Abstract Amphibious plants can thrive in both terrestrial and submerged environments, which are fundamentally distinct. Although morphological plasticity of leaf known as heterophylly has been well investigated, the morphological plasticity of root in amphibious plants remains poorly understood. In this study, we discovered that an amphibious plant Callitriche palustris (Plantaginaceae), which has significant heterophylly, has a remarkable morphological plasticity also in root in response to submergence. This species develops thin roots with abundant root hairs, fewer cortical and epidermal cells, and smaller aerenchyma in the terrestrial condition. On the other hand, it develops thicker roots with few root hairs, more cortical and epidermal cells, and larger aerenchyma in the submerged condition. We call this morphological plasticity of root as “heterorhizy”. Phytohormone perturbation experiments revealed that abscisic acid (ABA) and gibberellin regulate root hair development and root cell division respectively. We also found the possibility that heterorhizy was acquired in the genus Callitriche . Additionally, a similar form of root hair plasticity was also observed in the phylogenetically distinct amphibious species Ludwigia arcuata (Onagraceae). Furthermore, the absence of root hair development underwater and the similar structure of aerenchyma to C. palustris were broadly seen across diverse aquatic plants. This study provides new insights into the root morphological responses to submerged environments in aquatic plants.

Abstract Drought stress significantly threatens global food security. According to the FAO2024, agriculture absorbs up to 80% of drought impacts. Stomata are vital pores for gas exchange and transpiration in plants. Stomatal closure, which is crucial for drought tolerance, is regulated by ion transport systems. Here, we identify two inhibitors of plasma membrane voltage-dependent potassium (K + ) channels, NS5806 and UA49, that induce stomatal closure, reduce guard cell K + levels, and increase drought resistance in Arabidopsis plants. This chemical-induced stomatal closure pathway is distinct from the abscisic acid (ABA). Notably, K + channel inhibition led to increased cytosolic Ca 2+ , which was absent in K + inward channel mutants, highlighting the link between K + channel activity and cytosolic Ca 2+ elevation. These findings suggest that the chemical regulation of K + channels represents a strategy to induce stomatal closure, potentially improving plant drought tolerance through targeted interventions.

Karrikin Insensitive 2 (KAI2), identified as the receptor protein for karrikins (KARs), which are smoke-derived seed germination stimulants, belongs to the same α/β-hydrolase family as D14, the receptor for strigolactones (SLs). KAI2 is believed to recognize an endogenous butenolide (KAI2 ligand; KL), but the identity of this compound remains unknown. Recent studies have suggested that ligand hydrolysis by KAI2 is a prerequisite for receptor activation to induce interaction with the target proteins, similar to the situation with D14. However, direct experimental evidence has been lacking. Here, we designed KAI2 ligands (carba-dMGers) whose butenolide rings were modified so that they cannot be hydrolyzed or dissociated from the original ligand molecule by KAI2, by structurally modifying dMGer, a potent and selective KAI2 agonist. Using these dMGer analogs, we found that the strongly bioactive ligand, (+)-dMGer, was hydrolyzed by KAI2 at a lower enzymatic rate compared with the weakly bioactive ligand, (+)-1′-carba-dMGer, and the hydrolyzed butenolide ring of (+)-dMGer was transiently trapped in the catalytic pocket of KAI2. Additionally, structural analysis revealed that (+)-6′-carba-dMGer bound to the catalytic pocket of KAI2 in the unhydrolyzed state. However, this binding did not induce the interaction between KAI2 and SMAX1, indicating that ligand binding to the receptor alone was not sufficient for KAI2 signaling. This study showed experimental data from a ligand structure–activity study that ligand hydrolysis and subsequent covalent adduct formation with the catalytic triad plays a key role in KAI2 activation, providing insight into the chemical structure of the Arabidopsis KL.

Diclofenac etalhyaluronate combines the sustained-release properties of diclofenac with the therapeutic benefits of hyaluronic acid, providing extended analgesic effects for osteoarthritis management. This study investigated the effects of diclofenac etalhyaluronate and subsequent osteoarthritic changes in rat models of advanced and end-stage osteoarthritis. Monosodium iodoacetate (0.5 or 2.0 mg) was injected directly into the right hip joint of rats (n = 8 rats/group) using a posterior approach to induce osteoarthritis. Four weeks after monosodium iodoacetate administration, diclofenac etalhyaluronate (0.25 mg/25 µL) or 25 µL saline was administered in the same way. Pain behavior, number of microglia in the dorsal horn of the spinal cord, radiological features on microcomputed tomography, and histology of the hip joint were evaluated. Administration of diclofenac etalhyaluronate increased the pain threshold and reduced the number of microglia in the dorsal horn of the spinal cord in both models. However, radiological and histological examinations did not detect significant arthritic changes in either group that received diclofenac etalhyaluronate. Intra-articular administration, therefore, contributes to pain relief and improvement of central sensitization in advanced and end-stage osteoarthritis of the hip without subsequent progression of osteoarthritis. These findings highlight the potential of intra-articular administration of diclofenac etalhyaluronate as a conservative treatment option for advanced and end-stage hip osteoarthritis, particularly for patients who may be unsuitable for surgery or have limited response to oral nonsteroidal anti-inflammatory drugs.

