Receptor for advanced glycation end-products (RAGE) is a member of the immunoglobulin superfamily. RAGE binds and mediates cellular responses to a range of DAMPs (damage-associated molecular pattern molecules), such as AGEs, HMGB1, and S100/calgranulins, and as an innate immune sensor, can recognize microbial PAMPs (pathogen-associated molecular pattern molecules), including bacterial LPS, bacterial DNA, and viral and parasitic proteins. RAGE and its ligands stimulate the activations of diverse pathways, such as p38MAPK, ERK1/2, Cdc42/Rac, and JNK, and trigger cascades of diverse signaling events that are involved in a wide spectrum of diseases, including diabetes mellitus, inflammatory, vascular and neurodegenerative diseases, atherothrombosis, and cancer. Thus, the targeted inhibition of RAGE or its ligands is considered an important strategy for the treatment of cancer and chronic inflammatory diseases. RAGE (receptor for advanced glycation end-products) was first isolated from the human lung library in 1992 and noted for its ability to act as a receptor for advanced glycation end products (AGEs). RAGE is a transmembrane protein of the immunoglobulin (Ig) superfamily of cell surface molecules ,11,12. LRAGE is a 50–55 kDa glycosylated protein that contains an extracellular (amino acids 23–342), a hydrophobic transmembrane (residues 343–363), and a cytoplasmic (residues 363–404) domain. The extracellular structure of RAGE is composed of a variable (V) immunoglobulin (Ig) domain (residues 23–116) and two constant C1 (residues 124–221) connected to C2 (residues 227–317) Ig domains by a flexible seven amino acid linker . The molThe human RAGE gene is located on chromosome 6 in the MHC (major histocompatibility complex) class III region, which contains many genes that impact the adaptive and innate immune systems . More thRAGE binds diverse classes of ligands, such as HMGB1, S100 calcium-binding protein/calgranulin, amyloid-β, and lysophosphatidic acid (LPA) ,45,46,47ɛN-carboxymethyl-lysine (CML) and ɛN-carboxyethyl-lysine (CEL) are found in human tissue and blood plasma and bind to the V domain of RAGE. Interactions between AGEs and RAGE induce the expressions of pro-inflammatory cytokines and chemokines, such as TNF-α, IL-1β, and CCL2 [N-terminal kinase (JNK) in vitro [Many types of AGE have been identified, and aging leads to accumulations of AGEs in tissues and plasma . Nɛ-carband CCL2 ,73, and and CCL2 . Methylgin vitro .The S100 protein family contains 25 members with different expression patterns, functions, and oligomeric states, and S100B, S100A1, S100A2, S100A4, S100A5, S100A5, S100A6, S100A7, S100A8/A9, S100A11, S100A12, and S100P have been shown to interact with RAGE in vivo . S100 prHMGB1 is a highly conserved nuclear protein that acts as a chromatin-binding factor, and this binding leads to the bending of DNA and the promotion of DNA transcription . HMGB1 iQuinolinic acid, a neuroactive metabolite of the kynurenine pathway, is an agonist of the N-methyl-D-aspartate (NMDA) receptor and is normally present at nanomolar concentrations in human brain and cerebrospinal fluid (CSF). Excessive quinolinic acid levels have been implicated in a variety of human neurological diseases, including AD and Huntington’s disease . The dirEndothelial RAGE interacts with Mac-1 on leukocytes , and OrlLysophosphatidic acid (LPA) is a serum phospholipid with growth factor-like activities in many cell types. LPA stimulates cell migration, proliferation, and survival by acting on its cognate G-protein-coupled receptors . FurtherPhosphatidylserine (PS) is a structural component of nuclear envelopes, endoplasmic reticulum, the inner cytosolic region of plasma membranes, myelin, and Golgi apparatus . Cells uComplement protein C1q binds to microbial surfaces or immune complexes, and thus, stimulates the complementation system and the productions of membrane lytic complexes, opsonins, and anaphylatoxins. Surface plasmon resonance (SPR) showed RAGE and C1q interact directly with a Kd of 5.6 μM, and this interaction plays a role in adaptive immunity and promotes C1q-mediated phagocytosis .The recognition of DNA and RNA derived from hosts or pathogens is one way the innate immune system responds to infection and tissue damage. RAGE promotes DNA uptake by endosomes and augments TNR response through TLR9 . TIRAP aHaemaphysalis longicornis also binds to RAGE. Longistatin acts as a RAGE antagonist, suppresses NF-κB translocation, and thus, hosts immune response [RAGE plays a role in immune reactions by recognizing and responding to various PAMPs, including bacterial lipopolysaccharide (LPS), bacterial DNA, and viral and parasitic proteins. LPS is a major component of the cell walls of Gram-negative bacteria and initiates inflammatory cascades leading to sepsis. RAGE directly interacts with LPS and is involved in LPS-induced NF-κB activation and endothelial hyperpermeability ,153. Theresponse .Although many extracellular ligands interact with the VC1 domain of RAGE, the cytoplasmic domain of RAGE plays a vital role in RAGE-mediated signaling and overall RAGE function. RAGE activates diverse intracellular signaling pathways, including those of p38MAPK, AKT, ERK, mammalian diaphanous 1 (mDia1), and Rho GTPase , and these pathways activate cascade transcription factors, such as NF-κB, SP-1, STAT3, and EGR-1 ,51,154. The cytoplasmic domain of RAGE binds with formin homology domain-1 (FH-1) of mDia1 , and theIshihara et al. revealed by immunoprecipitation that ERK1/2 interacts with the cytoplasmic region of RAGE in HT1080 cells . The relRAGE overexpression and activation are hallmarks of various diseases, including neurodegenerative, cardiovascular, vascular, and coronary diseases and atherosclerosis, diabetes, retinopathy, and cancer ,167,168.RAGE and its ligands accumulate in acute inflammatory conditions, such as diabetes, atherosclerosis, and nondiabetic vascular disease ,171,172.RAGE has been reported to be elevated in human brain tissue in neurological disorders, including AD, Huntington’s disease, Parkinson’s disease, and schizophrenia ,180,181.RAGE has been implicated in the pathogeneses of breast, bladder, hepatic, pancreatic, colorectal, gastric, and lung cancer, glioma, and melanoma ,191,192.Endothelial dysfunctions involve of the extracellular matrix (ECM) enzymes lysyl oxidase (LOX) and endothelin-1 (ET-1). At the gene level, the expressions of these enzymes are regulated by transcription factors such as NF-κB and AP-1. In human endothelial cells, AGE/RAGE increased the expressions of LOX and ET-1 through the AGE/RAGE/MAPK signaling cascade, which disrupted endothelial homeostasis by promoting cellular proliferation, altering the biomechanical properties of ECM, and impairing endothelial barrier functions . In addiRAGE and its ligands are also involved in coronary artery disease (CAD). sRAGE elevates acute ischemia and acts as a potential biomarker of acute coronary syndrome (ACS) . MoreoveRetinal microvascular dysfunction is a major component of diabetic retinopathy. RAGE plays a critical role in Müller glial activation and the downstream cytokine production associated with diabetic retinopathy . In mousObesity increases the risks of cardiovascular disease, hypertension, diabetes, and cancer. S100A4, S100A8/9, S100A12, and S100B act as DAMPs, activate receptors such as RAGE and TLR-4, and promote macrophage-based inflammation . FurtherElevated RAGE expression in lung alveolar epithelial type 1 (AT1) cells may be involved in the proliferation and differentiation of pulmonary epithelial cells . RAGE is2α [1c (HbA1C) levels. In addition, the -374T/A allele has been shown to affect gene transcription and to be a potential marker of vascular disease [The gene coding for RAGE is located within the gene-dense major histocompatibility class III region on chromosome 6, which contains numerous genes involved in immune and inflammatory responses . At leas2α . The pro disease .Although