S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

The Na+/H+ antiporter SALT OVERLY SENSITIVE 1 regulates salt compensation of circadian rhythms by stabilizing GIGANTEA in Arabidopsis.

The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na+/H+ antiporter SOS1 specifically functions as a salt-specific circadian clock regulator via GIGANTEA (GI) in Arabidopsis thaliana. SOS1 directly interacts with GI in a salt-dependent manner and stabilizes this protein to sustain a proper clock period under salinity conditions. SOS1 function in circadian clock regulation requires the salt-mediated secondary messengers cytosolic free calcium and reactive oxygen species, pointing to a distinct regulatory role for SOS1 in addition to its function as a transporter to maintain Na+ homeostasis. Our results demonstrate that SOS1 maintains homeostasis of the salt response under high or daily fluctuating salt levels. These findings highlight the genetic capacity of the circadian clock to maintain timekeeping activity over a broad range of salinity levels.

The E3 ubiquitin ligase COP1 regulates salt tolerance via GIGANTEA degradation in roots.

Excess soil salinity significantly impairs plant growth and development. Our previous reports demonstrated that the core circadian clock oscillator GIGANTEA (GI) negatively regulates salt stress tolerance by sequestering the SALT OVERLY SENSITIVE (SOS) 2 kinase, an essential component of the SOS pathway. Salt stress induces calcium-dependent cytoplasmic GI degradation, resulting in activation of the SOS pathway; however, the precise molecular mechanism governing GI degradation during salt stress remains enigmatic. Here, we demonstrate that salt-induced calcium signals promote the cytoplasmic partitioning of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), leading to the 26S proteasome-dependent degradation of GI exclusively in the roots. Salt stress-induced calcium signals accelerate the cytoplasmic localization of COP1 in the root cells, which targets GI for 26S proteasomal degradation. Align with this, the interaction between COP1 and GI is only observed in the roots, not the shoots, under salt-stress conditions. Notably, the gi-201 cop1-4 double mutant shows an enhanced tolerance to salt stress similar to gi-201, indicating that GI is epistatic to COP1 under salt-stress conditions. Taken together, our study provides critical insights into the molecular mechanisms governing the COP1-mediated proteasomal degradation of GI for salt stress tolerance, raising new possibilities for developing salt-tolerant crops.

Orchardgrass ACTIVATOR OF HSP90 ATPASE possesses autonomous chaperone properties and activates Hsp90 transcription to enhance thermotolerance.

High temperature stress is an environmental factor that negatively affects the growth and development of crops. Hsp90 (90 kDa heat shock protein) is a major molecular chaperone in eukaryotic cells, contributing to the maintenance of cell homeostasis through interaction with co-chaperones. Aha1 (activator of Hsp90 ATPase) is well known as a co-chaperone that activates ATPase activity of Hsp90 in mammals. However, biochemical and physiological evidence relating to Aha has not yet been identified in plants. In this study, we investigated the heat-tolerance function of orchardgrass (Dactylis glomerata L.) Aha (DgAha). Recombinant DgAha interacted with cytosolic DgHsp90s and efficiently protected substrates from thermal denaturation. Furthermore, heterologous expression of DgAha in yeast (Saccharomyces cerevisiae) cells and Arabidopsis (Arabidopsis thaliana) plants conferred thermotolerance in vivo. Enhanced expression of DgAha in Arabidopsis stimulates the transcription of Hsp90 under heat stress. Our data demonstrate that plant Aha plays a positive role in heat stress tolerance via chaperone properties and/or activation of Hsp90 to protect substrate proteins in plants from thermal injury.

Nucleoredoxin 1 positively regulates heat stress tolerance by enhancing the transcription of antioxidants and heat-shock proteins in tomato.

Thioredoxins (TRXs) are small oxidoreductase proteins located in various subcellular compartments. Nucleoredoxin (NRX) is a nuclear-localized TRX and a key component for the integration of the antioxidant system with the immune response. Although NRX is well characterized in biotic stress responses, its functional role in abiotic stress responses is still elusive. To understand whether NRX contributes to heat stress response in tomato (Solanum lycopersicum), we generated CRISPR/Cas9-mediated mutations in SlNRX1 (slnrx1). Interestingly, the slnrx1 mutant was extremely sensitive to heat stress with higher electrolyte leakage, malondialdehyde contents, and H2O2 concentration compared to wild-type tomato plants, suggesting that SlNRX1 negatively regulates heat stress-induced oxidative damage. We also found that transcripts encoding antioxidant enzymes and Heat-Shock Proteins (HSPs) in slnrx1 were down-regulated either in the absence or presence of heat stress. These data suggest that NRX1 is a positive regulator for heat stress tolerance by elevating antioxidant capacity and inducing HSPs to protect cells against heat stress-induced oxidative damage and protein denaturation, respectively.

