responses conferred by phyE deficiency are typical of shade avoidance responses to the low red/far-red ratio. We conclude that in conjunction with phyB and to a lesser extent with phyD, phyE functions in the regulation of the shade avoidance syndrome.

Phytochrome (P) was characterized in etiolated seedlings of wild-type, mutant and transgenic strains of Arabidopsis with the use of low-temperature (85 K) fluorescence spectroscopy and photochemistry. The position (lambda max) of the Pr emission spectrum, its intensity (F0) proportional to [P tot] and the extent of the Pr-->lumi-R phototransformation at 85 K (gamma 1) were shown to vary depending on the plant strains and tissues used, while the extent of the Pr-->Pfr transformation at 273 K (gamma 2) remained relatively constant. Depletion of phyA (fre1-1 in Nagatani et al., Plant Physiol. 102 (1993) 269-277, and fhy2-2 in Whitelam et al., Plant Cell 5 (1993) 757-768) resulted in a steep decrease of F0 to approximately equal to 10%. The phyB mutant (hy3-B064 in Reed et al., Plant Cell 5 (1993) 147-157) revealed a slight reduction (by approximately equal to 20%) of F0 while lambda max and gamma 1 remained practically unaffected. In phyAphyB mutuant no P emmission was observed. Overexpression of oat phyA (13k7 and 21k15 in Boylan and Quail, Proc. Natl. Acad. Sci. USA 88 (1991) 10806-10810) brought about an increase of F0 by two or three times, a shift of lambda max to 685 nm and an increase of gamma 1 to 0.3-0.4. On the contrary, an increase of F0 (up to 40%) in Arabidopsis and rice phyB overexpressors (ABO and RBO in Wagner et al., Plant Cell 3 (1991) 1275-1288) was followed by a decrease of gamma 1 values to 0.13-0.14. These data together with the results on phyB (lh) mutant of cucumber prove the existence of the two phyA populations with high (phyA') and low (phyA") photochemical activity at low temperatures. PhyB emits maximally in the same region as phyA in Arabidopsis (approximately equal to 683 nm) and at shorter wavelength (< 680 nm) in rice. It is characterized by low photochemical activity at 85 K (gamma 1 < or = 0.05) and can be attributed in this respect to the same pigment type as phyA".

The elongated internode (ein) mutation of Brassica rapa leads to a deficiency in immunochemically detectable phytochrome B. Molecular analysis of the PHYB gene from ein indicates a deletion in the flanking DNA 5' of the ATG start codon, which could interfere either with PHYB transcription or processing of the PHYB transcript. Restriction fragment length polymorphisms and inverse PCR fragments generated from the PHYB gene of wild-type and ein seedlings demonstrate the deletion to be 500 bp in length. Seedlings of heterozygote, EIN/ein, contain about 50% of the level of immunochemically detectable phytochrome B of equivalent wild-type EIN/EIN seedlings. Etiolated seedlings of EIN/ein show a responsiveness to red light almost intermediate between that of ein/ein and EIN/EIN homozygotes. Furthermore, whereas the ein/ein homozygote is poorly responsive to low red/far-red ratio light, the presence of one functional allele of EIN in the heterozygote confers an elongation response intermediate between that of the homozygotes EIN/EIN and ein/ein in these light conditions. The partial dominance of ein indicates a close relationship between phytochrome B level and phenotype.

A major function of phytochromes in light-grown plants involves the perception of changes in the relative amounts of red and far-red light (R:FR ratio) and the initiation of the shade-avoidance response. In Arabidopsis thaliana, this response is typified by increased elongation growth of petioles and accelerated flowering and can be fully induced by end-of-day far-red light (EOD FR) treatments. Phytochrome B-deficient (phyB) mutants, which have a constitutive elongated-petiole and early-flowering pheno-type, do not display a petiole elongation growth response to EOD FR, but they do respond to EOD FR by earlier flowering. Seedlings deficient in both phytochrome A and phytochrome B (phyA phyB), have a greatly reduced stature compared with wild-type or either monogenic mutant. The phyA phyB double null mutants also respond to EOD FR treatments by flowering early, suggesting the operation of novel phytochromes. Contrary to the behaviour of wild-type or monogenic phyA or phyB seedlings, petiole elongation in phyA phyB seedlings is reduced in response to EOD FR treatments. This reduction in petiole elongation is accompanied by the appearance of elongated internodes such that under these conditions the plants no longer display a rosette habit.

A novel elongated mutant has been isolated from EMS-mutagenized populations of the Arabidopsis thaliana ga4 mutant. After backcrossing with the Landsberg erecta (Ler) wild-type (WT) followed by selfing, the mutant phenotype was identified in the GA4 background. Seedlings of the mutant, which has been named elg (elongated), are characterized by elongated hypocotyls and petioles, leaves that are narrow and somewhat epinastic and early flowering. Allelism tests with the hy1-hy5 mutants indicate that elg is not allelic with any of these long-hypocotyl mutants. From linkage analyses, the location of elg on chromosome 4, between cer2 and ap2 has been established. The pleiotropic phenotype of elg seedlings is suggestive of a disruption of phytochrome and/or gibberellin (GA) function. Although the elg mutant displays a light-dependent long-hypocotyl phenotype, elg seedlings retain a full range of photomorphogenic responses and the elg mutation acts additively with the photomorphogenic mutants phyB, hy1 and hy2. This suggests that ELG acts independently of phytochrome action. The elg mutation partially suppresses the effect of GA-deficiency on elongation growth, and, although elg ga1 seedlings are more elongated than ga1 seedlings, both genotypes respond in the same way to applied GA. That applied GA and the elg mutation interact additively suggests that ELG acts independently of GA action.

Several phytochrome-controlled processes have been examined in etiolated and light-grown seedlings of a normal genotype and the elongated internode (ein/ein) mutant of rapid-cycling Brassica rapa. Although etiolated ein seedlings displayed normal sensitivity to prolonged far-red light with respect to inhibition of hypocotyl elongation, expansion of cotyledons, and synthesis of anthocyanin, they displayed reduced sensitivity to prolonged red light for all three of these deetiolation responses. In contrast to normal seedlings, light-grown ein seedlings did not show a growth promotion in response to end-of-day far-red irradiation. Additionally, whereas the first internode of light-grown normal seedlings showed a marked increase in elongation in response to reduced ratio of red to far-red light, ein seedlings showed only a small elongation response. When blots of protein extracts from etiolated and light-treated ein and normal seedlings were probed with monoclonal antibody to phytochrome A, an immunostaining band at about 120 kD was observed for both extracts. The immunostaining intensity of this band was substantially reduced for extracts of light-treated normal and ein seedlings. A mixture of three monoclonal antibodies directed against phytochrome B from Arabidopsis thaliana immunostained a band at about 120 kD for extracts of etiolated and light-treated normal seedlings. This band was undetectable in extracts of ein seedlings. We propose that ein is a photoreceptor mutant that is deficient in a light-stable phytochrome B-like species.