Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis.

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1's involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.

Disrupting FKF1 homodimerization increases FT transcript levels in the evening by enhancing CO stabilization.

KEY MESSAGE: FKF1 dimerization is crucial for proper FT levels to fine-tune flowering time. Attenuating FKF1 homodimerization increased CO abundance by enhancing its COP1 binding, thereby accelerating flowering under long days. In Arabidopsis (Arabidopsis thaliana), the blue-light photoreceptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) plays a key role in inducing the expression of FLOWERING LOCUS T (FT), encoding the main florigenic signal in plants, in the late afternoon under long-day conditions (LDs) by forming dimers with FT regulators. Although structural studies have unveiled a variant of FKF1 (FKF1 I160R) that disrupts homodimer formation in vitro, the mechanism by which disrupted FKF1 homodimer formation regulates flowering time remains elusive. In this study, we determined that the attenuation of FKF1 homodimer formation enhances FT expression in the evening by promoting the increased stability of CONSTANS (CO), a primary activator of FT, in the afternoon, thereby contributing to early flowering. In contrast to wild-type FKF1, introducing the FKF1 I160R variant into the fkf1 mutant led to increased FT expression under LDs. In addition, the FKF1 I160R variant exhibited diminished dimerization with FKF1, while its interaction with GIGANTEA (GI), a modulator of FKF1 function, was enhanced under LDs. Furthermore, the FKF1 I160R variant increased the level of CO in the afternoon under LDs by enhancing its binding to COP1, an E3 ubiquitin ligase responsible for CO degradation. These findings suggest that the regulation of FKF1 homodimerization and heterodimerization allows plants to finely adjust FT expression levels around dusk by modulating its interactions with GI and COP1.

Low temperature-mediated repression and far-red light-mediated induction determine morning FLOWERING LOCUS T expression levels.

In order to flower in the appropriate season, plants monitor light and temperature changes and alter downstream pathways that regulate florigen genes such as Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT). In Arabidopsis, FT messenger RNA levels peak in the morning and evening under natural long-day conditions (LDs). However, the regulatory mechanisms governing morning FT induction remain poorly understood. The morning FT peak is absent in typical laboratory LDs characterized by high red:far-red light (R:FR) ratios and constant temperatures. Here, we demonstrate that ZEITLUPE (ZTL) interacts with the FT repressors TARGET OF EATs (TOEs), thereby repressing morning FT expression in natural environments. Under LDs with simulated sunlight (R:FR = 1.0) and daily temperature cycles, which are natural LD-mimicking environmental conditions, FT transcript levels in the ztl mutant were high specifically in the morning, a pattern that was mirrored in the toe1 toe2 double mutant. Low night-to-morning temperatures increased the inhibitory effect of ZTL on morning FT expression by increasing ZTL protein levels early in the morning. Far-red light counteracted ZTL activity by decreasing its abundance (possibly via phytochrome A (phyA)) while increasing GIGANTEA (GI) levels and negatively affecting the formation of the ZTL-GI complex in the morning. Therefore, the phyA-mediated high-irradiance response and GI play pivotal roles in morning FT induction. Our findings suggest that the delicate balance between low temperature-mediated ZTL activity and the far-red light-mediated functions of phyA and GI offers plants flexibility in fine-tuning their flowering time by controlling FT expression in the morning.

Responsiveness to long days for flowering is reduced in Arabidopsis by yearly variation in growing season temperatures.

Conservative flowering behaviours, such as flowering during long days in summer or late flowering at a high leaf number, are often proposed to protect against variable winter and spring temperatures which lead to frost damage if premature flowering occurs. Yet, due the many factors in natural environments relative to the number of individuals compared, assessing which climate characteristics drive these flowering traits has been difficult. We applied a multidisciplinary approach to 10 winter-annual Arabidopsis thaliana populations from a wide climactic gradient in Norway. We used a variable reduction strategy to assess which of 100 climate descriptors from their home sites correlated most to their flowering behaviours when tested for responsiveness to photoperiod after saturation of vernalization; then, assessed sequence variation of 19 known environmental-response flowering genes. Photoperiod responsiveness inversely correlated with interannual variation in timing of growing season onset. Time to flowering appeared driven by growing season length, curtailed by cold fall temperatures. The distribution of FLM, TFL2 and HOS1 haplotypes, genes involved in ambient temperature response, correlated with growing-season climate. We show that long-day responsiveness and late flowering may be driven not by risk of spring frosts, but by growing season temperature and length, perhaps to opportunistically maximize growth.

