Role of guard cell- or mesophyll cell-localized phytochromes in stomatal responses to blue, red, and far-red light.

MAIN CONCLUSION: Guard cell- or mesophyll cell-localized phytochromes do not have a predominant direct light sensory role in red- or blue-light-mediated stomatal opening or far-red-light-mediated stomatal closure of Arabidopsis. The role of phytochromes in blue- and red-light-mediated stomatal opening, and far-red-light- mediated decrease in opening, is still under debate. It is not clear whether reduced stomatal opening in a phytochrome B (phyB) mutant line, is due to phytochrome acting as a direct photosensor or an indirect growth effect. The exact tissue localization of the phytochrome photoreceptor important for stomatal opening is also not known. We studied differences in stomatal opening in an Arabidopsis phyB mutant, and lines showing mesophyll cell-specific or guard cell-specific inactivation of phytochromes. Stomatal conductance (gs) of intact leaves was measured under red, blue, and blue + far-red light. Lines exhibiting guard cell-specific inactivation of phytochrome did not show a change in gs under blue or red light compared to Col-0. phyB consistently exhibited a reduction in gs under both blue and red light. Addition of far-red light did not have a significant impact on the blue- or red-light-mediated stomatal response. Treatment of leaves with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a photosynthetic electron transport (PET) inhibitor, eliminated the response to red light in all lines, indicating that stomatal opening under red light is controlled by PET, and not directly by phytochrome. Similar to previous studies, leaves of the phyB mutant line had fewer stomata. Overall, phytochrome does not appear have a predominant direct sensory role in stomatal opening under red or blue light. However, phytochromes likely have an indirect effect on the degree of stomatal opening under light through effects on leaf growth and stomatal development.

The membrane-localized protein kinase MAP4K4/TOT3 regulates thermomorphogenesis.

Plants respond to mild warm temperature conditions by increased elongation growth of organs to enhance cooling capacity, in a process called thermomorphogenesis. To this date, the regulation of thermomorphogenesis has been exclusively shown to intersect with light signalling pathways. To identify regulators of thermomorphogenesis that are conserved in flowering plants, we map changes in protein phosphorylation in both dicots and monocots exposed to warm temperature. We identify MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4)/TARGET OF TEMPERATURE3 (TOT3) as a regulator of thermomorphogenesis that impinges on brassinosteroid signalling in Arabidopsis thaliana. In addition, we show that TOT3 plays a role in thermal response in wheat, a monocot crop. Altogether, the conserved thermal regulation by TOT3 expands our knowledge of thermomorphogenesis beyond the well-studied pathways and can contribute to ensuring food security under a changing climate.

An early-morning gene network controlled by phytochromes and cryptochromes regulates photomorphogenesis pathways in Arabidopsis.

Light perception at dawn plays a key role in coordinating multiple molecular processes and in entraining the plant circadian clock. The Arabidopsis mutant lacking the main photoreceptors, however, still shows clock entrainment, indicating that the integration of light into the morning transcriptome is not well understood. In this study, we performed a high-resolution RNA-sequencing time-series experiment, sampling every 2 min beginning at dawn. In parallel experiments, we perturbed temperature, the circadian clock, photoreceptor signaling, and chloroplast-derived light signaling. We used these data to infer a gene network that describes the gene expression dynamics after light stimulus in the morning, and then validated key edges. By sampling time points at high density, we are able to identify three light- and temperature-sensitive bursts of transcription factor activity, one of which lasts for only about 8 min. Phytochrome and cryptochrome mutants cause a delay in the transcriptional bursts at dawn, and completely remove a burst of expression in key photomorphogenesis genes (HY5 and BBX family). Our complete network is available online (http://www-users.york.ac.uk/∼de656/dawnBurst/dawnBurst.html). Taken together, our results show that phytochrome and cryptochrome signaling is required for fine-tuning the dawn transcriptional response to light, but separate pathways can robustly activate much of the program in their absence.

Hot topic: Thermosensing in plants.