RAGE is greatly expressed in adult lung tissues, RAGE knockout (RAGE-KO) mice do not exhibit pulmonary changes associated with life expectancy . HoweversRAGE administration dose-dependently reduced aortic atherosclerotic lesion sizes, numbers, and complexities ,218, andInterestingly, RAGE KO mice are healthy and developmentally normal, which suggests RAGE knockdown might be a safe therapeutic strategy . FurtherN-benzyl-N-cyclohexyl-4-chlorobenzamide) inhibits the interaction between the RAGE V domain and Aβ and the ability of Aβ to cross the BBB. FPS-ZM1 acts to block RAGE-mediated inflammatory signaling and inhibits nuclear NF-κB levels and BACE1, a key enzyme involved in the generation of Aβ in mouse brains. In addition, FPS-ZM1 had no toxic side effects in mice at doses up to 500 mg/kg [On the other hand, FPS-ZM1  is an AGE cross-link breaker, and treatment with alagebrium reduced AGE accumulation and atherosclerotic plaque formation and lesions . AGE-RAGThe intracellular domain of RAGE is required for many types of RAGE signals and for inducing downstream effects, and the disruption of RAGE-mDia1 binding is important for the therapeutic targeting of RAGE-mediated chronic inflammatory diseases. In one study, 13 compounds with high affinity for the cytoplasmic domain of RAGE (ctRAGE) inhibited the interaction between ctRAGE and mDia1 .Recently found is that Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP76) interacts with a cytosolic tail of RAGE, resulting in prompt activation of downstream signaling and gene transcription of pro-inflammatory mediators, such as TNF, CXCL10, HMGB1, and IL-6 in HEK 293 cells. Therapeutic targeting of interaction SLP76 with RAGE as a new approach for lethal sepsis .Zheng et al. reported that aptamer-based antagonist against RAGE inhibits tumor growth and microvasculature formation in colorectal tumor mice by suppression of the RAGE/NF-κB/VEGF-A signaling .RAGE is a multi-ligand receptor of the immunoglobulin family. RAGE and its ligands are present on most cell types and are involved in diabetes, diabetes complications, chronic inflammation, neurodegenerative disorders, and cancer. Recent advances have revealed the enormous breadth of the influence of RAGE and its ligands. Although, sRAGE and esRAGE act as biomarkers and endogenous protection factors against RAGE-mediated pathologies, sRAGE and esRAGE may not be ideal therapeutic means for targeting RAGE because they are large recombinant proteins that are difficult to produce at therapeutic levels. Accordingly, small-molecular inhibitors have been developed to target the extracellular ligand-binding site of RAGE and its intracellular signaling pathway. Further, there are critical issues that remain to be addressed of the understanding of RAGE-targeting therapy and the long-term impact of RAGE blockade in humans. Future investigations are required to improve understanding of the characteristics of efficient RAGE inhibitors to develop a significant understanding of the impact of RAGE blockage.


CFAP61 variants, c.451_452del (p.I151Nfs*4) in family 1 and c.847C > T (p.R283*) in family 2 and 3, were identified recessively co-segregating with the MMAF phenotype. Transmission electron microscopy analyses revealed severe disorganized axonemal ultrastructures, and missings of central pair, RSs, and inner dynein arms were also observed and confirmed by immunofluorescence staining in spermatozoa from patients. CFAP61 and CFAP251 signals were absent from sperm tails of the patients, which suggested the loss of functional CSC in sperm flagella. Altogether, our findings report that homozygous variants in CFAP61 are associated with MMAF and male infertility, demonstrating the essential role of this gene in normal sperm flagellum structure in humans.Multiple morphological abnormalities of the flagella (MMAF) can lead to male infertility due to impaired sperm motility and morphology. Calmodulin- and spoke-associated complex (CSC) are known for their roles in radial spoke (RS) assembly and ciliary motility in Chlamydomonas, while the role of cilia- and flagella-associated protein 61 (CFAP61), a mammalian ortholog of the CSC subunits, in humans is yet unknown. Here, we recruited three unrelated Pakistani families comprising of 11 infertile male patients diagnosed with MMAF.  CFAP43, CFAP44, CFAP47, CFAP65, CFAP69, CFAP70, CFAP91, CFAP251, DNAH17 etc . Disruption of CSC proteins in C. reinhardtii could lead to impaired flagellar motility and axoneme structure defects, including partially loss of radial spokes and inner dynein arms (IDAs)  is known for its essential role in radial spoke (RS) assembly and ciliary/flagellar motility . CSC pros (IDAs) . Cilia- proteins . All theproteins , suggestividuals . Moreoveertility . NonetheCFAP61 responsible for MMAF in one and two families, respectively. Transmission electron microscopy (TEM) analyses and immunofluorescence (IF) staining revealed that these variants led to severely disorganized ultrastructures of sperm flagella with loss of CP, RSs, and IDAs in patients. Interestingly, the signals of CSC proteins CFAP61 and CFAP251 were absent from flagella of the patients. These observations suggested that these variants in CFAP61 are pathogenic for MMAF and human male infertility.In this study, we recruited three unrelated Pakistani families, having 11 primary infertile individuals diagnosed with MMAF without manifesting any primary ciliary dyskinesia (PCD) symptoms. WES and bioinformatics analyses identified a homozygous frameshift variant  and a homozygous stop-gain variant  in This study was approved by the Ethical Committee of University of Science and Technology of China (USTC). Written informed consent forms from all participants were obtained at the beginning of the study.2O once. and put into 70% ethanol with 1% HCl for 2 s. After washing in tap water for 3 min, the slides were gradiently dehydrated by ethanol. Then the smears were stained with eosin and then placed in the baths of 100% ethanol and xylene. After sealed with neutral balsam, the smears were observed under a light microscope. Morphological analysis was performed for P1-P9 with at least 200 spermatozoa for each experiment examined . The semen samples for fertile control were obtained from the volunteers at the First Affiliated Hospital of USTC. A detailed questionnaire regarding PCD clinical features was obtained from all available family members and all excluded the history or presence of ciliary-related symptoms, including situs ambiguous, unexplained chronic airway infections, respiratory distress at newborn, chronic middle ear infections, clubbing, hydrocephalus etc .This study recruited three Pakistani families, family 1, 2 and 3, having four (P1-P4), four (P5-P8), and three (P9-P11) infertile brothers, respectively. Semen analysis, including semen volume, sperm concentration, and sperm motility, were performed at least twice for P1-P5, P7, P9, and P10 according to the World Health Organization (WHO) guidelines . Semen sQIAamp Blood DNA Mini Kit (QIAGEN) was used to extract the blood genomic DNA from available family members. For WES, AIExome Enrichment Kit V1 -captured libraries were constructed for family members of Family 1 , Family 2 , and Family 3 (III:2 and IV:1) as instructed by the manufacturer. Sequencing was carried out on a Hiseq2000 platform . Variants following recessive inheritance were kept for further screening following the strategy as we described previously , and as Fresh semen samples were obtained and centrifuged at 900 x g for 3 min. After washing with PBS for three times, spermatozoa were fixed in 0.1 M phosphate buffer (pH 7.4) containing 4% glutaraldehyde, 4% paraformaldehyde, and 0.2% picric acid at 4°C for at least overnight. Scanning electron microscopy (SEM) and TEM analyses were conducted as we previously described .IF staining were performed as we previously reported . BrieflyEleven infertile men from three unrelated Pakistani families were