The FLOWERING LOCUS T gene expression is controlled by high-irradiance response and external coincidence mechanism in long days in Arabidopsis.

In natural long days, the florigen gene FLOWERING LOCUS T (FT) shows a bimodal expression pattern with morning and dusk peaks in Arabidopsis. This pattern differs from the one observed in the laboratory, and little is known about underlying mechanisms. A red : far-red (R : FR) ratio difference between sunlight and fluorescent light causes this FT pattern mismatch. We showed that bimodal FT expression patterns were induced in a day longer than 14 h with sunlight R : FR (= c. 1) conditions. By circadian gating experiments, we found that cumulative exposure of R : FR-adjusted light (R : FR ratio was adjusted to 1 with FR supplement) spanning from the afternoon to the next morning required full induction of FT in the morning. Conversely, only 2 h of R : FR adjustment in the late afternoon was sufficient for FT induction at dusk. We identified that phytochrome A (phyA) is required for the morning FT expression in response to the R : FR adjustment on the previous day. As a part of this mechanism, we showed that PHYTOCHROME-INTERACTING FACTOR 7 contributes to FT regulation. Our results suggest that phyA-mediated high-irradiance response and the external coincidence mechanism contribute to morning FT induction under natural long-day conditions.

Photoperiodic flowering in Arabidopsis: Multilayered regulatory mechanisms of CONSTANS and the florigen FLOWERING LOCUS T.

The timing of flowering affects the success of sexual reproduction. This developmental event also determines crop yield, biomass, and longevity. Therefore, this mechanism has been targeted for improvement along with crop domestication. The underlying mechanisms of flowering are highly conserved in angiosperms. Central to these mechanisms is how environmental and endogenous conditions control transcriptional regulation of the FLOWERING LOCUS T (FT) gene, which initiates floral development under long-day conditions in Arabidopsis. Since the identification of FT as florigen, efforts have been made to understand the regulatory mechanisms of FT expression. Although many transcriptional regulators have been shown to directly influence FT, the question of how they coordinately control the spatiotemporal expression patterns of FT still requires further investigation. Among FT regulators, CONSTANS (CO) is the primary one whose protein stability is tightly controlled by phosphorylation and ubiquitination/proteasome-mediated mechanisms. In addition, various CO interaction partners, some of them previously identified as FT transcriptional regulators, positively or negatively modulate CO protein activity. The FT promoter possesses several transcriptional regulatory "blocks," highly conserved regions among Brassicaceae plants. Different transcription factors bind to specific blocks and affect FT expression, often causing topological changes in FT chromatin structure, such as the formation of DNA loops. We discuss the current understanding of the regulation of FT expression mainly in Arabidopsis and propose future directions related to this topic.

Clock-Controlled and Cold-Induced CYCLING DOF FACTOR6 Alters Growth and Development in Arabidopsis.

The circadian clock represents a critical regulatory network, which allows plants to anticipate environmental changes as inputs and promote plant survival by regulating various physiological outputs. Here, we examine the function of the clock-regulated transcription factor, CYCLING DOF FACTOR 6 (CDF6), during cold stress in Arabidopsis thaliana. We found that the clock gates CDF6 transcript accumulation in the vasculature during cold stress. CDF6 mis-expression results in an altered flowering phenotype during both ambient and cold stress. A genome-wide transcriptome analysis links CDF6 to genes associated with flowering and seed germination during cold and ambient temperatures, respectively. Analysis of key floral regulators indicates that CDF6 alters flowering during cold stress by repressing photoperiodic flowering components, FLOWERING LOCUS T (FT), CONSTANS (CO), and BROTHER OF FT (BFT). Gene ontology enrichment further suggests that CDF6 regulates circadian and developmental-associated genes. These results provide insights into how the clock-controlled CDF6 modulates plant development during moderate cold stress.

A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

BrABF3 promotes flowering through the direct activation of CONSTANS transcription in pak choi.