Plants alter their morphology and cellular homeostasis to promote resilience under a variety of heat regimes. Molecular processes that underlie these responses have been intensively studied and found to encompass diverse mechanisms operating across a broad range of cellular components, timescales and temperatures. This review explores recent progress throughout this landscape with a particular focus on thermosensing in the model plant Arabidopsis. Direct temperature sensors include the photosensors phytochrome B and phototropin, the clock component ELF3 and an RNA switch. In addition, there are heat-regulated processes mediated by ion channels, lipids and lipid-modifying enzymes, taking place at the plasma membrane and the chloroplast. In some cases, the mechanism of temperature perception is well understood but in others, this remains an open question. Potential novel thermosensing mechanisms are based on lipid and liquid-liquid phase separation. Finally, future research directions of high temperature perception and signalling pathways are discussed.

Phytochrome B and PCH1 protein dynamics store night temperature information.

Plants experience temperature fluctuations during the course of the daily cycle, and although stem growth responds rapidly to these changes we largely ignore whether there is a short-term memory of previous conditions. Here we show that nighttime temperatures affect the growth of the hypocotyl of Arabidopsis thaliana seedlings not only during the night but also during the subsequent photoperiod. Active phytochrome B (phyB) represses nighttime growth and warm temperatures reduce active phyB via thermal reversion. The function of PHOTOPERIODIC CONTROL OF HYPOCOTYL1 (PCH1) is to stabilise active phyB in nuclear bodies but, surprisingly, warmth reduces PCH1 gene expression and PCH1 stability. When phyB was active at the beginning of the night, warm night temperatures enhanced the levels of nuclear phyB and reduced hypocotyl growth rate during the following day. However, when end-of-day far-red light minimised phyB activity, warm night temperatures reduced the levels of nuclear phyB and enhanced the hypocotyl growth rate during the following day. This complex growth pattern was absent in the phyB mutant. We propose that temperature-induced changes in the levels of PCH1 and in the size of the physiologically relevant nuclear pool of phyB amplify the impact of phyB-mediated temperature sensing.

Phytochrome A inhibits shade avoidance responses under strong shade through repressing the brassinosteroid pathway in Arabidopsis.

In dense canopy, a reduction in red to far-red (R/FR) light ratio triggers shade avoidance responses (SARs) in Arabidopsis thaliana, a shade avoiding plant. Two red/far-red (R/FR) light photoreceptors, PHYB and PHYA, were reported to be key negative regulators of the SARs. PHYB represses the SARs under normal light conditions; however, the role of PHYA in the SARs remains elusive. We set up two shade conditions: Shade and strong Shade (s-Shade) with different R/FR ratios (0.7 and 0.1), which allowed us to observe phenotypes dominated by PHYB- and PHYA-mediated pathway, respectively. By comparing the hypocotyl growth under these two conditions with time, we found PHYA was predominantly activated in the s-Shade after prolonged shade treatment. We further showed that under s-Shade, PHYA inhibits hypocotyl elongation partially through repressing the brassinosteroid (BR) pathway. COP1 and PIF4,5 act downstream of PHYA. After prolonged shade treatment, the nuclear localization of COP1 was reduced, while the PIF4 protein level was much lower in the s-Shade than that in Shade. Both changes occurred in a PHYA-dependent manner. We propose that under deep canopy, the R/FR ratio is extremely low, which promotes the nuclear accumulation of PHYA. Activated PHYA reduces COP1 nuclear speckle, which may lead to changes of downstream targets, such as PIF4,5 and HY5. Together, these proteins regulate the BR pathway through modulating BES1/BZR1 and the expression of BR biosynthesis and BR target genes.

Effect of phyB and phyC loss-of-function mutations on the wheat transcriptome under short and long day photoperiods.

BACKGROUND: Photoperiod signals provide important cues by which plants regulate their growth and development in response to predictable seasonal changes. Phytochromes, a family of red and far-red light receptors, play critical roles in regulating flowering time in response to changing photoperiods. A previous study showed that loss-of-function mutations in either PHYB or PHYC result in large delays in heading time and in the differential regulation of a large number of genes in wheat plants grown in an inductive long day (LD) photoperiod. RESULTS: We found that under non-inductive short-day (SD) photoperiods, phyB-null and phyC-null mutants were taller, had a reduced number of tillers, longer and wider leaves, and headed later than wild-type (WT) plants. The delay in heading between WT and phy mutants was greater in LD than in SD, confirming the importance of PHYB and PHYC in accelerating heading date in LDs. Both mutants flowered earlier in SD than LD, the inverse response to that of WT plants. In both SD and LD photoperiods, PHYB regulated more genes than PHYC. We identified subsets of differentially expressed and alternatively spliced genes that were specifically regulated by PHYB and PHYC in either SD or LD photoperiods, and a smaller set of genes that were regulated in both photoperiods. We found that photoperiod had a contrasting effect on transcript levels of the flowering promoting genes VRN-A1 and PPD-B1 in phyB and phyC mutants compared to the WT. CONCLUSIONS: Our study confirms the major role of both PHYB and PHYC in flowering promotion in LD conditions. Transcriptome characterization revealed an unexpected reversion of the wheat LD plants into SD plants in the phyB-null and phyC-null mutants and identified flowering genes showing significant interactions between phytochromes and photoperiod that may be involved in this phenomenon. Our RNA-seq data provides insight into light signaling pathways in inductive and non-inductive photoperiods and a set of candidate genes to dissect the underlying developmental regulatory networks in wheat.