Background/Objectives: Previously, we reported that high molecular weight hyaluronic acid (HMW-HA) exerts chondroprotective effects by enhancing dual specificity protein phosphatase 10/mitogen-activated protein kinase (MAPK) phosphatase 5 (DUSP10/MKP5) expression and suppressing inflammatory cytokine-induced matrix metalloproteinase-13 (MMP13) expression in a human immortalized chondrocyte line (C28/I2 cells) via inhibition of MAPKs. The aim of this study was to elucidate the molecular mechanisms underlying the enhancement of DUSP10/MKP5 expression by HMW-HA in C28/I2 cells. Methods: C28/I2 cells were treated with HMW-HA, and the activation of intracellular signaling molecules was determined using Western blot analysis. The expression levels of mRNAs and microRNAs (miRNAs) were evaluated through real-time quantitative reverse transcription PCR analysis. Results: HMW-HA treatment induced Akt phosphorylation via interaction with CD44, and pretreatment with specific inhibitors of phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling attenuated the HMW-HA-induced expression of DUSP10/MKP5. HMW-HA suppressed the expression of miR-92a, miR-181a, and miR-181d. Loss-of-function and gain-of-function analyses of these miRNAs indicate that miR-92a, miR-181a, and miR-181d negatively regulate DUSP10/MKP5 expression. Moreover, HMW-HA-induced Akt phosphorylation was partially suppressed by miR-181a and miR-181d mimics. Finally, we found that HMW-HA activates RhoA-associated protein kinase (ROK) signaling, which contributes to Akt phosphorylation. Conclusions: These findings suggest that the induction of DUSP10/MKP5 expression by HMW-HA binding to CD44, leading to MMP13 suppression, involves multiple regulatory mechanisms, including PI3K/Akt and RhoA-activated ROK signaling, in addition to miRNA-mediated regulation. Elucidating these detailed molecular mechanisms may reveal novel biological activities that contribute to the therapeutic efficacy of HMW-HA against osteoarthritis.

BackgroundDiclofenac etalhyaluronate (DF-HA, SI-613/ONO-5704) is a conjugate of hyaluronic acid (HA) and diclofenac (DF), and its intra-articular injection is widely used for the treatment of osteoarthritis in Japan. While novel mechanisms of cartilage protection by DF-HA have been identified, a comprehensive analysis of the biological responses unique to DF-HA has not yet been conducted.DesignWe used an RNA sequencing (RNA-seq) method to comprehensively analyze gene expression in the knee joint cartilage of arthritic rats and cytokine-stimulated chondrocytes. For the mechanistic analysis of DF-HA, genes that were downregulated or upregulated by DF-HA, HA, or DF were extracted. Pathway analysis was then performed on genes that specifically varied with DF-HA treatment.ResultsIn the cartilage of rats with collagen-induced arthritis, treatment with DF-HA, but not DF or HA, suppressed the extracellular matrix (ECM) remodeling pathway and promoted the parathyroid hormone/parathyroid hormone-related peptide receptor-mediated pathway, which regulates chondrocyte differentiation and bone/cartilage development. In cytokine-stimulated chondrocytes, DF-HA similarly suppressed the ECM remodeling pathway; specifically, gene expression changes in IGFBP4, MMP10, MMP13, and TIMP1 were consistent with those observed in vivo.ConclusionRNA-seq analysis of cartilage in arthritic rats and cytokine-stimulated chondrocytes provided molecular mechanistic insights, indicating that DF-HA treatment induced cartilage protection through the suppression of ECM remodeling.

