Abstract Hygrophila difformis is an ornamental amphibious plant that develops distinct terrestrial (shallow-serrated) and submerged (deep-lobed) leaf forms in response to environmental conditions. Although previous studies have reported bicarbonate (HCO₃ −) utilization and highlighted the involvement of carbonic anhydrase in inorganic carbon uptake, the photosynthetic strategy and carbon assimilation pathway operating under submergence remain unclear. Here, we applied an integrated framework combining physiological measurements, enzyme activity assays, transcriptomic, proteomic, and metabolomic profiling, stable carbon isotope analysis, and ultrastructural observations to compare terrestrial and submerged leaves. Submerged leaves displayed a δ 13C value of −20.068‰, indicating a substantial shift toward C₄-like photosynthetic metabolism under aquatic conditions. Consistent with this signature, activities of key carboxylation and decarboxylation enzymes were significantly elevated, supporting a transition from C₃ photosynthesis toward an NAD-malic enzyme (NAD-ME)–type C₄ pathway. Multi-omics analyses revealed extensive reprogramming of photosynthetic and starch metabolic networks in response to submergence, with strong upregulation of genes and metabolites associated with the NAD-ME–type C₄ cycle, including cytosolic aspartate aminotransferase and alanine aminotransferase, accompanied by coordinated downregulation of Calvin cycle components. By contrast, terrestrial leaves retained high Rubisco activity and strong expression of Calvin cycle genes, consistent with classical C₃ photosynthesis. RT-qPCR analyses demonstrated marked induction of carbonic anhydrase genes under submerged conditions, with HdαCA9 showing a 194-fold increase in transcript abundance, highlighting its central role in underwater carbon utilization. Transmission electron microscopy further revealed dimorphic chloroplasts within submerged mesophyll cells. Collectively, results demonstrate plasticity in H. difformis and provide insights into C₄-like carbon assimilation strategies in amphibious plants.

While the epidermal cells of terrestrial plants typically lack chloroplasts, this is not the case for aquatic plants, a trait that likely evolved to enhance photosynthetic efficiency underwater. Amphibious plants display various response strategies to survive under both terrestrial and aquatic environments. In this study, we described an environmental response in the amphibious plant Rorippa aquatica that involves chloroplasts differentiating into epidermal cells upon submergence. This phenomenon has not been previously documented and was named environmentally responsive epidermal chloroplast differentiation (ECD). To elucidate the mechanisms underlying ECD, we conducted RNA-sequencing analysis. The results revealed ethylene signaling, hypoxia responses, and light-regulated chloroplast development as key pathways. Physiological experiments showed that ethylene is a central trigger; exogenous ethylene or 1-aminocyclopropane-1-carboxylic acid promote ECD, whereas silver nitrate inhibits it. Submergence alone was insufficient to fully induce ECD, with underwater light exposure accelerating chloroplast maturation. Comparative analysis of phylogenetically distant amphibious species revealed that ECD is not unique to R. aquatica, implying ECD occurs in other species as well, with its extent varying among them. These findings provide new insights on plant acclimation to aquatic environments and highlight the intricate interplay between submergence, ethylene, and light in regulating chloroplast differentiation.

Aquatic plants inhabit highly dynamic environments characterized by rapid and extreme fluctuations in CO2 availability, light intensity, hydrodynamics, temperature, and pH. These conditions have driven the evolution of exceptional photosynthetic plasticity. This review synthesizes recent advances in understanding how higher aquatic plants deploy a diverse array of carbon-concentrating mechanisms (CCMs), including canonical C3 and C4 pathways, C4-like and CAM-like metabolic flexibility, and efficient biophysical CCMs based on HCO3 - uptake, proton extrusion, and the coordinated action of internal and external carbonic anhydrases. We highlight key evolutionary innovations unique to aquatic lineages, such as single-cell C4 photosynthesis, dimorphic chloroplasts, and heterophylly, which together facilitate rapid optimization of carbon assimilation under shifting ecological conditions. A detailed comparison between terrestrial model species and aquatic macrophytes further illustrates how contrasting environmental pressures have shaped distinct morphological, anatomical, and physiological strategies. We also emphasize emerging model systems, including Ottelia ovalifolia, O. alismoides, Hygrophila difformis, and Rorippa aquatica, which provide powerful platforms for investigating the evolution, regulation, and integration of CCMs with phenotypic plasticity. Collectively, aquatic plants represent underexplored yet promising systems with significant potential for advancing carbon neutrality strategies, freshwater ecosystem management, crop improvement, and synthetic biology.