Light controls stamen elongation via cryptochromes, phytochromes and COP1 through HY5 and HYH.

In Arabidopsis, stamen elongation, which ensures male fertility, is controlled by the auxin response factor ARF8, which regulates the expression of the auxin repressor IAA19. Here, we uncover a role for light in controlling stamen elongation. By an extensive genetic and molecular analysis we show that the repressor of light signaling COP1, through its targets HY5 and HYH, controls stamen elongation, and that HY5 - oppositely to ARF8 - directly represses the expression of IAA19 in stamens. In addition, we show that in closed flower buds, when light is shielded by sepals and petals, the blue light receptors CRY1/CRY2 repress stamen elongation. Coherently, at flower disclosure and in subsequent stages, stamen elongation is repressed by the red and far-red light receptors PHYA/PHYB. In conclusion, different light qualities - sequentially perceived by specific photoreceptors - and the downstream COP1-HY5/HYH module finely tune auxin-induced stamen elongation and thus male fertility.

Oligomerization and Photo-Deoligomerization of HOOKLESS1 Controls Plant Differential Cell Growth.

Apical hook curvature is crucial for buried seedling survival and a superb model for dissecting differential cell growth. HOOKLESS1 (HLS1) is essential for apical hook formation, acting as a hub integrating various external and internal signals. However, its functional mechanism remains unclear. Here, we demonstrate that HLS1 protein is present as an oligomer in the nucleus of dark-grown seedlings. Oligomerization is required for HLS1 activation, as the mutated HLS1 protein abolishing self-association exists as nonfunctional monomers. Upon light exposure, photoreceptor phyB translocates into the nucleus and interacts with HLS1, disrupting the self-association and oligomerization of HLS1 to initiate hook unfolding. Remarkably, genetic expression of nuclear-localized phyB is sufficient to inactivate HLS1, resulting in compromised hook curvature in etiolated seedlings. Together, we conclude that HLS1 protein is active as oligomeric form in darkness and achieves allosteric photo-deactivation upon light, providing intriguing mechanistic insight into the molecular switch for developmental transition.

phyB Interacts with BES1 to Regulate Brassinosteroid Signaling in Arabidopsis.

Light is an important environmental factor, which mainly inhibits hypocotyl elongation through various photoreceptors. In contrast, brassinosteroids (BRs) are major hypocotyl elongation-promoting hormones in plants, which could optimize photomorphogenesis concurrent with external light. However, the precise molecular mechanisms underlying the antagonism of light and BR signaling remain largely unknown. Here we show that the Arabidopsis red light receptor phyB is involved in inhibition of BR signaling via its direct interaction with the BR transcription factor BES1. In our study, the phyB mutant displays BR hypersensitivity, which is repressed in transgenic plants overexpressing phyB, suggesting that phyB negatively regulates the BR signaling pathway. In addition, protein interaction results show that phyB directly interacts with dephosphorylated BES1, the physiologically active form of BES1 induced by BR, in a red light-dependent manner. Genetic analyses suggest that phyB may act partially through BES1 to regulate BR signaling. Transcriptomic data and quantitative real-time PCR assay further show that phyB-mediated red light inhibits BR signaling by repressing expression of BES1 target genes, including the BR biosynthesis genes DWF4, the SAUR family and the PRE family genes required for promoting cell elongation. Finally, we found that red light treatment inhibits the DNA-binding activity of BES1 and photoactivated phyB represses the transcriptional activity of BES1 under red light. Taken together, we suggest that the interaction of phyB with dephosphorylated BES1 may allow plants to balance light and BR signaling by repressing transcriptional activity of BES1 to regulate expression of its target genes.

