The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

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.

A chromosome-level genome assembly for the amphibious plant Rorippa aquatica reveals its allotetraploid origin and mechanisms of heterophylly upon submergence.

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.

Diving into the Water: Amphibious Plants as a Model for Investigating Plant Adaptations to Aquatic Environments.

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. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

A Small Compound, HYGIC, Promotes Hypocotyl Growth Through Ectopic Ethylene Response.

Plant seedlings adjust the growth of the hypocotyl in response to surrounding environmental changes. Genetic studies have revealed key players and pathways in hypocotyl growth, such as phytohormones and light signaling. However, because of genetic redundancy in the genome, it is expected that not-yet-revealed mechanisms can be elucidated through approaches different from genetic ones. Here, we identified a small compound, HYGIC (HG), that simultaneously induces hypocotyl elongation and thickening, accompanied by increased nuclear size and enlargement of cortex cells. HG-induced hypocotyl growth required the ethylene signaling pathway activated by endogenous ethylene, involving CONSTITUTIVE PHOTOMORPHOGENIC 1, ETHYLENE INSENSITIVE 2 (EIN2) and redundant transcription factors for ethylene responses, ETHYLENE INSENSITIVE 3 (EIN3) and EIN3 LIKE 1. By using EBS:GUS, a transcriptional reporter of ethylene responses based on an EIN3-binding-cis-element, we found that HG treatment ectopically activates ethylene responses at the epidermis and cortex of the hypocotyl. RNA-seq and subsequent gene ontology analysis revealed that a significant number of HG-induced genes are related to responses to hypoxia. Indeed, submergence, a representative environment where the hypoxia response is induced in nature, promoted ethylene-signaling-dependent hypocotyl elongation and thickening accompanied by ethylene responses at the epidermis and cortex, which resembled the HG treatment. Collectively, the identification and analysis of HG revealed that ectopic responsiveness to ethylene promotes hypocotyl growth, and this mechanism is activated under submergence.

Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion.

Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

Diversity of tomato leaf form provides novel insights into breeding.

Tomato (Solanum lycopersicum L.) is cultivated widely globally. The crop exhibits tremendous morphological variations because of its long breeding history. Apart from the commercial tomato varieties, wild species and heirlooms are grown in certain regions of the world. Since the fruit constitutes the edible part, much of the agronomical research is focused on it. However, recent studies have indicated that leaf morphology influences fruit quality. As leaves are specialized photosynthetic organs and the vascular systems transport the photosynthetic products to sink organs, the architectural characteristics of the leaves have a strong influence on the final fruit quality. Therefore, comprehensive research focusing on both the fruit and leaf morphology is required for further tomato breeding. This review summarizes an overview of knowledge of the basic tomato leaf development, morphological diversification, and molecular mechanisms behind them and emphasizes its importance in breeding. Finally, we discuss how these findings and knowledge can be applied to future tomato breeding.

Identification of a pluripotency-inducing small compound, PLU, that induces callus formation via Heat Shock Protein 90-mediated activation of auxin signaling.

Plants retain the ability to generate a pluripotent tissue called callus by dedifferentiating somatic cells. A pluripotent callus can also be artificially induced by culturing explants with hormone mixtures of auxin and cytokinin, and an entire body can then be regenerated from the callus. Here we identified a pluripotency-inducing small compound, PLU, that induces the formation of callus with tissue regeneration potency without the external application of either auxin or cytokinin. The PLU-induced callus expressed several marker genes related to pluripotency acquisition via lateral root initiation processes. PLU-induced callus formation required activation of the auxin signaling pathway though the amount of active auxin was reduced by PLU treatment. RNA-seq analysis and subsequent experiments revealed that Heat Shock Protein 90 (HSP90) mediates a significant part of the PLU-initiated early events. We also showed that HSP90-dependent induction of TRANSPORT INHIBITOR RESPONSE 1, an auxin receptor gene, is required for the callus formation by PLU. Collectively, this study provides a new tool for manipulating and investigating the induction of plant pluripotency from a different angle from the conventional method with the external application of hormone mixtures.