Seedlings are particularly vulnerable to herbivory because their defenses are underdeveloped and their capacity to tolerate damage is limited. However, how seedlings cope with such threats remains poorly understood. Animal feces may provide important chemical cues that influence plant responses to herbivory. We examined whether the presence of isopod feces affects the herbivory of Japanese plantain (Plantago asiatica) seedlings in the field. Laboratory experiments were also conducted to test the effects of herbivore feces on P. asiatica germination. Bioactive compounds in the feces that regulate seed germination were extracted and identified. The field experiments indicated that the presence of isopod feces induces seed germination on rainy days (when isopod activity is low), reduces herbivory, and triples the survival rate of P. asiatica seedlings. In the laboratory, feces from isopods that had recently fed on P. asiatica leaves suppressed seed germination. However, germination resumed after simulated rain washed them away. Chemical analyses revealed that trehalose and abscisic acid are the active compounds responsible for germination suppression. These findings demonstrate that the detection of chemical cues in animal feces by seeds alters their germination timing accordingly, thereby increasing their chances of survival in environments with high herbivore densities.

Effects of endogenous abscisic acid (ABA) and ABA signal transduction on anthocyanin and photoreceptors in grape berries (Vitis labruscana) were examined using NDGA, the inhibitor of the rate-limiting ABA biosynthesis enzyme in grapes, and PANMe, an ABA receptor interaction antagonist. Both NDGA and PANMe treatments decreased anthocyanin concentrations. The expressions of VvSnRK2.6, VvABF2, and VvPP2C9 in NDGA treatment were not significantly different from the untreated control; by contrast, PANMe treatment decreased the expressions of VvSnRK2.6, VvABF2, and VvPP2C9. Both NDGA and PANMe treatments decreased the expressions of VvPhot2 and VvCRYa. These results suggest that both endogenous ABA concentrations and ABA signaling affect anthocyanin synthesis and photoreceptor genes such as VvPhot2 and VvCRYa in grape skins.

The effects of blue-light irradiation on abscisic acid (ABA) signaling, sugar metabolism and translocation, and photoreceptors and gene expressions were investigated to clarify the mechanism by which blue-LED irradiation increases sugar concentrations in grape berries (Vitis labruscana L.). Blue light-emitting diode (LED) irradiation increased the portion of 13C-photosynthates in the grapevine clusters that were fed 13CO2; compared to the portion in the cluster in the untreated control. Fructose and glucose concentrations and the expressions of VvSWEET10, VvSUC11, and VvSUS4 in blue LED-irradiated berries were increased. The blue LED-irradiated berries’ sucrose concentrations were significantly lower than the untreated control at 14 days after treatment. We speculated that the blue LED-treated berries’ decreased sucrose was associated with the increased Sugars Will Eventually be Exported Transporter (VvSWEET10), sucrose transporter (VvSUC11), and sucrose synthase (VvSUS4) expressions and promoted the translocation of 13C-photosynthates from the leaves that were fed 13CO2. Blue-LED irradiation increased the expressions of SNF1-related protein kinases (VvSnRK2.6) and ABA responding element binding transcription factor (VvABF1), while decreasing the expression of protein phosphateses 2C9 (VvPP2C9) genes, which are related to ABA signaling. Blue-LED irradiation increased the expressions of cryptochrome (VvCRYa) and phototropin (VvPHOT2), which are photoreceptor genes. The application of the pyrabactin resistance-like (PYL)-PP2C ABA receptor interaction antagonist AS6 did not affect endogenous ABA concentrations in the grape berries, but it decreased sucrose concentrations at harvest. The application of ABA did not affect sucrose, glucose, or fructose concentrations or the expressions of VvSnRK2.6 and VvPP2C9. The application of nordihydroguaiaretic acid (NDGA, an inhibitor of 9-cis-epoxycarotenoid dioxygenase activity in ABA biosynthesis) did not affect sugar concentrations at harvest. These results suggest that upregulation of photoreceptor gene expressions and ABA signaling are associated with sugar concentrations in grape berries.