Orchids have evolved flowers with unique morphologies through coevolution with pollinators, such as insects. Among the floral organs, the lip (labellum), one of the three petals, exhibits a distinctive shape and plays a crucial role in attracting pollinators and facilitating pollination in many orchids. The lip of the terrestrial orchid Habenaria radiata is shaped like a flying white bird and is believed to attract and provide a platform for nectar-feeding pollinators, such as hawk moths. To elucidate the mechanism of lip morphogenesis, we conducted time-lapse imaging of blooming flowers to observe the extension process of the lip and analyzed the cellular morphology during the generation of serrations. We found that the wing part of the lip folds inward in the bud and fully expands in two hours after blooming. The serrations of the lip were initially formed through cell division and later deepened through polar cell elongation. Transcriptome analysis of floral buds revealed the expression of genes involved in floral organ development, cell division, and meiosis. Additionally, genes involved in serration formation are also expressed in floral buds. This study provides insights into the mechanism underlying the formation of the unique lip morphology in Habenaria radiata.

Cells sense and integrate multiple signals to coordinate a response. A receptor-kinase signaling pathway for plant stomatal development shares components with the immunity pathway. The mechanism ensuring their signal specificities remains unclear. Using chemical genetics, here, we report the identification of a small molecule, kC9, that triggers excessive stomatal differentiation by inhibiting the canonical ERECTA pathway. kC9 binds to and inhibits the downstream mitogen-activated protein kinase MPK6, perturbing its substrate interaction. Notably, activation of immune signaling by a bacterial flagellin peptide nullified kC9's effects on stomatal development. This cross-regulation depends on the immune receptor FLS2 (FLAGELLIN SENSING 2) and occurs even in the absence of kC9 if the ERECTA family receptor population becomes suboptimal. Proliferating stomatal lineage cells are vulnerable to this immune signal penetration. Our findings suggest that the signal specificity between development and immunity can be ensured by mitogen-activated protein kinase homeostasis, reflecting the availability of upstream receptors, thereby providing an unanticipated view on signal specificity.

Cadmium (Cd) is a toxic metal that poses an environmental risk, but its effects on amphibious plants like Hygrophila difformis, which thrive in both terrestrial and submerged conditions, remain unexplored. This study investigates the morphological, anatomical, and physiological responses of H. difformis to Cd exposure. H. difformis was exposed to 0, 1, 2, and 4 mg/L Cd for 30 days under both terrestrial and submerged conditions. Leaves were examined at 10, 20, and 30 days for morphological and physiological changes. At lower Cd concentration (1 mg/L), leaf morphology showed minimal changes compared to the control. Submerged control leaves were highly dissected (26.1 ± 0.45), whereas dissection was substantially reduced at 4 mg/L Cd (16.96 ± 0.67), indicating a profound impact on heterophylly. Cd stress affected leaf size significantly, particularly in submerged plants (17.8 ± 3.22 cm²) compared to controls (39.2 ± 2.84 cm²). Furthermore, compared to terrestrial leaves (4.27 ± 0.31 mg/kg), submerged leaves accumulated significantly higher content of Cd (45.2 ± 6.66 mg/kg), indicating higher absorption under aquatic conditions. Terrestrial leaves appeared more resistant; however, higher concentrations caused tissue damage. Following 30 days of treatment, qualitative TEM-based anatomical analysis revealed noticeable cell shrinkage and fewer visible chloroplasts in submerged leaves compared to controls, while terrestrial leaves exhibited thicker cell walls. Cd exposure also inhibited photosynthesis, reducing pigment levels and enzyme activity. Interestingly, Rubisco activity increased in submerged leaves after 30 days of high Cd exposure, preventing the transition from C3 to C4 photosynthesis. H. difformis exhibits poor growth under Cd stress and can serve as a bioindicator for heavy metal pollution.