Ab-GALFA, A bioassay for insect gall formation using the model plant Arabidopsis thaliana.

Insect galls are abnormal plant organs formed by gall-inducing insects to provide shelter and nutrients for themselves. Although insect galls are spatialized complex structures with unique shapes and functions, the molecular mechanism of the gall formation and the screening system for the gall inducing effectors remains unknown. Here, we demonstrate that an extract of a gall-inducing aphid, Schlechtendalia chinensis, induces an abnormal structure in the root-tip region of Arabidopsis seedlings. The abnormal structure is composed of stem-like cells, vascular, and protective tissues, as observed in typical insect galls. Furthermore, we confirm similarities in the gene expression profiles between the aphid-treated seedlings and the early developmental stages of Rhus javanica galls formed by S. chinensis. Based on the results, we propose a model system for analyzing the molecular mechanisms of gall formation: the Arabidopsis-based Gall-Forming Assay (Ab-GALFA). Ab-GALFA could be used not only as a model to elucidate the mechanisms underlying gall formation, but also as a bioassay system to isolate insect effector molecules of gall-induction.

Rewiring of hormones and light response pathways underlies the inhibition of stomatal development in an amphibious plant Rorippa aquatica underwater.

Land plants have evolved the ability to cope with submergence. Amphibious plants are adapted to both aerial and aquatic environments through phenotypic plasticity in leaf form and function, known as heterophylly. In general, underwater leaves of amphibious plants are devoid of stomata, yet their molecular regulatory mechanisms remain elusive. Using the emerging model of the Brassicaceae amphibious species Rorippa aquatica, we lay the foundation for the molecular physiological basis of the submergence-triggered inhibition of stomatal development. A series of temperature shift experiments showed that submergence-induced inhibition of stomatal development is largely uncoupled from morphological heterophylly and likely regulated by independent pathways. Submergence-responsive transcriptome analysis revealed rapid reprogramming of gene expression, exemplified by the suppression of RaSPEECHLESS and RaMUTE within 1 h and the involvement of light and hormones in the developmental switch from terrestrial to submerged leaves. Further physiological studies place ethylene as a central regulator of the submergence-triggered inhibition of stomatal development. Surprisingly, red and blue light have opposing functions in this process: blue light promotes, whereas red light inhibits stomatal development, through influencing the ethylene pathway. Finally, jasmonic acid counteracts the inhibition of stomatal development, which can be attenuated by the red light. The actions and interactions of light and hormone pathways in regulating stomatal development in R. aquatica are different from those in the terrestrial species, Arabidopsis thaliana. Thus, our work suggests that extensive rewiring events of red light to ethylene signaling might underlie the evolutionary adaption to water environment in Brassicaceae.

Transcriptomic heterochrony and completely cleistogamous flower development in the mycoheterotrophic orchid Gastrodia.

Cleistogamy, in which plants can reproduce via self-fertilization within permanently closed flowers, has evolved in > 30 angiosperm lineages; however, consistent with Darwin's doubts about its existence, complete cleistogamy - the production of only cleistogamous flowers - has rarely been recognized. Thus far, the achlorophyllous orchid genus, Gastrodia, is the only known genus with several plausible completely cleistogamous species. Here, we analyzed the floral developmental transcriptomes of two recently evolved, completely cleistogamous Gastrodia species and their chasmogamous sister species to elucidate the possible changes involved in producing common cleistogamous traits. The ABBA-BABA test did not support introgression and protein sequence convergence as evolutionary mechanisms leading to cleistogamy, leaving convergence in gene expression as a plausible mechanism. Regarding transcriptomic differentiation, the two cleistogamous species had common modifications in the expression of developmental regulators, exhibiting a gene family-wide signature of convergent expression changes in MADS-box genes. Our transcriptomic pseudotime analysis revealed a prolonged juvenile state and eventual maturation, a heterochronic pattern consistent with partial neoteny, in cleistogamous flower development. These findings indicate that transcriptomic partial neoteny, arising from changes in the expression of conserved developmental regulators, might have contributed to the rapid and repeated evolution of cleistogamous flowers in Gastrodia.

