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.

Rice OsGATA16 is a positive regulator for chlorophyll biosynthesis and chloroplast development.

Chloroplasts are essential organelles in plants that contain chlorophylls and facilitate photosynthesis for growth and development. As photosynthetic efficiency significantly impacts crop productivity, understanding the regulatory mechanisms of chloroplast development has been crucial in increasing grain and biomass production. This study demonstrates the involvement of OsGATA16, an ortholog of Arabidopsis GATA, NITRATE INDUCIBLE, CARBON-METABOLISM INVOLVED (GNC), and GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR 1 (GNL/CGA1), in chlorophyll biosynthesis and chloroplast development in rice (Oryza sativa). The osgata16-1 knockdown mutants produced pale-green leaves, while OsGATA16-overexpressed plants (OsGATA16-OE1) generated dark-green leaves, compared to their parental japonica rice. Reverse transcription and quantitative PCR analysis revealed downregulation of genes related to chloroplast division, chlorophyll biosynthesis, and photosynthesis in the leaves of osgata16-1 and upregulation in those of OsGATA16-OE1. Additionally, in vivo binding assays showed that OsGATA16 directly binds to the promoter regions of OsHEMA, OsCHLH, OsPORA, OsPORB, and OsFtsZ, and upregulates their expression. These findings indicate that OsGATA16 serves as a positive regulator controlling chlorophyll biosynthesis and chloroplast development in rice.

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.

Proteomic Analysis Reveals a Critical Role of the Glycosyl Hydrolase 17 Protein in Panax ginseng Leaves under Salt Stress.

Ginseng, an important crop in East Asia, exhibits multiple medicinal and nutritional benefits because of the presence of ginsenosides. On the other hand, the ginseng yield is severely affected by abiotic stressors, particularly salinity, which reduces yield and quality. Therefore, efforts are needed to improve the ginseng yield during salinity stress, but salinity stress-induced changes in ginseng are poorly understood, particularly at the proteome-wide level. In this study, we report the comparative proteome profiles of ginseng leaves at four different time points (mock, 24, 72, and 96 h) using a label-free quantitative proteome approach. Of the 2484 proteins identified, 468 were salt-responsive. In particular, glycosyl hydrolase 17 (PgGH17), catalase-peroxidase 2, voltage-gated potassium channel subunit beta-2, fructose-1,6-bisphosphatase class 1, and chlorophyll a-b binding protein accumulated in ginseng leaves in response to salt stress. The heterologous expression of PgGH17 in Arabidopsis thaliana improved the salt tolerance of transgenic lines without compromising plant growth. Overall, this study uncovers the salt-induced changes in ginseng leaves at the proteome level and highlights the critical role of PgGH17 in salt stress tolerance in ginseng.

Comparative transcriptome and metabolome analyses of four Panax species explore the dynamics of metabolite biosynthesis.

Background: The genus Panax in the Araliaceae family has been used as traditional medicinal plants worldwide and is known to biosynthesize ginsenosides and phytosterols. However, genetic variation between Panax species has influenced their biosynthetic pathways is not fully understood. Methods: Simultaneous analysis of transcriptomes and metabolomes obtained from adventitious roots of two tetraploid species (Panax ginseng and P. quinquefolius) and two diploid species (P. notoginseng and P. vietnamensis) revealed the diversity of their metabolites and related gene expression profiles. Results: The transcriptome analysis showed that 2,3-OXIDOSQUALENE CYCLASEs (OSCs) involved in phytosterol biosynthesis are upregulated in the diploid species, while the expression of OSCs contributing to ginsenoside biosynthesis is higher in the tetraploid species. In agreement with these results, the contents of dammarenediol-type ginsenosides were higher in the tetraploid species relative to the diploid species. Conclusion: These results suggest that a whole-genome duplication event has influenced the triterpene biosynthesis pathway in tetraploid Panax species during their evolution or ecological adaptation. This study provides a basis for further efforts to explore the genetic variation of the Panax genus.

Genes and Regulatory Mechanisms for Ginsenoside Biosynthesis.