HOS15 Interacts with the Histone Deacetylase HDA9 and the Evening Complex to Epigenetically Regulate the Floral Activator GIGANTEA.

In plants, seasonal inputs such as photoperiod and temperature modulate the plant's internal genetic program to regulate the timing of the developmental transition from vegetative to reproductive growth. This regulation of the floral transition involves chromatin remodeling, including covalent modification of histones. Here, we report that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), a WD40 repeat protein, associates with a histone deacetylase complex to repress transcription of the GIGANTEA (GI)-mediated photoperiodic flowering pathway in Arabidopsis (Arabidopsis thaliana). Loss of function of HOS15 confers early flowering under long-day conditions because elevated GI expression. LUX ARRHYTHMO (LUX), a DNA binding transcription factor and component of the Evening Complex (EC), is important for the binding of HOS15 to the GI promoter. In wild type, HOS15 associates with the EC components LUX, EARLY FLOWERING 3 (ELF3), and ELF4 and the histone deacetylase HDA9 at the GI promoter, resulting in histone deacetylation and reduced GI expression. In the hos15-2 mutant, the levels of histone acetylation are elevated at the GI promoter, resulting in increased GI expression. Our data suggest that the HOS15-EC-HDA9 histone-modifying complex regulates photoperiodic flowering via the transcriptional repression of GI.

Production of a Bacteria-like Particle Vaccine Targeting Rock Bream (Oplegnathus fasciatus) Iridovirus Using Nicotiana benthamiana.

Viral diseases are extremely widespread infections that change constantly through mutations. To produce vaccines against viral diseases, transient expression systems are employed, and Nicotiana benthamiana (tobacco) plants are a rapidly expanding platform. In this study, we developed a recombinant protein vaccine targeting the major capsid protein (MCP) of iridovirus fused with the lysine motif (LysM) and coiled-coil domain of coronin 1 (ccCor1) for surface display using Lactococcus lactis. The protein was abundantly produced in N. benthamiana in its N-glycosylated form. Total soluble proteins isolated from infiltrated N. benthamiana leaves were treated sequentially with increasing ammonium sulfate solution, and recombinant MCP mainly precipitated at 40-60%. Additionally, affinity chromatography using Ni-NTA resin was applied for further purification. Native structure analysis using size exclusion chromatography showed that recombinant MCP existed in a large oligomeric form. A minimum OD600 value of 0.4 trichloroacetic acid (TCA)-treated L. lactis was required for efficient recombinant MCP display. Immunogenicity of recombinant MCP was assessed in a mouse model through enzyme-linked immunosorbent assay (ELISA) with serum-injected recombinant MCP-displaying L. lactis. In summary, we developed a plant-based recombinant vaccine production system combined with surface display on L. lactis.

The GIGANTEA-ENHANCED EM LEVEL Complex Enhances Drought Tolerance via Regulation of Abscisic Acid Synthesis.

Drought is one of the most critical environmental stresses limiting plant growth and crop productivity. The synthesis and signaling of abscisic acid (ABA), a key phytohormone in the drought stress response, is under photoperiodic control. GIGANTEA (GI), a key regulator of photoperiod-dependent flowering and the circadian rhythm, is also involved in the signaling pathways for various abiotic stresses. In this study, we isolated ENHANCED EM LEVEL (EEL)/basic Leu zipper 12, a transcription factor involved in ABA signal responses, as a GI interactor in Arabidopsis (Arabidopsis thaliana). The diurnal expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), a rate-limiting ABA biosynthetic enzyme, was reduced in the eel, gi-1, and eel gi-1 mutants under normal growth conditions. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed that EEL and GI bind directly to the ABA-responsive element motif in the NCED3 promoter. Furthermore, the eel, gi-1, and eel gi-1 mutants were hypersensitive to drought stress due to uncontrolled water loss. The transcript of NCED3, endogenous ABA levels, and stomatal closure were all reduced in the eel, gi-1, and eel gi-1 mutants under drought stress. Our results suggest that the EEL-GI complex positively regulates diurnal ABA synthesis by affecting the expression of NCED3, and contributes to the drought tolerance of Arabidopsis.