Oxicam-type nonsteroidal anti-inflammatory drugs enhance Agrobacterium-mediated transient transformation in plants.

Agrobacterium-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of Agrobacterium-mediated transient transformation. TNX treatment increased the transformation efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana mature leaves by agroinfiltration. The increase of efficiency by TNX treatment was not observed in dde2/ein2/pad4/sid2 quadruple mutant, indicating that TNX inhibits the immune system mediated by jasmonic acid, ethylene, and salicylic acid against to Agrobacterium. We also found that TNX-treatment is applicable for the transient expression and subcellular localization analysis of fluorescent-tagged proteins in Arabidopsis leaf cells. In addition, we found that TNX increases the efficiency of Agrobacterium-mediated transient transformation of Jatropha. Given that treatment with oxicam compounds is a simple and cost effective method, our findings will provide a new option to overcome limitations associated with Agrobacterium-mediated transformation of various plant species.

Leaf Cell Morphology Alternation in Response to Environmental Signals in Rorippa aquatica.

Heterophylly, the phenomenon by which plants alter leaf forms to adapt to surrounding conditions, is apparent in amphibious plant species. In response to submergence, they emerge leaves with narrower blade areas. The pathway that receives the submergence signals and the mechanism regulating leaf form via cell proliferation and/or expansion systems have not yet been fully identified yet. Our anatomical study of Rorippa aquatica, an amphibious plant that exhibits heterophylly in response to various signals, showed that leaf thickness increased upon submergence; this was caused by the expansion of mesophyll cell size. Additionally, these submergence effects were inhibited under blue-light conditions. The ANGUSTIFOLIA3 (AN3)/GROWTH-REGULATING FACTOR (GRF) pathway regulating cell proliferation and cell expansion was downregulated in response to submergence; and the response was blocked under the blue-light conditions. These results suggest that submergence and light quality determine leaf cell morphology via the AN3/GRF pathway.

Non-cell-autonomous regulation of petal initiation in Arabidopsis thaliana.

In many flowering plants, petals initiate in alternate positions from first whorl sepals, suggesting possible signaling between sepal boundaries and petal initiation sites. PETAL LOSS (PTL) and RABBIT EARS (RBE) regulate petal initiation in Arabidopsis thaliana and their transcripts are expressed in sepal boundary and petal initiation sites, respectively, suggesting that PTL acts in a non-cell-autonomous manner. Here, we determined that cells expressing PTL and RBE fusion proteins did not overlap but were adjacent, confirming the non-cell-autonomous function of PTL. Genetic ablation of intersepal cells by expressing the diphtheria toxin-A chain gene driven by the PTL promoter resulted in flowers lacking petals, suggesting these cells are required for petal initiation. Transcriptome analysis combined with a PTL induction system revealed 42 genes that were upregulated under PTL activation, including UNUSUAL FLORAL ORGANS (UFO), which likely plays an important role in petal initiation. These findings suggest a molecular mechanism in which PTL indirectly regulates petal initiation and UFO mediates positional signaling between the sepal boundary and petal initiation sites.

SHOOT MERISTEMLESS participates in the heterophylly of Hygrophila difformis (Acanthaceae).