investigated in the current study. Family 1 has four infertile brothers, P1, P2, P3, and P4; family 2 has four infertile brothers, P5, P6, P7, and; family 3 has three infertile brothers, P9, P10, and P11, born to a consanguineous marriage . All theThe morphology of spermatozoa from P1-P9 was further assessed by H&E staining of semen smears. Comparing to the long and threadlike flagella observed in a fertile control, more than 82.2% of the spermatozoa in P1-P9 displayed malformed morphologies of flagella, including absent, short, coiled, bent, and irregular caliber, which are typical presentations of MMAF . MoreoveIn addition, all the patients declared they have no ciliary-related clinical features  and none of them wish to participate in any further related examinations. Altogether, all the 11 patients exhibited male infertility due to asthenozoospermia and/or teratozoospermia associated with MMAF.CFAP61 (M1), NM_015585.4: c.451_452del (NP_056400.3: p. I151Nfs*13), in family 1, and a homozygous CFAP61 stop-gain variant (M2), NM_015585.4: c.847C > T (NP_056400.3: p. R283*) in family 2 . Among the seven different ethnic groups of gnomAD, the allele frequency of M2 is 0.00023 in South Asians, 0.00016 in East Asians, and was absent in the other ethnic groups. Subsequent Sanger sequencing of all the available family members confirmed that the CFAP61 variants were recessively inherited from their heterozygous parents except P11 whose sperm morphology was not yet assessed , mitochondrial sheath (MS), and central axoneme (CFAP91 variants displayed defects in ultrastructure of sperm flagella including absence of CP, and IF staining also showed partially loss of CP protein SPAG6 and RS protein RSPH1 (CFAP61 variants showed severe disorganizations of the axonemal structures with loss of CP, RSs, and IDAs, which was confirmed by IF staining of SPAG6, TSGA2, and DNAH1. The IF results showed a total absence of SPAG6 and TSGA2, which is more severe than the observations in patients carrying CFAP91 variants (CFAP61 variants, indicating that CFAP61 is essential for the localization of CFAP251 in sperm flagella. Altogether, it is suggested that CFAP61 may play a more predominant role in CSC.Among the other three CSC protein members, variants in ith MMAF . Similar axoneme . Though in RSPH1 . In our variants . FurtherT. brucei cells (Interestingly, we found that about 17% of cross-sections displayed misoriented CPs, in contrast to CPs paralleling to the axis of the two longitudinal columns of the FS observed in controls. It was reported that RS is important for the anchorage of the CP, and mutation in RS proteins, like RSPH4A or RSPH9, will cause the rotation of CP, which might lead to the degeneration of CP . In the ei cells . Thus, wCFAP61 affect the normal assembly of CP, RSs, and IDAs, leading to MMAF in humans. These findings will expand the current understanding in the disease-causing mutations and the associated flagellar phenotypes of MMAF and provide guidance for genetic counseling and diagnosis of the disease.Altogether, our data demonstrate that CFAP61 is essential for normal sperm flagellum structure and function and variants in


The plant kingdom is a rich source of secondary metabolites with numerous properties, including the potential to modify keratinocyte biology. Keratinocytes are important epithelial cells that play a protective role against various chemical, physical and biological stimuli, and participate in reactive oxygen scavenging and inflammation and wound healing processes. The epidermal cell response may be modulated by phytochemicals via changes in signal transduction pathways. Plant extracts and single secondary compounds can possess a high antioxidant capacity and may suppress reactive oxygen species release, inhibit pro-apoptotic proteins and apoptosis and activate antioxidant enzymes in keratinocytes. Moreover, selected plant extracts and single compounds also exhibit anti-inflammatory properties and exposure may result in limited production of adhesion molecules, pro-inflammatory cytokines and chemokines in keratinocytes. In addition, plant extracts and single compounds may promote keratinocyte motility and proliferation via the regulation of growth factor production and enhance wound healing. While such plant compounds may modulate keratinocyte functions, further in vitro and in vivo studies are needed on their mechanisms of action, and more specific toxicity and clinical studies are needed to ensure their effectiveness and safety for use on human skin. Plants produce a range of secondary metabolites. These not only play a crucial role in the adaptation of plants to the environment but also have a strong impact on other living organisms, including humans. Various groups of molecules with a plant origin such as phenolics, alkaloids, saponins, terpenes, lipids and carbohydrates, are involved in a plethora of biological activities. Selected phytochemicals may modulate several cell-signaling pathways in different cell types, including those of epidermal cells .The skin creates a barrier protecting the host from the outside environment. The outer layer of the human skin, the epidermis, is formed by keratinocytes. These cells are on the first line of defense against various harmful chemical, physical and biological factors and are particularly vulnerable to ultraviolet (UV)-radiation, a strong source of reactive oxygen species (ROS). In response, keratinocytes produce a range of biologically active molecules that enhance ROS scavenging, inflammatory responses and wound healing .2O2) [Numerous plant extracts, as well as their component compounds, exert antioxidant activity. Phenolic compounds are particularly effective ROS scavengers. These chemicals are able to neutralize ROS via their ability to donate hydrogen atoms or electrons to radicals and chelate metal cations. Selected plant extracts and their component compounds have been found to suppress ROS release, decrease the activity of inducible nitric oxide synthase and nitric oxide, inhibit pro-apoptotic proteins and apoptosis and activate antioxidant enzymes in keratinocytes following exposure to hydrogen peroxide (H2O2) . A wide 2O2) . Moreove2O2) ,6. In ad2O2) .The aim of the present paper is to review the role of selected plant extracts and single compounds as modulators of keratinocyte biology, paying particular attention to stimuli, specific receptors, protein release and signal transduction pathways. It examines the potential of phytochemicals as ROS scavengers, important anti-inflammatory agents and as factors for mediating wound healing. The role, contributions and usefulness of plants were summarized.To give an overview of the current state of knowledge regarding the topic, i.e., in vitro treatment of human keratinocytes with oxidants, pro-inflammatory agents and UV radiation followed by exposure to different plant extracts, papers were selected from those included in the electronic databases PubMed/MEDLINE, Google Scholar, Scopus and Web of Science over the past 10 years. The following search terms were used: keratinocytes, plant extract, plant-derived compounds, reactive oxygen species, inflammation, UV-irradiation and wound healing. Moreover, the impact of plant secondary compounds on keratinocyte motility and proliferation rate was evaluated. Papers reporting articles published in languages other than English, those with only an abstract or lacking full-text access, those published earlier than 10 years ago, those without identified compounds in extracts and those examining cells other than human keratinocytes were excluded. Each selected document was analyzed and the following data were extracted and presented in tables: species name, plant part, type of extract, type of cell lines, identified compounds, mechanism of action and final effect. The main text includes a characterization of the signaling cascades involved in keratinocyte biology.The plant kingdom is a rich source of