Long-term abscisic acid promotes golden2-like1 degradation through constitutive photomorphogenic 1 in a light intensity-dependent manner to suppress chloroplast development.

Abiotic stress, a serious threat to plants, occurs for extended periods in nature. Abscisic acid (ABA) plays a critical role in abiotic stress responses in plants. Therefore, stress responses mediated by ABA have been studied extensively, especially in short-term responses. However, long-term stress responses mediated by ABA remain largely unknown. To elucidate the mechanism by which plants respond to prolonged abiotic stress, we used long-term ABA treatment that activates the signalling against abiotic stress such as dehydration and investigated mechanisms underlying the responses. Long-term ABA treatment activates constitutive photomorphogenic 1 (COP1). Active COP1 mediates the ubiquitination of golden2-like1 (GLK1) for degradation, contributing to lowering expression of photosynthesis-associated genes such as glutamyl-tRNA reductase (HEMA1) and protochlorophyllide oxidoreductase A (PORA), resulting in the suppression of chloroplast development. Moreover, COP1 activation and GLK1 degradation upon long-term ABA treatment depend on light intensity. Additionally, plants with COP1 mutation or exposed to higher light intensity were more sensitive to salt stress. Collectively, our results demonstrate that long-term treatment of ABA leads to activation of COP1 in a light intensity-dependent manner for GLK1 degradation to suppress chloroplast development, which we propose to constitute a mechanism of balancing normal growth and stress responses upon the long-term abiotic stress.

Epigenetic switch from repressive to permissive chromatin in response to cold stress.

Switching from repressed to active status in chromatin regulation is part of the critical responses that plants deploy to survive in an ever-changing environment. We previously reported that HOS15, a WD40-repeat protein, is involved in histone deacetylation and cold tolerance in Arabidopsis However, it remained unknown how HOS15 regulates cold responsive genes to affect cold tolerance. Here, we show that HOS15 interacts with histone deacetylase 2C (HD2C) and both proteins together associate with the promoters of cold-responsive COR genes, COR15A and COR47 Cold induced HD2C degradation is mediated by the CULLIN4 (CUL4)-based E3 ubiquitin ligase complex in which HOS15 acts as a substrate receptor. Interference with the association of HD2C and the COR gene promoters by HOS15 correlates with increased acetylation levels of histone H3. HOS15 also interacts with CBF transcription factors to modulate cold-induced binding to the COR gene promoters. Our results here demonstrate that cold induces HOS15-mediated chromatin modifications by degrading HD2C. This switches the chromatin structure status and facilitates recruitment of CBFs to the COR gene promoters. This is an apparent requirement to acquire cold tolerance.

A Significant Change in Free Amino Acids of Soybean (Glycine max L. Merr) through Ethylene Application.

In this study, the changes in free amino acids of soybean leaves after ethylene application were characterized based on quantitative and metabolomic analyses. All essential and nonessential amino acids in soybean leaves were enhanced by fivefold (250 to 1284 mg/100 g) and sixfold (544 to 3478 mg/100 g), respectively, via ethylene application. In particular, it was found that asparagine is the main component, comprising approximately 41% of the total amino acids with a twenty-five fold increase (78 to 1971 mg/100 g). Moreover, arginine and branched chain amino acids (Val, Leu, and Ile) increased by about 14 and 2-5 times, respectively. The increase in free amino acid in stem was also similar to the leaves. The metabolites in treated and untreated soybean leaves were systematically identified by gas chromatography-mass spectrometry (GC-MS), and partial variance discriminant analysis (PLS-DA) scores and heat map analysis were given to understand the changes of each metabolite. The application of ethylene may provide good nutrient potential for soybean leaves.

Transcriptome Changes Reveal the Molecular Mechanisms of Humic Acid-Induced Salt Stress Tolerance in Arabidopsis.

Humic acid (HA) is a principal component of humic substances, which make up the complex organic matter that broadly exists in soil environments. HA promotes plant development as well as stress tolerance, however the precise molecular mechanism for these is little known. Here we conducted transcriptome analysis to elucidate the molecular mechanisms by which HA enhances salt stress tolerance. Gene Ontology Enrichment Analysis pointed to the involvement of diverse abiotic stress-related genes encoding HEAT-SHOCK PROTEINs and redox proteins, which were up-regulated by HA regardless of salt stress. Genes related to biotic stress and secondary metabolic process were mainly down-regulated by HA. In addition, HA up-regulated genes encoding transcription factors (TFs) involved in plant development as well as abiotic stress tolerance, and down-regulated TF genes involved in secondary metabolic processes. Our transcriptome information provided here provides molecular evidences and improves our understanding of how HA confers tolerance to salinity stress in plants.