In heterophyllous plants, leaf shape shows remarkable plasticity in response to environmental conditions. However, transgenic studies of heterophylly are lacking and the molecular mechanism remains unclear. Here, we cloned the KNOTTED1-LIKE HOMEOBOX family gene SHOOT MERISTEMLESS (STM) from the heterophyllous plant Hygrophila difformis (Acanthaceae). We used molecular, morphogenetic, and biochemical tools to explore its functions in heterophylly. HdSTM was detected in different organs of H. difformis, and its expression changed with environmental conditions. Heterologous, ectopic expression of HdSTM in Arabidopsis (Arabidopsis thaliana) increased leaf complexity and CUP-SHAPED COTYLEDON (CUC) transcript levels. However, overexpression of HdSTM in H. difformis did not induce the drastic leaf change in the terrestrial condition. Overexpression of HdSTM in H. difformis induced quick leaf variations in submergence, while knockdown of HdSTM led to disturbed leaf development and weakened heterophylly in H. difformis. HdCUC3 had the same spatiotemporal expression pattern as HdSTM. Biochemical analysis revealed a physical interaction between HdSTM and HdCUC3. Our results provide genetic evidence that HdSTM is involved in regulating heterophylly in H. difformis.

Deceleration of the cell cycle underpins a switch from proliferative to terminal divisions in plant stomatal lineage.

Differentiation of specialized cell types requires precise cell-cycle control. Plant stomata are generated through asymmetric divisions of a stem-cell-like precursor followed by a single symmetric division that creates paired guard cells surrounding a pore. The stomatal-lineage-specific transcription factor MUTE terminates the asymmetric divisions and commits to differentiation. However, the role of cell-cycle machineries in this transition remains unknown. We discover that the symmetric division is slower than the asymmetric division in Arabidopsis. We identify a plant-specific cyclin-dependent kinase inhibitor, SIAMESE-RELATED4 (SMR4), as a MUTE-induced molecular brake that decelerates the cell cycle. SMR4 physically and functionally associates with CYCD3;1 and extends the G1 phase of asymmetric divisions. By contrast, SMR4 fails to interact with CYCD5;1, a MUTE-induced G1 cyclin, and permits the symmetric division. Our work unravels a molecular framework of the proliferation-to-differentiation switch within the stomatal lineage and suggests that a timely proliferative cell cycle is critical for stomatal-lineage identity.

Ribosome slowdown triggers codon-mediated mRNA decay independently of ribosome quality control.

The control of mRNA stability plays a central role in regulating gene expression patterns. Recent studies have revealed that codon composition in the open reading frame determines mRNA stability in multiple organisms. Based on genome-wide correlation approaches, this previously unrecognized role for the genetic code is attributable to the kinetics of the codon-decoding process by the ribosome. However, complementary experimental analyses are required to clarify the codon effects on mRNA stability and the related cotranslational mRNA decay pathways, for example, those triggered by aberrant ribosome stalling. In the current study, we performed a set of reporter-based analyses to define codon-mediated mRNA decay and ribosome stall-dependent mRNA decay in zebrafish embryos. Our analysis showed that the effect of codons on mRNA stability stems from the decoding process, independent of the ribosome quality control factor Znf598 and stalling-dependent mRNA decay. We propose that codon-mediated mRNA decay is rather triggered by transiently slowed ribosomes engaging in a productive translation cycle in zebrafish embryos.

Combination of genetic analysis and ancient literature survey reveals the divergence of traditional Brassica rapa varieties from Kyoto, Japan.

Since ancient times, humans have bred several plants that we rely on today. However, little is known about the divergence of most of these plants. In the present study, we investigated the divergence of Mibuna (Brassica rapa L. subsp. nipposinica L. H. Bailey), a traditional leafy vegetable in Kyoto (Japan), by combining genetic analysis and a survey of ancient literature. Mibuna is considered to have been bred 200 years ago from Mizuna, another traditional leafy vegetable in Kyoto. Mibuna has simple spatulate leaves, whereas Mizuna has characteristic serrated leaves. The quantitative trait loci (QTL) and gene expression analyses suggested that the downregulation of BrTCP15 expression contributed to the change in the leaf shape from serrated to simple spatulate. Interestingly, the SNP analysis indicated that the genomic region containing the BrTCP15 locus was transferred to Mibuna by introgression. Furthermore, we conducted a survey of ancient literature to reveal the divergence of Mibuna and found that hybridization between Mizuna and a simple-leaved turnip might have occurred in the past. Indeed, the genomic analysis of multiple turnip cultivars showed that one of the cultivars, Murasakihime, has almost the same sequence in the BrTCP15 region as Mibuna. These results suggest that the hybridization between Mizuna and turnip has resulted in the establishment of Mibuna.