secondary metabolites. Over 50,000 molecules have been discovered . These mSecondary metabolites may be obtained from the leaves, roots, stems, bark and aerial parts of the plants; however, the plants require careful preparation, extraction and quantitative and qualitative determination of active compounds. The first step involves the selection of an appropriate solvent. The most popular are polar ones such as water and alcohols, intermediate polar ones such as acetone and dichloromethane, and nonpolar ones such as chloroform, n-hexane and ether. Following this, an appropriate extraction method must be identified. Various options exist including maceration, decoction, digestion, percolation, infusion, Soxhlet extraction, superficial extraction, microwave-assisted extractions or ultrasound-assisted extraction. The isolated compounds must then be purified via chromatographic methods and identified via spectroscopy . HoweverThe skin is the largest organ in the human body. In addition to serving as a barrier for the inner environment, it also regulates body temperature, enhances metabolic functions and enables contact with the outer environment by its host of nerve endings.Human skin consists of three layers: an outer stratified epithelium, a middle dermis and an underlying subcutaneous tissue. The epidermis is formed mostly (at least 80%) by keratinocytes. Newly-synthesized keratinocytes build up a supply of keratin in the cytoplasm and then undergo structural and biochemical changes until terminal differentiation and cell death. Keratinocytes play numerous roles including the creation of a physical barrier. That barrier provides protection against water loss, biological agents such as pathogens, physical agents such as UV radiation and various chemical agents. Moreover, epithelial cells accumulate melanin derived from melanocytes, migrate and proliferate to heal wounds when skin integrity is disrupted and participate in skin immunity. In addition, keratinocytes are involved in molecular interactions between various surrounding skin cells via different signal transduction pathways ,22.During normal epidermal differentiation, the following types of keratinocytes are distinguished: basal, spinous, granular and cornified. These types vary in their phenotypic and biochemical properties based on changes in gene expression. Many of these genes are regulated by p63 transcription factors, including keratins, involucrin and loricrin . Basal cSpinous keratinocytes begin to express keratins K1 and K10, and this expression continues fully in the granular forms. They also express involucrin and loricrin, the precursors of cornified envelope proteins. These play a role in corneocyte formation, the final stage of differentiation preceded by loss of nuclei and organelles. The corneocytes are connected by corneodesmosomes and covered with lipid layers secured by a protein structure. These form a barrier to prevent water evaporation .The differentiation process of keratinocytes is regulated by the mitogen-activated protein kinase (MAPK) pathway, which is induced by various stimuli including epidermal growth factor (EGF), tumor necrosis factor (TNF) and calcium influx . The sigBeing located directly on the edge of the internal and external environment, epidermal keratinocytes are exposed to mechanical stress. Stretching the skin results in hyperproliferation via the induction of calcium influx followed by phosphorylation of essential growth-related factors, including the epidermal growth factor receptor (EGFR) . MoreoveUV radiation is the main source of ROS in keratinocytes, with heat shock and drugs being others. Oxidative stress may be involved in the inflammatory condition and apoptosis of the skin. In addition, ROS may modulate various signaling cascades in human cells in vitro. ROS have also been found to mediate MAPK activities, and an oxidant state has been associated with an elevated level of activator protein 1 (AP-1) and NFκB in mouse keratinocytes . However2O2-stimulated human keratinocytes. Cellular exposure to H2O2 is strictly connected with ROS induction; therefore, the presented phytochemicals exhibit important antioxidant properties such as suppressing ROS release, limiting damage to DNA and lipid oxidation, inhibiting iNOS and NO, decreasing the level of pro-apoptotic proteins and apoptosis and activating antioxidant enzymes.Many plants’ secondary metabolites, especially phenolic compounds, act as antioxidants. This has been attributed to their ability to donate hydrogen atoms or electrons to radicals to form nonreactive phenoxyl radicals or stable radical cations, respectively . They arUnperturbed keratinocytes do not release inflammatory mediators; however, their expression may be enhanced by various stimuli, including UV radiation, wounding, pathogens and pathogen products. ROS play a special role in skin inflammation and may trigger a chronic skin response. Following the activation, the upregulation of the adhesion molecule, cytokine and chemokine expression is observed .Adhesion molecules that belong to the immunoglobin superfamily, including intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) play an essential role in inflammatory mechanisms. ICAM-1 is a ligand for the B-2 integrin leukocyte function-associated antigen-1 (LFA-1). This molecule promotes cellular interaction in the immune response. ICAM-1 and LFA-1 interaction is essential for T lymphocytes activation. Elevated ICAM-1 expression is observed in keratinocytes after interferon-gamma (IFN-γ) exposure. Tumor necrosis factor-α (TNF-α) and IL-17 act synergistically with INF- γ in the induction of ICAM-1 expression. VCAM-1 interacts with various integrins .Cytokine action is initiated by the activation of NFκB pathways by Toll-Like Receptors (TLRs), which recognize pathogen-associated molecular patterns . It has IL-1, -6, -8 and TNF-α are well-known pro-inflammatory cytokines. IL-1 acts as a chemoattractant for keratinocytes , activatTNFα plays a differential role in epidermal keratinocytes. On the one hand, TNFα activates modulators of the actin cytoskeleton and integrins, regulates their degree of motility or attachment and stimulates the expression of collagen-degrading proteases and basement membrane components, followed by tissue repair. On the other hand, TNFα stimulates the nuclear translocation of NFκB. It is also believed to induce the expression of various genes, including those of cytokines, chemokines, growth factors and cell-surface receptors that may attract neutrophils, macrophages and T lymphocytes. Additionally, genes regulated by TNFα are involved in the modulation of the cell cycle and apoptosis . IL-7 and IL-15 are involved in T cell homeostasis. IL-7 participates in the development of mature T lymphocytes in the thymus. In addition, IL-7 also induces the expression of the anti-apoptotic protein Bcl-2, followed by the survival of both naïve and memory T lymphocytes. IL-15 stimulates the proliferation of CD8+ T cells in a process independent of antigens. While both cytokines may help to defend organisms against pathogens and tumors, elevated production may be linked to several autoimmune diseases . ResearcCytokines IL-10, IL-12 and IL-18 act as immunomodulators. IL-10 stimulates the Th2 immune response  and its IL-20 induces keratinocyte hyperproliferation and therefore may act as a modulator of skin inflammation . Its relPlant extracts and their component compounds may modulate the inflammatory keratinocyte response in vitro via different mechanisms. Keratinocytes exposed to TNF-α or IFN-γ exhibit an elevated production of pro-inflammatory cytokines. To counteract this, treatment with plant extracts may downregulate the expression of ICAM-1, IL-1, IL-6, IL-8, TNF-α and selected chemokines, and suppress the NFκB and MAPKs pathways. Ampelopsis glandulosa [andulosa  extract Cudrania tricuspidate: dihydrokaempferol, steppogenin, cudraflavanone D, cudraflavanone B, cudraflavone