Phytochrome B1-dependent control of SP5G transcription is the basis of the night break and red to far-red light ratio effects in tomato flowering.

BACKGROUND: Phytochromes are dimeric proteins with critical roles in perceiving day length and the environmental signals that trigger flowering. Night break (NB) and the red to far-red light ratio (R:FR) have been used extensively as tools to study the photoperiodic control of flowering. However, at the molecular level, little is known about the effect of NB and different R:FR values on flowering in day-neutral plants (DNPs) such as tomato. RESULTS: Here, we show that tomato SP5G, SP5G2, and SP5G3 are homologs of Arabidopsis thaliana FLOWERING LOCUS T (FT) that repress flowering in Nicotiana benthamiana. NB every 2 h at intensities of 10 µmol m- 2 s- 1 or lower R:FR (e.g., 0.6) caused a clear delay in tomato flowering and promoted SP5G mRNA expression. The promoted SP5G mRNA expression induced by red light NB and low R:FR treatments was reversed by a subsequent FR light stimulus or a higher R:FR treatment. The tomato phyB1 mutation abolished the effects of NB and lower R:FR treatments on flowering and SP5G mRNA expression, indicating that the effects were mediated by phytochrome B1 in tomato. CONCLUSION: Our results strongly suggest that SP5G mRNA suppression is the principal cause of NB and lower R:FR effects on flowering in tomato.

Phytochrome A and B Negatively Regulate Salt Stress Tolerance of Nicotiana tobacum via ABA-Jasmonic Acid Synergistic Cross-Talk.

Light signaling and phytohormones play important roles in plant growth, development, and biotic and abiotic stress responses. However, the roles of phytochromes and cross-talk between these two signaling pathways in response to salt stress in tobacco plants remain underexplored. Here, we explored the defense response in phytochrome-defective mutants under salt stress. We monitored the physiological and molecular changes of these mutants under salt stress conditions. The results showed that phytochrome A (phyA), phytochrome B (phyB) and phyAphyB (phyAB) mutants exhibited improved salt stress tolerance compared with wild-type (WT) plants. The mutant plants had a lower electrolyte leakage (EL) and malondialdehyde (MDA) concentration than WT plants, and the effect was clearly synergistic in the phyAB double mutant plants. Furthermore, the data showed that the transcript levels of defense-associated genes and the activities of some antioxidant enzymes in the mutant plants were much higher than those in WT plants. Additionally, the results indicated that phytochrome signaling strongly modulates the expression of endogenous abscisic acid (ABA) and jasmonic acid (JA) of Nicotiana tobacum in response to salt stress. To illustrate further the relationship between phytochrome and phytohormone, we measured the expression of defense genes and phytochrome. The results displayed that salt stress and application of methyl jasmonate (MeJA) or ABA up-regulated the transcript levels of salt response-associated genes and inhibited the expression of NtphyA and NtphyB. Foliar application of inhibitors of ABA and JA further confirmed that JA co-operated with ABA in phytochrome-mediated salt stress tolerance.

Root-expressed phytochromes B1 and B2, but not PhyA and Cry2, regulate shoot growth in nature.

Although photoreceptors are expressed throughout all plant organs, most studies have focused on their function in aerial parts with laboratory-grown plants. Photoreceptor function in naturally dark-grown roots of plants in their native habitats is lacking. We characterized patterns of photoreceptor expression in field- and glasshouse-grown Nicotiana attenuata plants, silenced the expression of PhyB1/B2/A/Cry2 whose root transcripts levels were greater/equal to those of shoots, and by micrografting combined empty vector transformed shoots onto photoreceptor-silenced roots, creating chimeric plants with "blind" roots but "sighted" shoots. Micrografting procedure was robust in both field and glasshouse, as demonstrated by transcript accumulation patterns, and a spatially-explicit lignin visual reporter chimeric line. Field- and glasshouse-grown plants with PhyB1B2, but not PhyA or Cry2, -blind roots, were delayed in stalk elongation compared with control plants, robustly for two field seasons. Wild-type plants with roots directly exposed to FR phenocopied the growth of irPhyB1B2-blind root grafts. Additionally, root-expressed PhyB1B2 was required to activate the positive photomorphogenic regulator, HY5, in response to aboveground light. We conclude that roots of plants growing deep into the soil in nature sense aboveground light, and possibly soil temperature, via PhyB1B2 to control key traits, such as stalk elongation.