Diclofenac etalhyaluronate (DF-HA) sustained diclofenac release with the effects of hyaluronic acid (HA), offering long-term analgesia in osteoarthritis. In this study, the effects of DF-HA on pain improvement and osteoarthritis were evaluated in a rat knee monoiodoacetate-induced osteoarthritis model compared to HA. Eight rats per group had been injected with monoiodoacetate (2.0 mg) or saline in the right knee for 4 weeks and were injected with either DF-HA (1.25 mg/kg; 0.5 mg), HA (0.5 mg), vehicle which was a substrate without DF-HA (50 μL), or saline and followed for 4 weeks. Mechanical plantar skin sensitivity was assessed weekly using the von Frey assay. Osteoarthritis changes were monitored with Larsen scores via CT imaging at every 2 weeks. The articular cartilage was analyzed using OARSI scores through H&E, Safranin-O staining at 8 weeks. The percentage of Iba-1 positive microglia in the spinal dorsal horn and of FG + CGRP-labeled cells among FG-positive cells in the dorsal root ganglion were evaluated by immunohistochemical staining. TNF-α and IL-6 mRNA expression levels in the knee synovium were evaluated by PCR. The DF-HA showed significantly improved pain hypersensitivity compared with the HA at 6-8 weeks. The percentage of Iba-1-positive microglia was significantly lower than that in the vehicle and the percentage of FG + CGRP/FG was significantly lower than that in the HA. OARSI scores did not differ among treatment groups, Larsen scores indicated lower in the DF-HA than in the vehicle. DF-HA was as effective as HA in joint protection and significantly improved inflammatory pain compared to HA.

The plant hormone abscisic acid (ABA) plays a critical role in various environmental stress responses and has long been expected to be used in agriculture. However, the practical use of ABA has been limited, mainly because of its photoinstability and rapid biodegradation. We previously developed photostable ABA agonists, BP2A and Me 1',4'-trans-diol BP2A, in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. This finding validated our structure-based approach in designing photostable agonists and provided a basis for developing a more potent or long-lasting ABA agonist. In this study, we synthesized novel BP2A analogs in which the cyclohexenone ring was modified to avoid catabolism by the ABA metabolic enzyme, ABA 8'-hydroxylase. All synthesized analogs showed higher photostability than BP2A under sunlight. In an Arabidopsis seed germination assay, (+)-compounds 5 and 6 with a tetralone ring displayed significantly stronger ABA agonist activity than (+)-BP2A. In contrast, in the in vitro phosphatase assays, both compounds showed comparable or weaker ABA receptor (PYL1) agonistic activity than (+)-BP2A, suggesting that the stronger ABA-like activity of (+)-5 and (+)-6 may arise from their metabolic stability in vivo. This study provides data relevant to designing photostable and persistent ABA agonists.

The plant hormone abscisic acid (ABA) plays a central role in adaptive responses to abiotic stresses that adversely affect crop growth and productivity. However, ABA photoinstability limits its use in agriculture. To overcome this drawback, in this study, we developed photostable ABA analogues, the (+)-BP2A compound series (compounds 5-9), in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. All BP2A analogues showed higher stability against UV-B irradiation at 302 nm than ABA, and compounds 6 and 7 barely decomposed even under sunlight. In physiological assays, (+)-BP2A and (+)-compound 7, in which the α,β-unsaturated carbonyl group of BP2A was reduced, exhibited ABA-like activities, including inhibition of seed germination and induced drought tolerance in Arabidopsis. Biochemical studies revealed that (+)-compound 7, unlike (+)-BP2A, did not activate pyrabactin resistance-like (PYL) receptors in vitro and was converted to (+)-BP2A in plants, suggesting that it functions as a prodrug PYL agonist. Furthermore, (+)-compound 7 inhibited seed germination of tomato, lettuce, and rice. Thus, this compound represents a potential plant growth regulator that induces ABA-type responses in agricultural fields.

Abstract Background Diclofenac etalhyaluronate (DF-HA) is a recently developed analgesic conjugate of diclofenac and hyaluronic acid that has analgesic and anti-inflammatory effects on acute arthritis. In this study, we investigated its analgesic effect on osteoarthritis, using a rat model of monoiodoacetate (MIA). Methods We injected MIA into the right knees of eight 6-weeks-old male Sprague–Dawley rats. Four weeks later, rats were randomly injected with DF-HA or vehicle into the right knee. Seven weeks after the MIA injection, fluorogold (FG) and sterile saline were injected into the right knees of all the rats. We assessed hyperalgesia with weekly von Frey tests for 8 weeks after MIA administration. We took the right knee computed tomography (CT) as radiographical evaluation every 2 weeks. All rats were sacrificed 8 weeks after administration of MIA for histological evaluation of the right knee and immunohistochemical evaluation of the DRG and spinal cord. We also evaluated the number of FG-labeled calcitonin gene-related peptide (CGRP)-immunoreactive(ir) neurons in the dorsal root ganglion (DRG) and ionized calcium-binding adapter molecule 1 (Iba1)-ir microglia in the spinal cord. Results Administration of DF-HA significantly improved pain sensitivity and reduced CGRP and Iba1 expression in the DRG and spinal cord, respectively. However, computed tomography and histological evaluation of the right knee showed similar levels of joint deformity, despite DF-HA administration. Conclusion DF-HA exerted analgesic effects on osteoarthritic pain, but did not affect joint deformity.