C, kuwanon C, cudraxanthone L, macluraxanthone B, 1,6,7-trihydroxy-2--3- methoxyxanthone, cudratricusxanthone L, cudracuspixanthone A [Ambrosia arborescens [Artemisia princeps [Studies based on single compounds indicate that HaCaT cells stimulated with TNF-α/IFN-γ exhibit a decreased IL-6, IL-8, ICAM-1 and selected chemokine production, suppression of NFκB translocation to the nucleus and lowered p38 and ERK1/2 activity following treatment with selected compounds derived from nthone A . Elsewheorescens . In addiprinceps .UV radiation is an environmental-damaging agent that causes oxidative stress, resulting in damage to cellular components and apoptosis. It is also known to induce inflammatory conditions that lead to skin disorders. Keratinocytes are the first line of defense against UV radiation . ExposurEgfr knockout mice are able to stay alive for only a few days after birth [Following exposure to UV radiation , keratinocytes prepared from newborn CD-1 mouse skin demonstrated EGFR receptor activation by phosphorylation of Tyr-992/Tyr-1045/Tyr-1068/Tyr-1173 residues via mechanisms associated with ROS. The EGFR receptor is strongly mitogenic and directly regulates the response of keratinocytes to UV radiation . These cUVA and UVB exposure induces different groups of factors. UVB exposure induces the activation of c-Jun amino-terminal kinases (JNK), a member of the MAPKs in NHEKs , as wellNHEKs and immortalized human HaCaT keratinocytes also exhibit MAPKs signaling activation following exposure to both UVA and UVB radiation. The HaCaT cells demonstrate two-fold or even four-fold higher activity of p38 and JNK than NHEK, but the latter release more TNFα. The data also indicate that immortalized cells do not demonstrate NFκB pathway induction . HaCaT keratinocytes exposed to both UVA and UVB radiation exhibit different gene expression profiles depending on a single or repetitive dose. Exposure to a single dose was associated with upregulation of the G/S checkpoint of the cell cycle, as well as NFκB, IL-1, death receptor and p38 signaling and downregulation of genes that regulate G2/M checkpoint of cell cycle and ATM serine/threonine kinase signaling. Additionally, numerous regulators including forkhead box M1 (FOXM1), forkhead box O1 (FOXO1), activating transcription factor 4 (ATF4), ATF6, melanocyte inducing transcription factor (MITF) and ETS homologous factor (EHF) were inhibited, while lysine demethylase 5B (KDM5B), SMAD family member 4 (SMAD4), tumor protein p63 (TP63), nuclear protein 1, transcriptional regulator (NUPR1), cyclin-dependent kinase inhibitor 2A (CDKN2A), NOTCH11 and STAT3 were activated. After repeated doses, interferon signaling was downregulated. Specific regulators such as FOXM1, TP73, NFE2 like bZIP transcription factor 2 (NFE2L2), MITF and EHF were inhibited, whereas STAT3, NUPR1, chromobox 5 (CBX5), ATF3, SMAD4 and MYC proto-oncogene, bHLH transcription factor (MYC), were activated . In addiExposure to UV radiation also causes collagen destruction. The enzymes responsible for such effects are named matrix metalloproteinases (MMPs). In vivo data indicate that UV irradiation induces the expression of MMP-1, MMP-3 and MMP-9 mainly in the human epidermis . KeratinHuman keratinocytes exposed to UV radiation result in ROS accumulation, as well as apoptosis by the intrinsic and extrinsic pathways . InductiUV-induced HaCaT cells express the TNF-family receptor CD95 and may undergo apoptosis via the extrinsic pathway . The datw/w) Euterpe oleracea extract demonstrated a PF-UVA value  of about 15 and a ratio of SPF  to PF-UVA of about 1.5 [Helichrysum arenarium, Sambucus nigra and Crataegus monogyna. All of them exhibit SPF values above 6 when used alone in O/W emulsion at 10% (wt.%), and above 16.5 and PF-UVA above 7.5 when used in combination [Hylocereus polyrhizus at 1 mg/mL demonstrated an SPF of about 35 [Pterodon emarginatus extract at 10% (w/w) in three different formulations  exhibited an SPF value of about 8 [w/w) extract of Olea europaea standardized to 20% oleuropein exhibited an SPF value of about 21 used in the special formulation [Washingtonia filifera exhibits an SPF value of about 3.4 [Salicornia ramosissima extract at 10% (w/w) was found to have both SPF and PF-UVA values of about 13.5 when applied as O/W emulsion [Vitis vinifera extract at a concentration of 250 μg/mL had an SPF of about 18.6 and a PF-UVA of about 3.2 [Plant-derived compounds may counteract the detrimental effects of UV exposure on human skin. Data indicate that oil in water (O/W) emulsion containing 5%  O/W emulsion, whereas PF-UVA provides a value of about 14.5 [w/w) rutin with 6% benzophenone, a synthetic organic filter, increased the SPF value from about 24 to 33 [w/w), 1% benzophenone (w/w) and 3.5% (w/w) ethylhexyl methoxycinnamate [The most important group of compounds that may be used as sunscreen agents are phenolic compounds. These molecules equipped with aromatic rings are able to absorb UVA and UVB radiation at wavelengths of 200–400 nm and scavenge ROS and modulate signaling processes. Two flavonoids, rutin and quercitin, provide SPF values of about 12 when used in 10% (out 14.5 . Combini24 to 33 . Similarinnamate . Anotherinnamate . w/v) concentrations , the highest SPF value was observed for apigenin, i.e., about 29 [Choquenet tested twelve phenolic compounds at various concentrations, including myricetin, luteolin, apigenin, puerarin, baicalin, baicalein, hesperidin, hesperetin, naringenin, diosmin, caffeic acid and chlorogenic acid. Of these, chlorogenic acid and apigenin were found to be the best UVB and UVA filters, with SPF values at about 10 and 7 and PF-UVA values at about 9 and 6, respectively . Similarabout 29 . The plant extracts and single compounds may also modulate the UV-radiated keratinocytes response via different mechanisms. Both may downregulate the ROS level and upregulate the antioxidative enzyme level in keratinocytes exposed to UV. Typically, the nuclear factor erythroid 2–related factor 2 (Nrf-2) pathway is induced, with NF-κB, p53, MAPK and Akt signaling being reduced. In addition, MMPs and prooxidative cytokine expression are suppressed. Rhus javanica extract suppresses COX-2 expression in mice exposed to UVB and has anti-wrinkle effects [Rosa multiflora extract reduces TNF-α and MMP-13 production [Paeonia × suffruticosa extract reduces MMP-1 production in mice exposed to UVB [Moringa oleifera extract is found to protect against UVB-induced oxidative stress injuries in the epidermis of the mouse in vivo. PPARα induction is involved in the protective effect of the extract [Mice treated orally with a red raspberry extract prior to exposure to UVB irradiation demonstrate less skin wrinkling, water loss and epidermal thickening in comparison to those that were not treated . Rhus ja effects . In addioduction , and Paed to UVB . Moringa extract .Atriplex gmelinii [Nymphoides indica [Youngia denticulatum [Ixeris dentata [Garcinia mangostana [Artocarpus communis exhibited reduced cell damage in contrast to untreated ones [Studies have examined the protective roles of single compounds on epidermal cells, including 3,5-dicaffeoyl-epiquinic acid obtained from gmelinii , quercets indica , youngiaiculatum , ixeriso dentata , α-, β-,ngostana . Of thested ones .Wound healing occurs as a consequence of skin barrier disruption. It is a complex process consisting of a series of phases, including hemostasis, inflammation, proliferation and remodeling . The firKeratinocytes migrate, proliferate, and cross-talk with fibroblasts following wound contraction . ActivatDesmosomes are structures that are essential for cell–cell adhesion and skin integrity. The core desmosome protein is named desmoplakin. Desmosomal adhesion in response to wounding is regulated by PKCα . It has Hemidesmosomes are structures that link cells into the basal layer and are mediated by integrins . IntegriNumerous other regulators take part in the process of keratinocyte