The evening complex coordinates environmental and endogenous signals in Arabidopsis.

Plants maximize their fitness by adjusting their growth and development in response to signals such as light and temperature. The circadian clock provides a mechanism for plants to anticipate events such as sunrise and adjust their transcriptional programmes. However, the underlying mechanisms by which plants coordinate environmental signals with endogenous pathways are not fully understood. Using RNA-sequencing and chromatin immunoprecipitation sequencing experiments, we show that the evening complex (EC) of the circadian clock plays a major role in directly coordinating the expression of hundreds of key regulators of photosynthesis, the circadian clock, phytohormone signalling, growth and response to the environment. We find that the ability of the EC to bind targets genome-wide depends on temperature. In addition, co-occurrence of phytochrome B (phyB) at multiple sites where the EC is bound provides a mechanism for integrating environmental information. Hence, our results show that the EC plays a central role in coordinating endogenous and environmental signals in Arabidopsis.

Shade Promotes Phototropism through Phytochrome B-Controlled Auxin Production.

Phototropism is an asymmetric growth response enabling plants to optimally position their organs. In flowering plants, the phototropin (phot) blue light receptors are essential to detect light gradients. In etiolated seedlings, the phototropic response is enhanced by the red/far-red (R/FR)-sensing phytochromes (phy) with a predominant function of phyA. In this study, we analyzed the influence of the phytochromes on phototropism in green (de-etiolated) Arabidopsis seedlings. Our experiments in the laboratory and outdoors revealed that, in open environments (high R/FR ratio), phyB inhibits phototropism. In contrast, under foliar shade, where access to direct sunlight becomes important, the phototropic response was strong. phyB modulates phototropism, depending on the R/FR ratio, by controlling the activity of three basic-helix-loop-helix (bHLH) transcription factors of the PHYTOCHROME INTERACTING FACTORs (PIFs) family. Promotion of phototropism depends on PIF-mediated induction of several members of the YUCCA gene family, leading to auxin production in the cotyledons. Our study identifies PIFs and YUCCAs as novel molecular players promoting phototropism in photoautotrophic, but not etiolated, seedlings. Moreover, our findings reveal fundamental differences in the phytochrome-phototropism crosstalk in etiolated versus green seedlings. We propose that in natural conditions where the light environment is not homogeneous, the uncovered phytochrome-phototropin co-action is important for plants to adapt their growth strategy to optimize photosynthetic light capture.

Phytochrome B integrates light and temperature signals in Arabidopsis.

Ambient temperature regulates many aspects of plant growth and development, but its sensors are unknown. Here, we demonstrate that the phytochrome B (phyB) photoreceptor participates in temperature perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state. Increased rates of thermal reversion upon exposing Arabidopsis seedlings to warm environments reduce both the abundance of the biologically active Pfr-Pfr dimer pool of phyB and the size of the associated nuclear bodies, even in daylight. Mathematical analysis of stem growth for seedlings expressing wild-type phyB or thermally stable variants under various combinations of light and temperature revealed that phyB is physiologically responsive to both signals. We therefore propose that in addition to its photoreceptor functions, phyB is a temperature sensor in plants.

Signalling to the nucleus under the control of light and small molecules.

One major regulatory mechanism in cell signalling is the spatio-temporal control of the localization of signalling molecules. We synthetically designed an entire cell signalling pathway, which allows controlling the transport of signalling molecules from the plasma membrane to the nucleus, by using light and small molecules.

Illuminating cell signalling with optogenetic tools.

The light-based control of ion channels has been transformative for the neurosciences, but the optogenetic toolkit does not stop there. An expanding number of proteins and cellular functions have been shown to be controlled by light, and the practical considerations in deciding between reversible optogenetic systems (such as systems that use light-oxygen-voltage domains, phytochrome proteins, cryptochrome proteins and the fluorescent protein Dronpa) are well defined. The field is moving beyond proof of concept to answering real biological questions, such as how cell signalling is regulated in space and time, that were difficult or impossible to address with previous tools.

A red and far-red light receptor mutation confers resistance to the herbicide glyphosate.

Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.