Light-dependent suppression of COP1 multimeric complex formation is determined by the blue-light receptor FKF1 in Arabidopsis.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a multifunctional E3 ligase protein with many target proteins, is involved in diverse developmental processes throughout the plant's lifecycle, including seed germination, the regulation of circadian rhythms, photomorphogenesis, and the control of flowering time. To function, COP1 must form multimeric complexes with SUPPRESSOR OF PHYA1 (SPA1), i.e., [(COP1)2(SPA1)2] tetramers. We recently reported that the blue-light receptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX1) represses COP1 activity by inhibiting its homodimerization, but it is not yet clear whether FKF1 affects the formation of COP1-containing multimeric complexes. To explore this issue, we performed size exclusion chromatography (SEC) of Arabidopsis thaliana proteins and found that the levels and composition of COP1-containing multimeric complexes varied throughout a 24-h period. The levels of 440-669 kDa complexes were dramatically reduced in the late afternoon compared to the morning and at night in wild-type plants. During the daytime, the levels of these complexes were reduced in FKF1-overexpressing plants but not in fkf1-t, a loss-of-function mutant of FKF1, suggesting that FKF1 is closely associated with the destabilization of COP1 multimeric protein complexes in a light-dependent manner. We also analyzed the SEC patterns of COP1 multimeric complexes in transgenic plants overexpressing mutant COP1 variants, including COP1L105A (which forms homodimers) and COP1L170A (which cannot form homodimers), and found that COP1 multimeric complexes were scarce in plants overexpressing COP1L170A. These results indicate that COP1 homodimers serve as basic building blocks that assemble into COP1 multimeric complexes with diverse target proteins. We propose that light-activated FKF1 inhibits COP1 homodimerization, mainly by destabilizing 440-669 kDa COP1 complexes, resulting in the repression of CONSTANS-degrading COP1 activity in the late afternoon in long days, but not in short days, thereby regulating photoperiodic flowering in Arabidopsis.

Arabidopsis GIGANTEA negatively regulates chloroplast biogenesis and resistance to herbicide butafenacil.

KEY MESSAGE: Arabidopsis GI negatively regulates chloroplast biogenesis and resistance to the herbicide butafenacil by enhanced activity and transcriptional levels of antioxidant enzymes Chloroplast biogenesis is blocked by retrograde signaling triggered by diverse internal and external cues, including sugar, reactive oxygen species (ROS), phytohormones, and abiotic stress. Efficient chloroplast biogenesis is essential for crop productivity due to its effect on photosynthetic efficiency, and is associated with agronomic traits such as insect/disease resistance, herbicide resistance, and abiotic stress tolerance. Here, we show that the circadian clock-controlled gene GIGANTEA (GI) regulates chloroplast biogenesis in Arabidopsis thaliana. The gi-2 mutant showed reduced sensitivity to the chloroplast biogenesis inhibitor lincomycin, maintaining high levels of photosynthetic proteins. By contrast, wild-type and GI-overexpressing plants were sensitive to lincomycin, with variegated leaves and reduced photosynthetic protein levels. GI is degraded by lincomycin, suggesting that GI is genetically linked to chloroplast biogenesis. The GI mutant alleles gi-1 and gi-2 were resistant to the herbicide butafenacil, which inhibits protoporphyrinogen IX oxidase activity and triggers ROS-mediated cell death via the accumulation of chlorophyll precursors. Butafenacil-mediated accumulation of superoxide anions and H2O2 was not detected in gi-1 or gi-2, as revealed by histochemical staining. The activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase were 1.2-1.4-fold higher in both gi mutants compared to the wild type. Finally, the expression levels of antioxidant enzyme genes were 1.5-2-fold higher in the mutants than in the wild type. These results suggest that GI negatively regulates chloroplast biogenesis and resistance to the herbicide butafenacil, providing evidence for a genetic link between GI and chloroplast biogenesis, which could facilitate the development of herbicide-resistant crops.

A chaperone function of NO CATALASE ACTIVITY1 is required to maintain catalase activity and for multiple stress responses in Arabidopsis.