migration and proliferation, including various growth factors from the tyrosine kinase, insulin, FGF, vascular endothelial growth factor and scatter factor families . In addiThe epidermal growth factor family of receptor tyrosine kinases (HER) are also known to play a role in keratinocyte activity. Keratinocytes express 1, 2 and 3 HER receptors. Their ligand, including epidermal growth factor (EGF), heparin-binding EGF like growth factor (HB-EGF) and TGF-α stimulate keratinocyte migration  and prolMoreover, insulin secreted by β-cells in the pancreas is able to circulate into the skin, where it can then bind to insulin receptors (IR) expressed in skin keratinocytes. Insulin mediates keratinocyte migration and proliferation. A combination of insulin and TGF-α was found to act synergistically in this regard. An in vivo study in a mouse model found that topical application of insulin results in accelerated wound healing . A similAdditionally, keratinocyte insulin-like growth factor 1 (IGF-1) receptor and its ligand, IGF-1, synthesized by fibroblasts or hepatocytes, has been found to stimulate the motogenic effect in keratinocytes. That signaling is downregulated in diabetic wounds. IGF-1 and EGF act synergistically; the first by induction of the PI3K pathway, the second by the MAPK/ERK pathway . IGF-1 aFibroblast growth factor receptor 1 (FGFR1) and FGFR2 have been shown to stimulate keratinocyte migration and proliferation following binding by several ligands, including fibroblast growth factor 1 (FGF-1), -2, -7, -10 and FGF-1 -10, respectively. FGFR1 and FGFR2 knockout keratinocytes have reduced the motility that may result from a lack of expression of focal adhesion components due to the absence of an FGFR signal transduction pathway . FGF-7 iKeratinocytes also express VEGFR-1, -2, and -3. Their ligand, VEGF-A, is produced by keratinocytes and macrophages. In addition to granulation tissue formation and angiogenesis, that factor also promotes keratinocyte migration . Keratinocytes also express the MET receptor tyrosine kinase (RTK). Their ligand, hepatocyte growth factor (HGF), is produced by fibroblasts and by keratinocytes after skin injury. Throughout the stimulation of STAT3 signaling, HGF induces keratinocyte motility. In addition, HGF activates VEGF-A expression and influences cell proliferation. The RON RTK receptor expressed by keratinocytes is activated by a macrophage-stimulating protein (MSP) produced by hepatocytes. RON activates the PI3K/Akt pathway; it also induces phosphorylation of both RON and integrin α6β4 at 14-3-3 binding sites, allowing the formation of RON and α6β4 complex by 14-3-3. Following this, α6β4 is relocated to the lamellipodia from hemidesmosomes. The induction of α3β1 is associated with keratinocyte spread on laminin-5, as well as activation of p38 and NF-κB signaling, which is required for cell migration . Keratinocyte proliferation is also stimulated by granulocyte macrophage-colony stimulating factor (GM-CSF) produced by keratinocytes, among others. Following binding to the CD116 receptor, GM-CSF activates the JAK/STAT signaling pathway. During wound healing, GM-CSF plays an indirect role by the induction of secondary cytokines. Studies of transgenic mice overexpressing the GM-SCF factor indicate that keratinocyte-derived GM-CSF overexpression takes place in the basal layer and is related to accelerated wound contracting. In the first phase of that process, significant mitogenic keratinocyte growth and granulation tissue formation were observed, as well as different regulation of TGF-β, IFN-γ, and iIL-6 release . Another factor, angiopoietin-related growth factor (AGF), is able to bind to keratinocytes αv integrin via RGD-motif. Transgenic mice overexpressing AGF revealed increased proliferation of keratinocytes and more rapid wound closure compared to wild-type controls. AGF is also produced by platelets and mast cells; however, AGF release takes place only on wounded skin. AGF is profusely expressed by hepatocytes .High mobility group protein β1 (HMGB1) enhances keratinocyte migration by an unknown receptor. That factor is released by macrophages and monocytes. HaCaT keratinocytes exposed to HMGB1 exhibit accelerated migration and proliferation, as well as ERK1/2 pathway induction . An in vHypoxia-inducible factor-1 (HIF-1), a regulator of oxygen homeostasis, is released during the hypoxia accompanying wound healing. The presence of HIF-1 results in elevated expression of heat shock protein 90 (HSP90) by keratinocytes and binding to LDL receptor-related protein 1 (LRP-1) followed by an increase in cell migration. HSP90 regulates the initial phase of wound healing ,208 and Cytokines including IL-1, IL-6 and TNF-α also regulate keratinocyte migration. IL-1 promotes the secretion of FGF-7 , while IChemokines produced by various types of cells may also propagate keratinocyte motility. The N terminus takes part in receptor activation. Keratinocytes express CXCL1, 8, 10, 11 and CCR14, 17, 27, which stimulate their migration, whereas CXCL1, 8 and 12 accelerate their proliferation. Reduced keratinocyte motility and proliferation has been observed in CXCR2 knockout mice. CXCR2 is a receptor for CXCL1 (growth-related oncogene-α) and CXCL8 (IL-8) chemokines, which are essential for keratinocyte activity . CXCL10 Neuropeptides, molecules released, among others, by sensory neurons during cutaneous injury, can also accelerate keratinocyte motility and proliferation . The G pKeratinocytes also express cholinergic receptors, including nicotinic (nAChR), including α3, α5, α7, α9, α10, β1, β2, and β4 and muscarinic acetylcholine (ACh) (mAChR) forms, including M1, 2, 3, 4, and 5. Increased keratinocyte migration is observed after the activation of the following receptors: α3 by PKCδ and RhoA signaling; α7 by PI3K and Rac/Cdc42 signaling; α9 by modulating adhesion between cells and between the cell and extracellular matrix (ECM) and M4 by integrins α5β1, αvβ5 and αvβ6. Conversely, M3 activation upregulates integrins α2β1 and α3β1, related to adhesion, and suppresses keratinocyte migration ,236,237.In wounds, keratinocyte migration onto the dermal matrix and MMP-1 expression is mediated by native type I collagen . In vitrThe plant extracts and their component compounds may modulate keratinocyte migration and proliferation rates via various mechanisms, including stimulation of the MAPK and PI3K/AKT signal transduction pathways. In some studies, a higher expression of β1-, α6-, β4-integrin and E-cadherin were observed after extract treatment. Aegle marmelos [Boerhavia diffusa [Stellera chamaejasme [Centella asiatica [A study of the abovementioned marmelos , Boerhav diffusa  and Stelmaejasme  extractsmaejasme  and Centasiatica  also demParrotia persica [Finally, cyanidin-3-glucoside derived from blackberry  and chlo persica  significPlants are key sources of secondary metabolites that exert various antioxidant and anti-inflammatory effects, among others. These compounds are able to modulate signaling pathways in numerous cells, including epidermal cells. Keratinocytes placed in the outer layer of the skin create a physical barrier against harmful stimuli. They are particularly vulnerable to UV-radiation, related oxidative stress and inflammation. Any disruption of the epidermis stimulates cell migration, proliferation and participation in wound healing. It is suggested that such compounds of plant origin may be used to modulate keratinocyte function by improving ROS scavenging, inhibiting inflammation and accelerating wound healing via influencing signal transduction pathways. Hence, plant extracts and their component compounds may have an impact on keratinocyte biology and its ability to maintain homeostasis. However, further in vitro and in vivo studies of the mechanisms of the action of phytochemicals, and more specific toxicity and clinical studies are needed to ensure the effectiveness and safety of plant compounds for use on human skin.