Tissue-residing mesenchymal stromal/stem cells (MSCs) have multipotent characteristics that are important for adult tissue homeostasis and tissue regeneration after injury. We previously reported that fibroblastic cells isolated from the synovial membrane in the knee joint give rise to cells with MSC characteristics in a two-dimensional culture. To explore the molecular mechanisms underlying these hyperplastic properties, we performed time-course surface antigen expression analyses during in vitro culture. Cells freshly isolated from the synovial membrane rarely contained cells that met the criteria (CD45-CD73+CD90+CD105+). However, the number of cells expressing MSC antigens increased on day 7. Flow cytometric analysis indicated that cells positive for either CD73 or CD90 were specifically derived from cells positive for CD44. CD44 expression was upregulated during culture, and CD105+ cells were specifically derived from the CD44 highly expressing cells. In addition, depletion of hyaluronic acid (HA), a major ligand of CD44, decreased the number of CD105+ cells, whereas supplementation with HA increased their number. These data suggest that intracellular signals activated by CD44 play an important role in the formation and/or maintenance of MSCs.

The molecular mechanisms controlling underwater elongation are based extensively on studies on internode elongation in the monocot rice (Oryza sativa) and petiole elongation in Rumex rosette species. Here, we characterize underwater growth in the dicot Nasturtium officinale (watercress), a wild species of the Brassicaceae family, in which submergence enhances stem elongation and suppresses petiole growth. We used a genome-wide transcriptome analysis to identify the molecular mechanisms underlying the observed antithetical growth responses. Though submergence caused a substantial reconfiguration of the petiole and stem transcriptome, only little qualitative differences were observed between both tissues. A core submergence response included hormonal regulation and metabolic readjustment for energy conservation, whereas tissue-specific responses were associated with defense, photosynthesis, and cell wall polysaccharides. Transcriptomic and physiological characterization suggested that the established ethylene, abscisic acid (ABA), and GA growth regulatory module for underwater elongation could not fully explain underwater growth in watercress. Petiole growth suppression is likely attributed to a cell cycle arrest. Underwater stem elongation is driven by an early decline in ABA and is not primarily mediated by ethylene or GA. An enhanced stem elongation observed in the night period was not linked to hypoxia and suggests an involvement of circadian regulation.

Significance Abscisic acid (ABA) is a phytohormone that plants utilize to coordinate responses to abiotic stress, modulate seed dormancy, and is central to plant development in several contexts. Chemicals that activate or block ABA signaling are useful as research tools and as potential agrochemical leads. Many successes have been reported for ABA activators (agonists), but existing ABA blockers (antagonists) are limited by modest in vivo activity. Here we report antabactin (ANT), a potent ABA blocker developed using “click chemistry”–based diversification of a known ABA activator. Structural studies reveal, ANT disrupts signaling by stabilizing ABA receptors in an unproductive form. ANT can accelerate seed germination in multiple species, making it a chemical tool for improving germination.

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.

Our previous study has shown that Tbx5-Gata4-Baf60c-induced functional cardiomyocytes via the ectopic expression of Nkx2-5/Islet1 in the mesodermal cells, but not in the endodermal/ectodermal cells [1, 2] (Fig. 57.1). Mesp1 is one of the major transcriptional regulators specifying the mesodermal lineage, but it also induces skeletal muscle, hematopoietic and vascular cells as well as cardiac cells [3–5]. Eomesodermin (Eomes), an upstream player of Mesp1, regulates mesodermal cell lineages, but it does not have a potential for specification of cardiac cell fate from cardiovascular lineages either [6]. Therefore, the study of cardiac cell fate specification from the embryonic stem cells by the defined factors still remains at least two major questions.

Small-molecule plant hormones principally control plant growth, development, differentiation, and environmental responses. Nine types of plant hormones are ubiquitous in angiosperms, and the molecular mechanisms of their hormone actions have been elucidated during the last two decades by genomic decoding of model plants with genetic mutants. In particular, the discovery of hormone receptors has greatly contributed to the understanding of signal transduction systems. The three-dimensional structure of the ligand-receptor complex has been determined for eight of the nine hormones by X-ray crystal structure analysis, and ligand perception mechanisms have been revealed at the atomic level. Collective research has revealed the molecular function of plant hormones that act as either molecular glue or an allosteric regulator for activation of receptors. In this review, we present an overview of the respective hormone signal transduction and describe the structural bases of ligand-receptor interactions.