Catalases are key regulators of reactive oxygen species homeostasis in plant cells. However, the regulation of catalase activity is not well understood. In this study, we isolated an Arabidopsis thaliana mutant, no catalase activity1-3 (nca1-3) that is hypersensitive to many abiotic stress treatments. The mutated gene was identified by map-based cloning as NCA1, which encodes a protein containing an N-terminal RING-finger domain and a C-terminal tetratricopeptide repeat-like helical domain. NCA1 interacts with and increases catalase activity maximally in a 240-kD complex in planta. In vitro, NCA1 interacts with CATALASE2 (CAT2) in a 1:1 molar ratio, and the NCA1 C terminus is essential for this interaction. CAT2 activity increased 10-fold in the presence of NCA1, and zinc ion binding of the NCA1 N terminus is required for this increase. NCA1 has chaperone protein activity that may maintain the folding of catalase in a functional state. NCA1 is a cytosol-located protein. Expression of NCA1 in the mitochondrion of the nca1-3 mutant does not rescue the abiotic stress phenotypes of the mutant, while expression in the cytosol or peroxisome does. Our results suggest that NCA1 is essential for catalase activity.

N-terminal arm of orchardgrass Hsp17.2 (DgHsp17.2) is essential for both in vitro chaperone activity and in vivo thermotolerance in yeast.

Small heat shock proteins are well-known to function as chaperone in the protection of proteins and subcellular structures against stress-induced denaturation in many cell compartments. Irrespective of such general functional assignment, a proof of function in a living organism is missing. Here, we used heat-induced orchardgrass small Hsp17.2 (DgHsp17.2). Its function in in vitro chaperone properties has shown in protecting the model substrate, malate dehydrogenase (MDH) and citrate synthase (CS). Overexpression of DgHsp17.2 triggering strong chaperone activity enhanced in vivo thermotolerance of yeast cells. To identify the functional domain on DgHsp17.2 and correlationship between in vitro chaperone property and in vivo thermotolerance, we generated truncation mutants of DgHsp17.2 and showed essentiality of the N-terminal arm of DgHsp17.2 for the chaperone function. In addition, beyond for acquisition of thermotolerance irrespective of sequences are diverse among the small Hsps. However, any truncation mutants of DgHsp17.2 did not exhibit strong interaction with orchardgrass heat shock protein 70 (DgHsp70) different from mature DgHsp17.2, indicating that full-length DgHsp17.2 is necessary for cooperating with Hsp70 protein. Our study indicates that the N-terminal arm of DgHsp17.2 is an important region for chaperone activity and thermotolerance.

Identification and functional characterization of Siberian wild rye (Elymus sibiricus L.) small heat shock protein 16.9 gene (EsHsp16.9) conferring diverse stress tolerance in prokaryotic cells.

Small heat shock proteins (Hsps) protect against stress-inducible denaturation of substrates. Our objectives were to clone and examine the mRNA expression of the Hsp16.9 gene from Siberian wild rye grown under diverse stress treatments. We characterized EsHsp16.9 from Elymus sibiricus L. EsHsp16.9 has a 456-bp open reading frame that encodes a 151-amino acid protein with a conserved α-crystallin domain. Northern blot analysis showed that EsHsp16.9 transcripts were enhanced by heat, drought, arsenate, methyl viologen, and H2O2 treatment. In addition, recombinant EsHsp16.9 protein acts as a molecular chaperone to prevent the denaturation of malate dehydrogenase. Growth of cells overexpressing EsHsp16.9 was up to 200% more rapid in the presence of NaCl, arsenate, and polyethylene glycol than that of cells harboring an empty vector. These data suggest that EsHsp16.9 acts as a molecular chaperone that enhances stress tolerance in living organisms.

Analysis of Arabidopsis thioredoxin-h isotypes identifies discrete domains that confer specific structural and functional properties.