Panax ginseng is a medicinal plant belonging to the Araliaceae family. Ginseng is known as the king of oriental medicine, which has been practiced since ancient times in East Asian countries and globally in the modern era. Ginseng is used as an adaptogen, and research shows that it has several pharmacological benefits for various ailments such as cancer, inflammation, diabetes, and neurological symptoms. The pharmacological benefits of ginseng are attributed to the triterpenoid saponin ginsenosides found throughout the Panax ginseng species, which are abundant in its root and are found exclusively in P. ginseng and Panax quinquefolius. Recently, with the completion of the entire ginseng genome sequencing and the construction of the ginseng genome database, it has become possible to access information about many genes newly predicted to be involved in ginsenoside biosynthesis. This review briefly summarizes the current progress in ginseng genome analysis and genes involved in ginsenoside biosynthesis, proposing directions for functional studies of the predicted genes related to ginsenoside production and its regulation.

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.

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.

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.

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.

Kinetics of the LOV domain of ZEITLUPE determine its circadian function in Arabidopsis.

A LOV (Light, Oxygen, or Voltage) domain containing blue-light photoreceptor ZEITLUPE (ZTL) directs circadian timing by degrading clock proteins in plants. Functions hinge upon allosteric differences coupled to the ZTL photocycle; however, structural and kinetic information was unavailable. Herein, we tune the ZTL photocycle over two orders of magnitude. These variants reveal that ZTL complexes with targets independent of light, but dictates enhanced protein degradation in the dark. In vivo experiments definitively show photocycle kinetics dictate the rate of clock component degradation, thereby impacting circadian period. Structural studies demonstrate that photocycle dependent activation of ZTL depends on an unusual dark-state conformation of ZTL. Crystal structures of ZTL LOV domain confirm delineation of structural and kinetic mechanisms and identify an evolutionarily selected allosteric hinge differentiating modes of PAS/LOV signal transduction. The combined biochemical, genetic and structural studies provide new mechanisms indicating how PAS/LOV proteins integrate environmental variables in complex networks.

The Effect of Fluctuations in Photoperiod and Ambient Temperature on the Timing of Flowering: Time to Move on Natural Environmental Conditions.

Plants have become physiologically adapted to a seasonally shifting environment by evolving many sensory mechanisms. Seasonal flowering is a good example of adaptation to local environmental demands and is crucial for maximizing reproductive fitness. Photoperiod and temperature are major environmental stimuli that control flowering through expression of a floral inducer, FLOWERING LOCUS T (FT) protein. Recent discoveries made using the model plant Arabidopsis thaliana have shown that the functions of photoreceptors are essential for the timing of FT gene induction, via modulation of the transcriptional activator CONSTANS (CO) at transcriptional and posttranslational levels in response to seasonal variations. The activation of FT transcription by the fine-tuned CO protein enables plants to switch from vegetative growth to flowering under inductive environmental conditions. The present review briefly summarizes our current understanding of the molecular mechanisms by which the information of environmental stimuli is sensed and transduced to trigger FT induction in leaves.

Cool night-time temperatures induce the expression of CONSTANS and FLOWERING LOCUS T to regulate flowering in Arabidopsis.

Day length and ambient temperature are major stimuli controlling flowering time. To understand flowering mechanisms in more natural conditions, we explored the effect of daily light and temperature changes on Arabidopsis thaliana. Seedlings were exposed to different day/night temperature and day-length treatments to assess expression changes in flowering genes. Cooler temperature treatments increased CONSTANS (CO) transcript levels at night. Night-time CO induction was diminished in flowering bhlh (fbh)-quadruple mutants. FLOWERING LOCUS T (FT) transcript levels were reduced at dusk, but increased at the end of cooler nights. The dusk suppression, which was alleviated in short vegetative phase (svp) mutants, occurred particularly in younger seedlings, whereas the increase during the night continued over 2 wk. Cooler temperature treatments altered the levels of FLOWERING LOCUS M-ß (FLM-ß) and FLM-δ splice variants. FT levels correlated strongly with flowering time across treatments. Day/night temperature changes modulate photoperiodic flowering by changing FT accumulation patterns. Cooler night-time temperatures enhance FLOWERING BHLH (FBH)-dependent induction of CO and consequently increase CO protein. When plants are young, cooler temperatures suppress FT at dusk through SHORT VEGETATIVE PHASE (SVP) function, perhaps to suppress precocious flowering. Our results suggest day length and diurnal temperature changes combine to modulate FT and flowering time.

Linked circadian outputs control elongation growth and flowering in response to photoperiod and temperature.