Allium sativum L.), which is often infected with soil Fusarium fungi, is very limited. In the present study, we identified 32 TLP homologs in the A. sativum cv. Ershuizao genome, which may function in the defense against Fusarium attack. The promoters of A. sativumTLP (AsTLP) genes contained cis-acting elements associated with hormone signaling and response to various types of stress, including those caused by fungal pathogens and their elicitors. The expression of AsTLP genes in Fusarium-resistant and -susceptible garlic cultivars was differently regulated by F. proliferatum infection. Thus, in the roots the mRNA levels of AsTLP7–9 and 21 genes were increased in resistant and decreased in susceptible A. sativum cultivars, suggesting the involvement of these genes in the garlic response to F. proliferatum attack. Our results provide insights into the role of TLPs in garlic and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.Plant antifungal proteins include the pathogenesis-related (PR)-5 family of fungi- and other stress-responsive thaumatin-like proteins (TLPs). However, the information on the TLPs of garlic ( Thaumatococcus danielli Benth [Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium solani, Verticillium spp., and Phytophtora spp. [Plant antifungal proteins provide resistance to fungal pathogens and are a focus of agricultural biotechnology. Overall, 13 classes of antifungal proteins are distinguished based on structural similarity and functional activity [li Benth . Thaumatora spp. ,5,6. TLPora spp. ,13,14,15TLP genes enhances plant tolerance to fungal pathogens [Botrytis cinerea, R. solani, Fusarium oxysporum, F. solani [A. alternata [Verticillium dahliae [Camellia sinensis TLP gene are resistant to Phytophthora infestans [Constitutive expression of athogens ,22,23,24lternata , and Vernfestans . It has nfestans ,26, whicnfestans ,18,24.Bursaphelenchus xylophilus (Bx-TH1 and Bx-TH2) transiently expressed in Nicotiana benthamiana induce plant cell death [Interestingly, TLPs can also be expressed by plant pathogens as a part of their molecular mimicry strategy to evade the plant’s defense system and promote colonization of the host . For exall death ,29.TLPs are also known as pollen and fruit allergens, which stimulate IgE production, thus predisposing people to allergy ,31,32.As a group, TLPs represent highly soluble low molecular weight proteins classified into L-type (22–26 kDa) and S-type (<18 kDa) ,34; accoIn total, 344 plant TLPs have been identified through analysis of NCBI, EMBL, DDBJ, UniProt, and other databases . Most ofTLP genes in garlic (Allium sativum L.), one of the important bulb crops highly susceptible to infection with F. oxysporum f. sp. cepae  and F. proliferatum (bulb rot), which are responsible for more than 60% of the world’s garlic production losses at both pre- and post-harvest stages [A. sativum PR5 (AsPR5) gene (AKU38392.1), which is upregulated by plant hormones methyl jasmonate (MeJA), abscisic acid (ABA), and ethylene, is also induced in response to F. oxysporum [Arabidopsis, PR5 expression confers resistance to a necrotrophic fungus B. cinerea, it can be suggested that AsPR5 plays a similar role in garlic through regulation of signaling pathways associated with antifungal defense. The other three identified garlic TLP-coding genes, AsPR5a (Asa2G01043.1), AsPR5b (Asa4G02099.1), and AsPR5c (Asa4G02100.1) have been reported to be differentially expressed in the roots of FBR-resistant and -susceptible garlic cultivars in response to F. proliferatum attack [The aim of this study was to characterize t stages ,46,47,48xysporum . Given tm attack .AsTLP genes in the genome of A. sativum cultivar (cv.) Ershuizao, performed their structural characterization, analyzed phylogeny, and cloned AsTLP homologs from FBR-resistant and -susceptible garlic cultivars. Comparative expression profiling of AsTLP1–32 in various tissues of these cultivars infected with F. proliferatum suggests a role of AsTLPs in the mechanisms underlying garlic defense against fungal pathogens. Our results provide new insights into the function of A. sativum TLPs, which can be used in breeding programs to increase the resistance to Fusarium in cultivated Allium spp.Here, we identified in silico 32 putative TLP gene sequences were detected in the genome and transcriptome of A. sativum cv. Ershuizao  and denoted AsTLP1–32 (AsTLP3–20) are located on chromosome 2 and seven —on chromosomes 1, 4, 6, 7, and 8 (AsTLP26–32) have been found in scaffolds and do not match any chromosomes  did not have introns, and the rest contained one  or two  introns   .The search for allergenic motifs disclosed statistically significant sequence similarity between AsTLPs and thaumatin-like allergens from apple  , peach  terms predicted their extracellular localization (GO:0005576), as well as a role in defense response (GO:0006952) and response to fungal infection (GO:0009620).AsTLP genes in various tissues  of A. sativum cv. Ershuizao was evaluated based on transcriptomics data (PRJNA607255). As the bulb is the part of garlic most susceptible to Fusarium infection and shows visible symptoms, gene expression analysis in this organ was more detailed and included eight developmental stages (from day 192 to day 227 after planting). AsTLP genes, for which Fragments Per Kilobase of transcript per Million mapped reads (FPKM) was ≥ 10 in at least one of the organs. AsTLP1–3, 5–11, and 16 had overall similar expression patterns , whereas those of AsTLP20, 21, and 23–25 were different.The expression of AsTLP genes  were strongly expressed in the stems and leaves. In the roots, the highest mRNA levels were observed for AsTLP1, 9, and 23 and the lowest—for AsTLP21, 24, and 25; the latter had the highest expression in the floral buds and stage 1 bulbs, which gradually decreased during bulb development. The expression of AsTLP20 and 23 in the bulbs showed bell-shape changes, with the maximum at stages 3 and 6, respectively. According to the expression profiles, the AsTLP genes could be divided into two groups: AsTLP1–3, 5–11, and 16 with the highest expression in the stems, leaves, and roots, and AsTLP20, 21, 24, and 25 with that in the developing bulbs, floral buds, and flowers. The first group includes all genes of phylogenetic clades V and VI, as well as one gene each from clades I and III; members of the second group are phylogenetically heterogeneous (AsTLP23 gene had the features of both groups (About a third of the ogeneous a. The exh groups .cis-acting elements in the 5′-UTR and promoter regions (1 kb upstream of the initiation codon) of AsTLP genes. The results indicated that the AsTLP regulatory regions contained 11 hormone-responsive and 10 stress-responsive elements , AsTLP17 (4 ERE and 3 ARE), AsTLP1, 24, and 25 , and AsTLP6 and 10 . The promoters of five genes  contained low-temperature-responsive (LTR) elements; among them, AsTLP23 had the highest number (6). The W-box, which responds to fungal elicitors and wounding, was found in only three genes . The rarest elements were ABA-responsive CARE (in AsTLP1 and 21) and auxin-responsive AuxRE (in AsTLP8 and 9) (AsTLP5 and 6 (clade V), 8 and 9 (clade VI), or 22, 28, and 29 (clade I).Considering the stress- and hormone-responding role of TLPs , we searelements . Among t8 and 9) . No pronAsTLP1-32 genes in the garlic response to fungal infection, we performed AsTLP expression profiling in garlic cultivars resistant (cv. Sarmat) and susceptible (cv. Strelets) to FBR [AsTLP7 corresponds to clade V group of genes (AsTLP5–7 and 11), AsTLP8—to set of AsTLP3, 8, and 10 genes, AsTLP12—to set of AsTLP12, 15, 17, 22, and 26-29 genes, AsTLP19—to AsTLP13, 18, 19, and 30 genes, and AsTLP31—to AsTLP31 and 32 (clade II) genes (F. proliferatum and compared with uninfected control (To elucidate the role of the identified ) to FBR . The AsTI) genes . Gene ex control .AsTLP genes were transcribed in all analyzed organs, except for AsTLP24 not expressed in the cloves  and AsTLP24 (decreased). Similar dynamics were observed in the cloves, except for AsTLP12 and 31, which were downregulated with time. In the stems, the expression of AsTLP7–9 and 31 decreased and that of AsTLP19 and 24 increased from 24 to 96 hpi, whereas that of AsTLP16 was unchanged . Among them, non-synonymous SNPs leading to amino acid substitutions were found in AsTLP7 (4), AsTLP16 , AsTLP20 , AsTLP21 (1), and AsTLP23 (1) of both cultivars, whereas a cultivar-specific non-synonymous SNP  was detected in AsTLP20 of cv. Strelets  and 20 SNPs in AsTLP7, 8 SNPs in AsTLP20, 3 indels  and 7 SNPs in AsTLP21, and 5 SNPs in AsTLP23.The regulatory sequences (promoter and 5′-UTR) of the elements . The regelements . In geneAsTLP7, 20, and 21 regulatory sequences did not affect cis-elements, whereas those in the AsTLP23 sequences did. Thus, two SNPs (at positions -674G>A and -676A>G) resulted in the loss of the CGTCA motif associated with MeJA response, another SNP (-521C>T) created the GARE motif (gibberellin-responsive element), and two SNPs (-640G>A and -641C>A) eliminated an LTR element (The mutations found in the  element .A. sativum L.) belongs to the Allium genus, which comprises 971 species, thus being one of the largest genera of the Amaryllidaceae family widely distributed in the Northern Hemisphere from dry subtropics to boreal zones [Fusarium spp. [Allium crop breeding programs.Garlic  had no introns or contained only a few (1–2) of them  were found to be homologous to motin 34 , which