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.

Phosphorylation of auxin signaling repressor IAA8 by heat-responsive MPKs causes defective flower development.

Heat stress is a substantial and imminent threat to plant growth and development. Understanding its adverse effects on plant development at the molecular level is crucial for sustainable agriculture. However, the molecular mechanism underlying how heat stress causes developmental defects in flowers remains poorly understood. Here, we identified Indole-3-Acetic Acid 8 (IAA8), a repressor of auxin signaling, as a substrate of mitogen-activated protein kinases (MPKs) in Arabidopsis thaliana, and found that MPK-mediated phosphorylation of IAA8 inhibits flower development. MPKs phosphorylated three residues of IAA8: S74, T77, and S135. Interestingly, transgenic plants overexpressing a phospho-mimicking mutant of IAA8 (IAA8DDD OX) exhibited defective flower development due to high IAA8 levels. Furthermore, MPK-mediated phosphorylation inhibited IAA8 polyubiquitination, thereby significantly increasing its stability. Additionally, the expression of key transcription factors involved in flower development, such as bZIP and MYB genes, was significantly perturbed in the IAA8DDD OX plants. Collectively, our study demonstrates that heat stress inhibits flower development by perturbing the expression of flower development genes through the MPK-mediated phosphorylation of IAA8, suggesting that Aux/IAA phosphorylation enables plants to fine-tune their development in response to environmental stress.

ASYMMETRIC LEAVES1 promotes leaf hyponasty in Arabidopsis by light-mediated auxin signaling.

In plants, balancing growth and environmental responses is crucial for maximizing fitness. Close proximity among plants and canopy shade, which negatively impacts reproduction, elicits morphological adjustments such as hypocotyl growth and leaf hyponasty, mainly through changes in light quality and auxin levels. However, how auxin, synthesized from a shaded leaf blade, distally induces elongation of hypocotyl and petiole cells remains to be elucidated. We demonstrated that ASYMMETRIC LEAVES1 (AS1) promotes leaf hyponasty through the regulation of auxin biosynthesis, polar auxin transport, and auxin signaling genes in Arabidopsis (Arabidopsis thaliana). AS1 overexpression leads to elongation of the abaxial petiole cells with auxin accumulation in the petiole, resulting in hyponastic growth, which is abolished by the application of an auxin transport inhibitor to the leaf blade. In addition, the as1 mutant exhibits reduced hypocotyl growth under shade conditions. We observed that AS1 protein accumulates in the nucleus in response to shade or far-red light. Chromatin immunoprecipitation analysis identified the association of AS1 with the promoters of YUCCA8 (YUC8) and INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19). In addition, AS1 forms complexes with PHYTOCHROME INTERACTING FACTORs in the nucleus and synergistically induces YUC8 and IAA19 expression. Our findings suggest that AS1 plays a crucial role in facilitating phenotypic plasticity to the surroundings by connecting light and phytohormone action.

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.

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.

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.

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.

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.

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.

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.

FLOWERING BHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS in Arabidopsis.

Many plants monitor day-length changes throughout the year and use the information to precisely regulate the timing of seasonal flowering for maximum reproductive success. In Arabidopsis thaliana, transcriptional regulation of the CONSTANS (CO) gene and posttranslational regulation of CO protein are crucial mechanisms for proper day-length measurement in photoperiodic flowering. Currently, the CYCLING DOF FACTOR proteins are the only transcription factors known to directly regulate CO gene expression, and the mechanisms that directly activate CO transcription have remained unknown. Here we report the identification of four CO transcriptional activators, named FLOWERING BHLH 1 (FBH1), FBH2, FBH3, and FBH4. All FBH proteins are related basic helix-loop-helix-type transcription factors that preferentially bind to the E-box cis-elements in the CO promoter. Overexpression of all FBH genes drastically elevated CO levels and caused early flowering regardless of photoperiod, whereas CO levels were reduced in the fbh quadruple mutants. In addition, FBH1 is expressed in the vascular tissue and bound near the transcription start site of the CO promoter in vivo. Furthermore, FBH homologs in poplar and rice induced CO expression in Arabidopsis. These results indicate that FBH proteins positively regulate CO transcription for photoperiodic flowering and that this mechanism may be conserved in diverse plant species. Our results suggest that the diurnal CO expression pattern is generated by a concert of redundant functions of positive and negative transcriptional regulators.

UV-B increases active phytochrome B to suppress thermomorphogenesis and enhance UV-B stress tolerance at high temperatures.

Plants respond to small increases in ambient temperature by changing their architecture, a response collectively termed thermomorphogenesis. Thermomorphogenesis is considered to attenuate the damage caused by potentially harmful high-temperature conditions, and multiple environmental factors can modulate this process. Among these factors, ultraviolet-B (UV-B) light has been shown to strongly suppress this response. However, the molecular mechanisms through which it regulates thermomorphogenesis and the physiological roles of the UV-B-mediated suppression of thermomorphogenesis remain poorly understood. Here, we show that UV-B inhibits thermomorphogenesis through the UVR8-COP1-phyB/HFR1 signaling module. We found that cop1 mutants maintain high levels of active phyB at high temperatures. Extensive genetic analyses revealed that the increased phyB, HFR1, and CRY1 in cop1 mutants redundantly reduce both the level and activity of a key positive regulator in thermomorphogenesis, PIF4, thereby repressing this growth response. Additionally, we found that UV-B light increases phyB stability and its photobody number through the inactivation of COP1. The UV-B-stabilized active phyB, together with HFR1, inhibits thermomorphogenesis by interfering with PIF4. We further show that the increased active phyB enhances UV-B tolerance by activating flavonoid biosynthesis and inhibiting thermomorphogenic growth. Taken together, our study demonstrates that UV-B increases the levels of active phyB and HFR1 by inhibiting COP1 to suppress PIF4-mediated growth responses, which is essential for plant tolerance to UV-B stress at high temperatures.

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.

CONSTANS and ASYMMETRIC LEAVES 1 complex is involved in the induction of FLOWERING LOCUS T in photoperiodic flowering in Arabidopsis.

Plants monitor changes in day length to coordinate flowering with favorable seasons to increase their fitness. The day-length specific induction of FLOWERING LOCUS T (FT) regulated by CONSTANS (CO) is the crucial aspect of photoperiodic flowering in Arabidopsis thaliana. Recent studies have elucidated some mechanisms of CO-dependent FT induction. Here, we demonstrate another mechanism of CO-dependent FT regulation. Our results indicate that CO protein partially regulates FT transcription by forming a complex with ASYMMETRIC LEAVES 1 (AS1) protein, which regulates leaf development partly by controlling gibberellin (GA) levels. We identified AS1 as a CO-interacting protein in yeast and verified their interaction in vitro and in planta. We also showed that the temporal and spatial expression pattern of AS1 overlapped with that of CO. In addition, as1 mutants showed GA-independent delayed flowering under different light/dark conditions. FT expression levels in the as1 mutants and the SUC2:CO-HA/as1 line under long-day and 12-h light/12-h dark conditions were reduced compared with wild-type plants and the SUC2:HA-CO line, respectively. Moreover, AS1 bound directly to the specific regions of the FT promoter in vivo. These results indicate that CO forms a functional complex with AS1 to regulate FT expression and that AS1 plays different roles in two regulatory pathways, both of which concomitantly regulate the precise timing of flowering.

F-box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control Arabidopsis clock progression.

Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.

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.

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.

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.