Clock-regulated pathways coordinate the response of many developmental processes to changes in photoperiod and temperature. We model two of the best-understood clock output pathways in Arabidopsis, which control key regulators of flowering and elongation growth. In flowering, the model predicted regulatory links from the clock to cycling DOF factor 1 (CDF1) and flavin-binding, KELCH repeat, F-box 1 (FKF1) transcription. Physical interaction data support these links, which create threefold feed-forward motifs from two clock components to the floral regulator FT. In hypocotyl growth, the model described clock-regulated transcription of phytochrome-interacting factor 4 and 5 (PIF4, PIF5), interacting with post-translational regulation of PIF proteins by phytochrome B (phyB) and other light-activated pathways. The model predicted bimodal and end-of-day PIF activity profiles that are observed across hundreds of PIF-regulated target genes. In the response to temperature, warmth-enhanced PIF4 activity explained the observed hypocotyl growth dynamics but additional, temperature-dependent regulators were implicated in the flowering response. Integrating these two pathways with the clock model highlights the molecular mechanisms that coordinate plant development across changing conditions.

Photoperiodic flowering: time measurement mechanisms in leaves.

Many plants use information about changing day length (photoperiod) to align their flowering time with seasonal changes to increase reproductive success. A mechanism for photoperiodic time measurement is present in leaves, and the day-length-specific induction of the FLOWERING LOCUS T (FT) gene, which encodes florigen, is a major final output of the pathway. Here, we summarize the current understanding of the molecular mechanisms by which photoperiodic information is perceived in order to trigger FT expression in Arabidopsis as well as in the primary cereals wheat, barley, and rice. In these plants, the differences in photoperiod are measured by interactions between circadian-clock-regulated components, such as CONSTANS (CO), and light signaling. The interactions happen under certain day-length conditions, as previously predicted by the external coincidence model. In these plants, the coincidence mechanisms are governed by multilayered regulation with numerous conserved as well as unique regulatory components, highlighting the breadth of photoperiodic regulation across plant species.

Distinct roles of FKF1, Gigantea, and Zeitlupe proteins in the regulation of Constans stability in Arabidopsis photoperiodic flowering.

Many plants measure changes in day length to synchronize their flowering time with appropriate seasons for maximum reproductive success. In Arabidopsis, the day-length-dependent regulation of Constans (CO) protein stability is crucial to induce flowering locus T (FT) expression for flowering in long days. The flavin-binding, KELCH repeat, F-box1 (FKF1) protein binds to CO protein specifically in the long-day afternoon and stabilizes it, although the mechanism remains unknown. Here we demonstrated that the FKF1-interacting proteins Gigantea (GI) and Zeitlupe (ZTL) are involved in CO stability regulation. First, our immunoprecipitation-mass spectrometry analysis of FKF1 revealed that FKF1 forms an S-phase kinase-associated protein 1 (Skp1)/Cullin(CUL)/F-box complex through interactions with Arabidopsis Skp1-like 1 (ASK1), ASK2, and CUL1 proteins and mainly interacts with GI protein in vivo. GI interacts with CO directly and indirectly through FKF1. Unexpectedly, the gi mutation increases the CO protein levels in the morning in long days. This gi-dependent destabilization of CO protein was cancelled by the fkf1 mutation. These results suggest that there are other factors likely influenced by both gi and fkf1 mutations that also control CO stability. We found that ZTL, which interacts with GI and FKF1, may be one such factor. ZTL also interacts with CO in vivo. The CO protein profile in the ztl mutant resembles that in the gi mutant, indicating that ZTL activity also may be changed in the gi mutant. Our findings suggest the presence of balanced regulation among FKF1, GI, and ZTL on CO stability regulation for the precise control of flowering time.

Photoperiodic flowering regulation in Arabidopsis thaliana.

Photoperiod, or the duration of light in a given day, is a critical cue that flowering plants utilize to effectively assess seasonal information and coordinate their reproductive development in synchrony with the external environment. The use of the model plant, Arabidopsis thaliana, has greatly improved our understanding of the molecular mechanisms that determine how plants process and utilize photoperiodic information to coordinate a flowering response. This mechanism is typified by the transcriptional activation of FLOWERING LOCUS T (FT) gene by the transcription factor CONSTANS (CO) under inductive long-day conditions in Arabidopsis. FT protein then moves from the leaves to the shoot apex, where floral meristem development can be initiated. As a point of integration from a variety of environmental factors in the context of a larger system of regulatory pathways that affect flowering, the importance of photoreceptors and the circadian clock in CO regulation throughout the day has been a key feature of the photoperiodic flowering pathway. In addition to these established mechanisms, the recent discovery of a photosynthate derivative trehalose-6-phosphate as an activator of FT in leaves has interesting implications for the involvement of photosynthesis in the photoperiodic flowering response that were suggested from previous physiological experiments in flowering induction.