Reduction in organ-organ friction is critical for corolla elongation in morning glory.

In complex structures such as flowers, organ-organ interactions are critical for morphogenesis. The corolla plays a central role in attracting pollinators: thus, its proper development is important in nature, agriculture, and horticulture. Although the intraorgan mechanism of corolla development has been studied, the importance of organ-organ interactions during development remains unknown. Here, using corolla mutants of morning glory described approximately 200 years ago, we show that glandular secretory trichomes (GSTs) regulate floral organ interactions needed for corolla morphogenesis. Defects in GST development in perianth organs result in folding of the corolla tube, and release of mechanical stress by sepal removal restores corolla elongation. Computational modeling shows that the folding occurs because of buckling caused by mechanical stress from friction at the distal side of the corolla. Our results suggest a novel function of GSTs in regulating the physical interaction of floral organs for macroscopic morphogenesis of the corolla.

SOG1, a plant-specific master regulator of DNA damage responses, originated from nonvascular land plants.

The suppressor of gamma response 1 (SOG1), a NAM, ATAF1, 2, and CUC2 (NAC)-type transcription factor found in seed plants, is a master regulator of DNA damage responses (DDRs). Upon DNA damage, SOG1 regulates the expression of downstream DDR genes. To know the origin of the DDR network in land plants, we searched for a homolog(s) of SOG1 in a moss Physcomitrium (Physcomitrella) patens and identified PpSOG1a and PpSOG1b. To assess if either or both of them function(s) in DDR, we knocked out the PpSOG1s using CRISPR/Cas9-mediated gene editing and analyzed the responses to DNA-damaging treatments. The double-knockout (KO) sog1a sog1b plants showed resistance to γ-rays, bleomycin, and ultraviolet B (UVB) treatments similarly seen in Arabidopsis sog1 plants. Next, we irradiated wild-type (WT) and KO plants with γ-rays and analyzed the whole transcriptome to examine the effect on the expression of DDR genes. The results revealed that many P. patens genes involved in the checkpoint, DNA repair, replication, and cell cycle-related genes were upregulated after γ-irradiation, which was not seen in sog1a sog1b plant. These results suggest that PpSOG1a and PpSOG1b work redundantly on DDR response in P. patens; in addition, plant-specific DDR systems had been established before the emergence of vascular plants.

Molecular and Biochemical Differences in Leaf Explants and the Implication for Regeneration Ability in Rorippa aquatica (Brassicaceae).

Plants have a high regeneration capacity and some plant species can regenerate clone plants, called plantlets, from detached vegetative organs. We previously outlined the molecular mechanisms underlying plantlet regeneration from Rorippa aquatica (Brassicaceae) leaf explants. However, the fundamental difference between the plant species that can and cannot regenerate plantlets from vegetative organs remains unclear. Here, we hypothesized that the viability of leaf explants is a key factor affecting the regeneration capacity of R. aquatica. To test this hypothesis, the viability of R. aquatica and Arabidopsis thaliana leaf explants were compared, with respect to the maintenance of photosynthetic activity, senescence, and immune response. Time-course analyses of photosynthetic activity revealed that R. aquatica leaf explants can survive longer than those of A. thaliana. Endogenous abscisic acid (ABA) and jasmonic acid (JA) were found at low levels in leaf explant of R. aquatica. Time-course transcriptome analysis of R. aquatica and A. thaliana leaf explants suggested that senescence was suppressed at the transcriptional level in R. aquatica. Application of exogenous ABA reduced the efficiency of plantlet regeneration. Overall, our results propose that in nature, plant species that can regenerate in nature can survive for a long time.