Heterophylly, the plasticity of leaf form in response to environmental conditions, widely occurs in aquatic and amphibious plants. Rorippa aquatica produces simple or shallow-serrated leaves in terrestrial conditions but deep dissected leaves under submerged conditions. Regulation of CIN-TCP transcription factors by miR319 controls leaf complexity in several species and here we provide evidence that this regulatory module acts in the heterophylly of R. aquatica. RaTCP1, one of the orthologs of AtTCP4 in R. aquatica, was identified as the most likely target of Raq-miR319b. Under submerged conditions that induced increased leaf complexity, RaTCP1 expression was reduced whereas Raq-miR319b expression was increased. Overexpressing Raq-miR319b in Arabidopsis thaliana reduced TCP gene expression and increased leaf serration. Ectopic expression of RaTCP1 rescued the phenotype of crinkled leaf form in tcp mutants of A. thaliana. The phytohormone abscisic acid (ABA) accumulated in terrestrial leaves of R. aquatica, while it was absent in submerged conditions. We found that the expression of Raq-miR319b can be induced by submergence, while it was repressed by ABA. Our results indicate that the environments regulated heterophylly in R. aquatica occur through the miR319-TCP module. These findings provide novel insights into how the plasticity of leaf shape is established in aquatic plants.

The formation of galls by gall-inducing insects on their host plants demonstrates the concept of 'extended phenotype', referring to traits expressed in a host organism manipulated by a parasite. Phytohormones, amino acids, and proteins have been suggested as compound types involved in gall formation, and a few specific molecules have been proposed as candidate effector molecules; however, no specific gall-inducing effector molecules have been identified. Hence, in this study, we identified the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) peptide from horned gall aphid Schlechtendalia chinensis transcripts through in silico screening and the Arabidopsis-based gall-forming assay, a bioassay system for analysing the molecular mechanisms of gall formation. Furthermore, we successfully generated the insect-gall-like structure in Veronica peregrina-the host plant of the gall-forming weevil Gymnaetron miyoshii Miyoshi-without insect parasitism, using three minimal effector elements: the CAP peptide, auxin, and cytokinin. Given the similarity of the gall structure to the native gall, both in gene expression profile and morphology, we experimentally demonstrated that the gall-like structure was artificially generated using effector molecules, the CAP peptide, auxin, and cytokinin.

Galls caused by gall-inducing insects in their host plants clearly illustrate the concept of ‘extended phenotype’, which refers to traits expressed in a host organism when manipulated by a parasite. Candidate effector molecules involved in gall formation, such as phytohormones, amino acids, and proteins, have been reported in numerous studies. However, to date, no attempts to artificially regenerate gall structures using effector candidates have been reported. In this study, we tested the peptide from Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins, CAP peptide as a gall-inducing effector candidate obtained from transcripts isolated from the horned gall aphid, ( Schlechtendalia chinensis ) through in silico screening and the Arabidopsis-based gall-forming assay, which is a bioassay system for analysing the molecular mechanisms of gall formation. Furthermore, we succeeded in generating an artificial gall in the host plant Veronica peregrina, without any insect parasitism, using three minimal effector elements: CAP peptide, auxin, and cytokinin. Given the strong similarities observed in organ structure with a central cavity and three types of tissue and gene expression patterns between the native and artificial galls, we concluded that CAP peptide is a general gall-inducing effector peptide secreted by gall-inducing insects.### Competing Interest StatementThe authors have declared no competing interest.