Drought stress triggers the accumulation of the phytohormone abscisic acid (ABA), which in turn activates the expression of the floral integrator gene CONSTANS (CO), accelerating flowering. However, the molecular mechanism of ABA-induced CO activation remains elusive. Here, we conducted a yeast one-hybrid assay using the CO promoter from Brassica campestris (syn. Brassica rapa) ssp. chinensis (pak choi) to screen the ABA-induced pak choi library and identified the transcription activator ABF3 (BrABF3). BrABF3, the expression of which was induced by ABA in pak choi, directly bound to the CO promoter from both pak choi and Arabidopsis. The BrABF3 promoter is specifically active in the Arabidopsis leaf vascular tissue, where CO is mainly expressed. Impaired BrABF3 expression in pak choi decreased BrCO expression levels and delayed flowering, whereas ectopic expression of BrABF3 in Arabidopsis increased CO expression and induced earlier flowering under the long-day conditions. Electrophoretic mobility shift assay analysis showed that BrABF3 was enriched at the canonical ABA-responsive element-ABRE binding factor (ABRE-ABF) binding motifs of the BrCO promoter. The direct binding of BrABF3 to the ABRE elements of CO was further confirmed by chromatin immunoprecipitation quantitative PCR. In addition, the induction of BrCO transcription by BrABF3 could be repressed by BrCDF1 in the morning. Thus, our results suggest that ABA could accelerate the floral transition by directly activating BrCO transcription through BrABF3 in pak choi.

Simple Nuclei Preparation and Co-immunoprecipitation Procedures for Studying Protein Abundance and Interactions in Plant Circadian Time Courses.

The plant circadian clock regulates multiple developmental and physiological events that occur at specific times and seasons. As many of the currently known clock proteins and clock-associated regulators are transcription factors, analyzing molecular events in the nuclei is crucial. In addition, long-time course analyses of protein abundance and interactions are often required to assess the role of the circadian clock on clock-regulated phenomena. Here we introduce a simple procedure to prepare nuclear-enriched tissues, which we routinely use to study time-resolved accumulation changes in low-abundance nuclear proteins (i.e., transcription factors). In addition to measuring changes in abundance, investigating the protein-protein interaction dynamics at specific times of day or under certain environmental conditions is needed for plant chronobiology studies. Therefore, we also present our co-immunoprecipitation method for studying diurnal/circadian protein-protein interactions, tailored to nuclear-localized proteins in Arabidopsis and tobacco.

Flowering Times of Wild Arabidopsis Accessions From Across Norway Correlate With Expression Levels of FT, CO, and FLC Genes.

Temperate species often require or flower most rapidly in the long daylengths, or photoperiods, experienced in summer or after prolonged periods of cold temperatures, referred to as vernalization. Yet, even within species, plants vary in the degree of responsiveness to these cues. In Arabidopsis thaliana, CONSTANS (CO) and FLOWERING LOCUS C (FLC) genes are key to photoperiod and vernalization perception and antagonistically regulate FLOWERING LOCUS T (FT) to influence the flowering time of the plants. However, it is still an open question as to how these genes vary in their interactions among wild accessions with different flowering behaviors and adapted to different microclimates, yet this knowledge could improve our ability to predict plant responses in variable natural conditions. To assess the relationships among these genes and to flowering time, we exposed 10 winter-annual Arabidopsis accessions from throughout Norway, ranging from early to late flowering, along with two summer-annual accessions to 14 weeks of vernalization and either 8- or 19-h photoperiods to mimic Norwegian climate conditions, then assessed gene expression levels 3-, 5-, and 8-days post vernalization. CO and FLC explained both FT levels and flowering time (days) but not rosette leaf number at flowering. The correlation between FT and flowering time increased over time. Although vernalization suppresses FLC, FLC was high in the late-flowering accessions. Across accessions, FT was expressed only at low FLC levels and did not respond to CO in the late-flowering accessions. We proposed that FT may only be expressed below a threshold value of FLC and demonstrated that these three genes correlated to flowering times across genetically distinct accessions of Arabidopsis.

Low nitrogen conditions accelerate flowering by modulating the phosphorylation state of FLOWERING BHLH 4 in Arabidopsis.