Multiple isoforms of Arabidopsis thaliana h-type thioredoxins (AtTrx-hs) have distinct structural and functional specificities. AtTrx-h3 acts as both a disulfide reductase and as a molecular chaperone. We prepared five representative AtTrx-hs and compared their protein structures and disulfide reductase and molecular chaperone activities. AtTrx-h2 with an N-terminal extension exhibited distinct functional properties with respect to other AtTrx-hs. AtTrx-h2 formed low-molecular-mass structures and exhibited only disulfide reductase activity, whereas the other AtTrx-h isoforms formed high-molecular-mass complexes and displayed both disulfide reductase and molecular chaperone activities. The domains that determine the unique structural and functional properties of each AtTrx-hs protein were determined by constructing a domain-swap between the N- and C-terminal regions of AtTrx-h2 and AtTrx-h3 (designated AtTrx-h-2N3C and AtTrx-h-3N2C respectively), an N-terminal deletion mutant of AtTrx-h2 [AtTrx-h2-N(∆19)] and site-directed mutagenesis of AtTrx-h3. AtTrx-h2-N(∆19) and AtTrx-h-3N2C exhibited similar properties to those of AtTrx-h2, but AtTrx-h-2N3C behaved more like AtTrx-h3, suggesting that the structural and functional specificities of AtTrx-hs are determined by their C-terminal regions. Hydrophobicity profiling and molecular modelling revealed that Ala100 and Ala106 in AtTrx-h3 play critical roles in its structural and functional regulation. When these two residues in AtTrx-h3 were replaced with lysine, AtTrx-h3 functioned like AtTrx-h2. The chaperone function of AtTrx-hs conferred enhanced heat-shock-resistance on a thermosensitive trx1/2-null yeast mutant.

HSP90 functions in the circadian clock through stabilization of the client F-box protein ZEITLUPE.

The autoregulatory loops of the circadian clock consist of feedback regulation of transcription/translation circuits but also require finely coordinated cytoplasmic and nuclear proteostasis. Although protein degradation is important to establish steady-state levels, maturation into their active conformation also factors into protein homeostasis. HSP90 facilitates the maturation of a wide range of client proteins, and studies in metazoan clocks implicate HSP90 as an integrator of input or output. Here we show that the Arabidopsis circadian clock-associated F-box protein ZEITLUPE (ZTL) is a unique client for cytoplasmic HSP90. The HSP90-specific inhibitor geldanamycin and RNAi-mediated depletion of cytoplasmic HSP90 reduces levels of ZTL and lengthens circadian period, consistent with ztl loss-of-function alleles. Transient transfection of artificial microRNA targeting cytoplasmic HSP90 genes similarly lengthens period. Proteolytic targets of SCF(ZTL), TOC1 and PRR5, are stabilized in geldanamycin-treated seedlings, whereas the levels of closely related clock proteins, PRR3 and PRR7, are unchanged. An in vitro holdase assay, typically used to demonstrate chaperone activity, shows that ZTL can be effectively bound, and aggregation prevented, by HSP90. GIGANTEA, a unique stabilizer of ZTL, may act in the same pathway as HSP90, possibly linking these two proteins to a similar mechanism. Our findings establish maturation of ZTL by HSP90 as essential for proper function of the Arabidopsis circadian clock. Unlike metazoan systems, HSP90 functions here within the core oscillator. Additionally, F-box proteins as clients may place HSP90 in a unique and more central role in proteostasis.

Effect of transcription factor MEOX on insulin gene expression in glucagon-like peptide 1-secreting cells.

Currently, the supply of beta cells for islet transplantation in the treatment of type 1 diabetes is limited. Enteroendocrine cells (EECs) are believed to have high potential as stem cells because they share significant developmental similarities with beta cells. In a previous study, we derived EEC cells that secrete individual gut hormones from STC-1 cells. This study aimed to examine intestinal hormone secretion and expression, investigate the expression of developmental-related transcription factors, and analyze the effect of MEOX on insulin gene expression in isolated EECs. The expression and secretion of enteroendocrine hormones were evaluated in L6 and K34 cells from STC-1 cells. Expression patterns of beta cell- and development-related genes in L6 and K34 cells were compared with beta cells. Comparisons of the MEOX-induced expression of Ins in beta cells and GLP-1-secreting cells were investigated. Both L6 and K34 cells predominantly expressed Glp1 and Gip, respectively. The secretion pattern of GLP-1 in L6 cells was similar to that of GLUTag cells. Previous microarray analysis confirmed MEOX as developmentally relevant transcription factors expressed in beta cells. Overexpression of MEOX showed a tendency to increase Ins expression in L6 and GLUTag cells, but not in MIN6 cells. However, when PDX1 and MEOX were co-expressed in GLUTag cells, insulin expression was suppressed, similar to that observed in MIN6 cells. These findings suggest a potential role for MEOX in regulating the expression of the Ins gene in both beta cells and GLP-1-secreting cells. Further studies are warranted to elucidate the underlying mechanism.