Amphibious plants can grow and survive in both aquatic and terrestrial environments. This review explores the diverse adaptations that enable them to thrive in such contrasting habitats. Plants with amphibious lifestyles possess fascinating traits, and their phenotypic plasticity plays an important role in adaptations. Heterophylly, the ability to produce different leaf forms, is one such trait, with submerged leaves generally being longer, narrower, and thinner than aerial leaves. In addition to drastic changes in leaf contours, amphibious plants display significant anatomical and physiological changes, including a reduction in stomatal number and cuticle thickness and changes in photosynthesis mode. This review summarizes and compares the regulatory mechanisms and evolutionary origins of amphibious plants based on molecular biology studies actively conducted in recent years using novel model amphibious plant species. Studying amphibious plants will enhance our understanding of plant adaptations to aquatic environments.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

The ability to respond to varying environments is crucial for sessile organisms such as plants. The amphibious plant Rorippa aquatica exhibits a striking type of phenotypic plasticity known as heterophylly, a phenomenon in which leaf form is altered in response to environmental factors. However, the underlying molecular mechanisms of heterophylly are yet to be fully understood. To uncover the genetic basis and analyze the evolutionary processes driving heterophylly in R. aquatica, we assembled the chromosome-level genome of the species. Comparative chromosome painting and chromosomal genomics revealed that allopolyploidization and subsequent post-polyploid descending dysploidy occurred during the speciation of R. aquatica. Based on the obtained genomic data, the transcriptome analyses revealed that ethylene signaling plays a central role in regulating heterophylly under submerged conditions, with blue light signaling acting as an attenuator of ethylene signal. The assembled R. aquatica reference genome provides insights into the molecular mechanisms and evolution of heterophylly.

Quality control of translation is crucial for maintaining cellular and organismal homeostasis. Obstacles in translation elongation induce ribosome collision, which is monitored by multiple sensor mechanisms in eukaryotes. The E3 ubiquitin ligase Znf598 recognizes collided ribosomes, triggering ribosome-associated quality control (RQC) to rescue stalled ribosomes and no-go decay (NGD) to degrade stall-prone mRNAs. However, the impact of RQC and NGD on maintaining the translational homeostasis of endogenous mRNAs has remained unclear. In this study, we investigated the endogenous substrate mRNAs of NGD during the maternal-to-zygotic transition (MZT) of zebrafish development. RNA-Seq analysis of zebrafish znf598 mutant embryos revealed that Znf598 down-regulates mRNAs encoding the C2H2-type zinc finger domain (C2H2-ZF) during the MZT. Reporter assays and disome profiling indicated that ribosomes stall and collide while translating tandem C2H2-ZFs, leading to mRNA degradation by Znf598. Our results suggest that NGD maintains the quality of the translatome by mitigating the risk of ribosome collision at the abundantly present C2H2-ZF sequences in the vertebrate genome.

AimLong-distance dispersal (LDD) plays an important role in shaping the distribution of global biodiversity. Polyploidy could favour invasion and thereby facilitate LDD. However, how and to what extent polyploidy interacts with LDD remain unclear. Here, we test the putative role of polyploidy in the global dispersal of a cosmopolitan genus Rorippa.LocationGlobal.Time PeriodLate Miocene to present.Major Taxa StudiedRorippa Scop., Brassicaceae.MethodsWe traced the biogeographical and speciation history for 17 diploids and 41 polyploids of Rorippa using variation from plastid genomes and multiple nuclear loci. The ploidy role in dispersal rate difference was demonstrated using trait-dependent biogeographical modelling.ResultsLDD shaped the amphitropical disjunction of Rorippa, during which polyploids showed higher dispersal rates than those of diploids, with 5.6x increase under the best-fitted model. Five diploids and 21 polyploids were identified as products of transoceanic speciation events. Polyploidy-involved LDD was more common in terms of polyploidization following LDD than those preceding LDD.Main ConclusionsWe demonstrate that polyploidy would be not only a driver but also a responder of LDD in Rorippa, highlighting a synergistic relationship between them. Our results provide a framework to uncover the biogeographical consequences of polyploidization and the joint roles of polyploidy and LDD in shaping the distribution of biodiversity.