Nitrogen (N) is an essential nutrient that affects multiple plant developmental processes, including flowering. As flowering requires resources to develop sink tissues for reproduction, nutrient availability is tightly linked to this process. Low N levels accelerate floral transition; however, the molecular mechanisms underlying this response are not well understood. Here, we identify the FLOWERING BHLH 4 (FBH4) transcription factor as a key regulator of N-responsive flowering in Arabidopsis Low N-induced early flowering is compromised in fbh quadruple mutants. We found that FBH4 is a highly phosphorylated protein and that FBH4 phosphorylation levels decrease under low N conditions. In addition, decreased phosphorylation promotes FBH4 nuclear localization and transcriptional activation of the direct target CONSTANS (CO) and downstream florigen FLOWERING LOCUS T (FT) genes. Moreover, we demonstrate that the evolutionarily conserved cellular fuel sensor SNF1-RELATED KINASE 1 (SnRK1), whose kinase activity is down-regulated under low N conditions, directly phosphorylates FBH4. SnRK1 negatively regulates CO and FT transcript levels under high N conditions. Together, these results reveal a mechanism by which N levels may fine-tune FBH4 nuclear localization by adjusting the phosphorylation state to modulate flowering time. In addition to its role in flowering regulation, we also showed that FBH4 was involved in low N-induced up-regulation of nutrient recycling and remobilization-related gene expression. Thus, our findings provide insight into N-responsive growth phase transitions and optimization of plant fitness under nutrient-limited conditions.

Steric and Electronic Interactions at Gln154 in ZEITLUPE Induce Reorganization of the LOV Domain Dimer Interface.

Plants measure light quality, intensity, and duration to coordinate growth and development with daily and seasonal changes in environmental conditions; however, the molecular details linking photochemistry to signal transduction remain incomplete. Two closely related light, oxygen, or voltage (LOV) domain-containing photoreceptor proteins, ZEITLUPE (ZTL) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), divergently regulate the protein stability of circadian clock and photoperiodic flowering components to mediate daily and seasonal development. Using structural approaches, we identified that mutations at the Gly46 position led to global rearrangements of the ZTL dimer interface in the isolated ZTL-LOV domain. Specifically, G46S and G46A variants induce a 180° rotation about the ZTL-LOV dimer interface that is coupled to ordering of N- and C-terminal signaling elements. These conformational changes hinge upon rotation of a C-terminal Gln residue (Gln154) analogous to that present in light-state structures of ZTL. In contrast to other LOV proteins, a Q154L variant retains light-state interactions with GIGANTEA (GI), thereby indicating N5 protonation is not required for ZTL signaling. The results presented herein confirm a divergent signaling mechanism within ZTL, whereby steric and electronic effects following adduct formation can be sufficient for signal propagation in LOV proteins containing a Gly residue at position 46. Examination of bacterial LOV structures with Gly residues at the equivalent position suggests that mechanisms of signal transduction in LOV proteins may be fluid across the LOV protein family.

Building customizable auto-luminescent luciferase-based reporters in plants.

Bioluminescence is a powerful biological signal that scientists have repurposed as a reporter for gene expression in plants and animals. However, there are downsides associated with the need to provide a substrate to these reporters, including its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using a composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for future construction of programmable auto-luminescent plant traits, such as light driven plant-pollinator interactions or light emitting plant-based sensors.

Many animals have evolved the capacity to produce light from chemical reactions. For example, an enzyme known as luciferase in fireflies produces light by acting on a molecule called luciferin. Scientists have identified the enzymes that drive several of these systems and used them to build reporters that can study the activity of genes in the tissues of plants and other lifeforms over space and time. However, these reporters often require chemicals to be added to the tissues to produce light. These chemicals tend to be expensive and may not penetrate evenly into the tissues of interest, limiting the potential applications of the reporters in research studies. Recently, it has been discovered that fungi have a bioluminescence pathway that converts a molecule known as caffeic acid into luciferin. Caffeic acid is a common molecule in plants, therefore, it is possible the fungal bioluminescence pathway could be used to build reporters that produce light without needing the addition of chemicals. Now, Khakhar et al. have inserted the genes that encode the enzymes of the fungal bioluminescence pathway into tobacco plants. The experiments found that this was sufficient to turn caffeic acid into molecules of luciferin which are able to produce light. Inserting the same genes into several other plant species, including tomatoes and dahlias, produced similar results. Further experiments showed that the fungal bioluminescence pathway can be used to build reporters that monitor the activity of plant genes throughout living tissues and over a period of several days as well as examine the response to plant hormones. Alongside studying the activities of genes in plants, Khakhar et al. propose that the toolkit developed in this work could be used to generate plants with luminescence that can be switched on or off as desired. This could have many uses including helping plants attract insects to pollinate flowers and building plant biosensors that emit light in response to environmental signals.

GIGANTEA Regulates the Timing Stabilization of CONSTANS by Altering the Interaction between FKF1 and ZEITLUPE.