Plant galls generated by insects have highly organized structures, providing nutrients and shelter to the insects living within them. Most research on the physiological and molecular mechanisms of gall development has focused on single galls. To understand the diversity of gall development, we examined five galls with different morphologies generated by distinct species of Rhopalomyia (gall midge; Diptera: Cecidomyiidae) on a single host plant of Artemisia indica var. maximowiczii (Asteraceae). Vasculature developed de novo within the galls, indicating active transport of nutrients between galls and the host plant. Each gall had a different pattern of vasculature and lignification, probably due to differences in the site of gall generation and the gall midge species. Transcriptome analysis indicated that photosynthetic and cell wall-related genes were down-regulated in leaf and stem galls, respectively, compared with control leaf and stem tissues, whereas genes involved in floral organ development were up-regulated in all types of galls, indicating that transformation from source to sink organs occurs during gall development. Our results help to understand the diversity of galls on a single herbaceous host plant.

Heterophylly is a phenomenon whereby an individual plant dramatically changes leaf shape in response to the surroundings. Hygrophila difformis (Acanthaceae; water wisteria), has recently emerged as a model plant to study heterophylly because of its striking leaf shape variation in response to various environmental factors. When submerged, H. difformis often develops complex leaves, but on land it develops simple leaves. Leaf complexity is also influenced by other factors, such as light density, humidity, and temperature. Here, we sequenced and assembled the H. difformis chromosome-level genome (scaffold N50: 60.43 Mb, genome size: 871.92 Mb), which revealed 36 099 predicted protein-coding genes distributed over 15 pseudochromosomes. H. difformis diverged from its relatives during the Oligocene climate-change period and expanded gene families related to its amphibious habit. Genes related to environmental stimuli, leaf development, and other pathways were differentially expressed in submerged and terrestrial conditions, possibly modulating morphological and physiological acclimation to changing environments. We also found that auxin plays a role in H. difformis heterophylly. Finally, we discovered candidate genes that respond to different environmental conditions and elucidated the role of LATE MERISTEM IDENTITY 1 (LMI1) in heterophylly. We established H. difformis as a model for studying interconnections between environmental adaptation and morphogenesis.

Heterophylly, the plasticity of leaf form in response to environmental conditions, occurs in aquatic and amphibious plants where it modulates water availability, gas exchange and photosynthesis to maximize plant productivity. Rorippa aquatica produces simple leaves in terrestrial conditions but dissected leaves under submerged conditions. Regulation of CIN-TCP transcription factors by miR319 controls leaf complexity in a number of species and we provide evidence that this regulatory module acts in R. aquatica heterophylly. RaTCP1 was identified as the most likely target of Raq-miR319b . Under conditions that induced increased leaf complexity, RaTCP1 expression was reduced whereas Raq-miR319b expression was increased. Overexpression of Raq-miR319 in Arabidopsis thaliana reduced TCP gene expression and increased leaf serration. Similarly, a repressive form of RaTCP1 expressed in either A. thaliana or R. aquatica resulted in more complex leaves. Our results indicate that the environmental induction of dissected leaves in R. aquatica occurs through down-regulation of RaTCP by Raq-miR319 .

At the beginning of ovarian aging, the ovulation of immature oocytes is accelerated, leading to the arrest of ovulation despite the remaining oocytes. Here, RNA expression in the ovarian aging of mice is comprehensively analyzed during the estrous cycle after ovulation stimulation. The aldo-keto reductase Akr1b7 pathway transiently activated in the ovaries of young mice disappears in those of old mice. Akr1b7 —/— mice attenuate oocyte Akt activation essential for the follicular development in primordial follicles, and enhanced ovulation in immature oocytes. The estrous cycle is extended because of the prolonged diestrous stage by a sustained progesterone level in Akr1b7 —/— mice ovaries, which is caused by the decline of Cyp17a1 , a major metabolic enzyme of progesterone in Akr1b7 -expressed theca cell layers. In summary, the decreased Akr1b7 pathway causes ovulation of immature oocytes and a prolonged estrous cycle, typical symptoms of ovarian aging.