Plants monitor changes in day length to coordinate their flowering time with appropriate seasons. In Arabidopsis , the diel and seasonal regulation of CONSTANS (CO) protein stability is crucial for the induction of FLOWERING LOCUS T (FT) gene in long days. FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and ZEITLUPE (ZTL) proteins control the shape of CO expression profile antagonistically, although regulation mechanisms remain unknown. In this study, we show that GIGANTEA (GI) protein modulates the stability and nuclear function of FKF1, which is closely related to the stabilization of CO in the afternoon of long days. The abundance of FKF1 protein is decreased by the gi mutation, but increased by GI overexpression throughout the day. Unlike the previous report, the translocation of FKF1 to the nucleus was not prevented by ZTL overexpression. In addition, the FKF1-ZTL complex formation is higher in the nucleus than in the cytosol. GI interacts with ZTL in the nucleus, implicating the attenuation of ZTL activity by the GI binding and, in turn, the sequestration of FKF1 from ZTL in the nucleus. We also found that the CO-ZTL complex presents in the nucleus, and CO protein abundance is largely reduced in the afternoon by ZTL overexpression, indicating that ZTL promotes CO degradation by capturing FKF1 in the nucleus under these conditions. Collectively, our findings suggest that GI plays a pivotal role in CO stability for the precise control of flowering by coordinating balanced functional properties of FKF1 and ZTL.

An explanatory model of temperature influence on flowering through whole-plant accumulation of FLOWERING LOCUS T in Arabidopsis thaliana.

We assessed mechanistic temperature influence on flowering by incorporating temperature-responsive flowering mechanisms across developmental age into an existing model. Temperature influences the leaf production rate as well as expression of FLOWERING LOCUS T (FT), a photoperiodic flowering regulator that is expressed in leaves. The Arabidopsis Framework Model incorporated temperature influence on leaf growth but ignored the consequences of leaf growth on and direct temperature influence of FT expression. We measured FT production in differently aged leaves and modified the model, adding mechanistic temperature influence on FT transcription, and causing whole-plant FT to accumulate with leaf growth. Our simulations suggest that in long days, the developmental stage (leaf number) at which the reproductive transition occurs is influenced by day length and temperature through FT, while temperature influences the rate of leaf production and the time (in days) the transition occurs. Further, we demonstrate that FT is mainly produced in the first 10 leaves in the Columbia (Col-0) accession, and that FT accumulation alone cannot explain flowering in conditions in which flowering is delayed. Our simulations supported our hypotheses that: (i) temperature regulation of FT, accumulated with leaf growth, is a component of thermal time, and (ii) incorporating mechanistic temperature regulation of FT can improve model predictions when temperatures change over time.

Time-resolved interaction proteomics of the GIGANTEA protein under diurnal cycles in Arabidopsis.

The plant-specific protein GIGANTEA (GI) controls many developmental and physiological processes, mediating rhythmic post-translational regulation. GI physically binds several proteins implicated in the circadian clock, photoperiodic flowering, and abiotic stress responses. To understand GI's multifaceted function, we aimed to comprehensively and quantitatively identify potential interactors of GI in a time-specific manner, using proteomics on Arabidopsis plants expressing epitope-tagged GI. We detected previously identified (in)direct interactors of GI, as well as proteins implicated in protein folding, or degradation, and a previously uncharacterized transcription factor, CYCLING DOF FACTOR6 (CDF6). We verified CDF6's direct interaction with GI, and ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LIGHT KELCH PROTEIN 2 proteins, and demonstrated its involvement in photoperiodic flowering. Extending interaction proteomics to time series provides a data resource of candidate protein targets for GI's post-translational control.

Molecular basis of flowering under natural long-day conditions in Arabidopsis.

Plants sense light and temperature changes to regulate flowering time. Here, we show that expression of the Arabidopsis florigen gene, FLOWERING LOCUS T (FT), peaks in the morning during spring, a different pattern than we observe in the laboratory. Providing our laboratory growth conditions with a red/far-red light ratio similar to open-field conditions and daily temperature oscillation is sufficient to mimic the FT expression and flowering time in natural long days. Under the adjusted growth conditions, key light signalling components, such as phytochrome A and EARLY FLOWERING 3, play important roles in morning FT expression. These conditions stabilize CONSTANS protein, a major FT activator, in the morning, which is probably a critical mechanism for photoperiodic flowering in nature. Refining the parameters of our standard growth conditions to more precisely mimic plant responses in nature can provide a powerful method for improving our understanding of seasonal response.