fAsTLP genes contained cis-regulatory elements associated with the activation of immune mechanisms through responses to stresses , elicitors, and hormones, , and stresses.It is known that fungi-mediated biotic stress activates plant immune system through perception of pathogen-associated molecular patterns  with folormones, . A similLP genes ,74 and o–5 genes ,60. AccoAsTLPs  may be involved in the response to cold stress   .AsTLP genes within the same clade   a tended patterns , suggestTLP genes with the established function in plant defense has predicted two unique combinations of regulatory elements to distinguish between abiotic and biotic stress responses: genes regulated by abiotic stresses contain the ABRE element, whereas those responding to fungal attack do not [AsTLP3, 4, 7, 14–17, 20–23, 26–29, and 31, whose promoters do not have ABRE  and AsTLP21 in the roots of cv. Sarmat and their downregulation in those of cv. Strelets  and negative (miR394) regulation of resistance to Fusarium [Allium roots produce antifungal volatiles such as 2-methyl-2-pentenal and organosulfur compounds, as well as non-volatiles such as spirostanol, furostanol, and steroidal saponins, which inhibit Fusarium growth [It has been suggested that diseases . Thus, oFusarium ,50,60,78m growth ,80.TLP genes was performed in the A. sativum cv. Ershuizao whole-genome  and transcriptome (PRJNA607255) sequences [http://pfam.xfam.org/family/PF00314; accessed on 25 October 2021) was used as reference. All the selected sequences contained start and stop codons and full-length catalytic domains.The search for equences . The thaAsTLP genes and encoded proteins were conducted with MEGA 7.0.26 [Multiple sequence alignment and structural analyses of the A 7.0.26 . The phyAsTLP genes and CDSs were analyzed with GSDS v2.0 [https://web.expasy.org/protparam/; accessed on 30 October 2021; GRAVY indexes were calculated as the sum of hydrophobicity values of all residues divided by sequence length), conserved domains, sites, and motifs , http://meme-suite.org/tools/meme; accessed on 30 October 2021), biological processes , subcellular localization , functional importance of residue substitutions , and signal peptide cleavage sites . The chromosomal localization map was drawn using MG2C v. 2.1 . The search for allergenic motifs in AsTLPs was conducted using the Structural Database of Allergenic Proteins (SDAP) .To predict exon–intron composition, SDS v2.0 . PutativAsTLP genes in the roots, bulbs, stems , leaves, buds, flowers, and sprouts was analyzed based on A. sativum cv. Ershuizao RNA-Seq data , normalized as FPKM [The expression of  as FPKM , and vis as FPKM . Only trAsTLP CDSs from garlic cultivars, gene-specific primers were designed based on A. sativum cv. Ershuizao transcriptomic data (NCBI project accession number: PRJNA607255) , cloned in the pGEM®-T Easy vector , and sequenced (3–5 clones for each accession) on ABI Prism 3730 DNA Sequencer  using the designed primers.To amplify the A607255) . CDNA . The strain was originally isolated from the bulbs of garlic cv. Strelets; according to the pathogenicity test, the first signs of the disease appeared on the clove surface 5 days after infection [nfection .A. sativum cv. Sarmat and cv. Strelets (winter garlic of Russian breeding) resistant and susceptible to FBR, respectively, were kindly provided by the Federal Scientific Vegetable Center . The number of cloves used per biological replicate in the Fusarium infection assay was based on that of cloves in the bulb (5–7 for cv. Strelets and 7–11 for cv. Sarmat). In total, 12 cloves of each cultivar (6 infected and 6 uninfected) were processed . Cloves were surface-sterilized in 70% ethanol for 3 min, rinsed with sterile water, placed in Petri dishes with wet filter paper, and incubated at +25 °C in the dark. After 72 h, active root growth was observed, and half of the cloves were infected by soaking in F. proliferatum conidial suspension (~106 conidia ml−1) for 5 min as previously described [n = 3 cloves per each time point).The roots, stems, and cloves of the infected and uninfected samples were collected at each time point, frozen in liquid nitrogen, and stored at −80 °C. The time points were chosen according to the expression peaks of some PR genes, which were observed 1–3 days after inoculation with hemibiotrophic pathogens [Accessions of escribed ), transfathogens .® Fluorometer, Thermo Fisher Scientific, Waltham, MA, USA) and qRT-PCR was performed in a CFX96 Real-Time PCR Detection System  with 3.0 ng cDNA, SYBR Green RT-PCR mixture , and specific primers  using the RNeasy Plant Mini Kit , purified from genomic DNA , qualified by gel electrophoresis, and used for first-strand cDNA synthesis  with an oligo-dT primer. RNA and cDNA concentrations were quantified by fluorimetry ). The data were expressed as the mean ± standard deviation (SE) based on three technical replicates of three biological replicates for each combination of cDNA and primer pairs. The unequal variance (Welch’s) t-test was applied to assess differences in gene expression; p < 0.01 was considered to indicate statistical significance. and UBQ , and thecis-elements in the promoters and 5′-UTRs (1.0 kb regions upstream of the initiation codon) was performed using the PlantCARE database, which provides evaluation of cis-regulatory elements, enhancers, and repressors; .The search for specific A. sativum cv. Ershuizao genome. The AsTLP genes were distributed among six chromosomes and four scaffolds and might have been evolutionary originated from segmental or tandem duplications. AsTLP7, 16, 20, 21, and 23 homologs were amplified from garlic cultivars resistant and susceptible to Fusarium infection. The promoters of AsTLP genes contained distinct sets of cis-acting elements associated with hormone and stress reactivity, suggesting differential transcriptional regulation of garlic TLPs in response to pathogens and abiotic stresses, which was consistent with specific expression patterns of AsTLP genes in garlic cultivars infected with F. proliferatum. The transcription of AsTLP7–9, and 21 genes in the roots was downregulated in FBR-susceptible and upregulated in FBR-resistant cultivars, suggesting their particular involvement in the sensitivity of garlic to fungal infection. Our results provide the foundation for further functional characterization of the AsTLP genes using a reverse genetics strategy, and may contribute in the breeding of A. sativum cultivars with increased resistance to Fusarium infections as well as various abiotic stresses.We identified and characterized 32 genes encoding thaumatin-like proteins in


In recent years, studies on early plant defense signaling in response to herbivory have been gaining momentum with the application of genetically encoded GFP-based sensors for real-time monitoring of early signaling events and genetic tools to manipulate ion channels involved in plant-herbivore interactions. In this review, we provide an update on recent developments and advances on early signaling events in plant-herbivore interactions, with an emphasis on the role of ion channels in early plant defense signaling.Plants and insect herbivores are in a relentless battle to outwit each other. Plants have evolved various strategies to detect herbivores and mount an effective defense system against them. These defenses include physical and structural barriers such as spines, trichomes, cuticle, or chemical compounds, including secondary metabolites such as phenolics and terpenes. Plants perceive herbivory by both mechanical and chemical means. Mechanical sensing can occur through the perception of insect biting, piercing, or chewing, while chemical signaling occurs through the perception of various herbivore-derived compounds such as oral secretions (OS) or regurgitant, insect excreta (frass), or oviposition fluids. Interestingly, ion channels or transporters are the first responders for the perception of these mechanical and chemical cues. These transmembrane pore proteins can play an important role in plant defense through the induction of early signaling components such as plasma transmembrane potential (V Abiotic stress includes drought, salinity, extreme temperatures, radiation, floods, and heavy metals, whereas biotic stressors include insect, animal herbivores, and microbial pathogens. Plant and insect-herbivore interactions are among the most significant species interactions found in nature ,2, and ic levels ,7,8,9,10c levels ,24,25,26During herbivory, the damaged areas of the plant need to inform the rest of the plant to keep them ready for the imminent herbivory threat. Therefore, plants need to alert their unaffected parts by sending long-distance signals from the site of damage to various parts of the plant to appraise the threat. Plants respond to diverse stimuli by communicating amongst cells from distinct tissues or organs, a process called systemic signaling . Studies2+ [Considerable progress has been made in understanding this intricate relationship between plants and herbivores with a plethora of field and lab studies. These include studies that have dissected pairwise interactions between a specific herbivore and its host; interactions at species, genus, and community levels with multiple hosts and herbivores; and studies examining plant defense signaling networks through molecular genetics genomics, to name a few ,30. Howe2+ , reactiv2+ ,33,34,35m, Ca2+, and ROS  and Ca2+ flux. Variation in Vm is critical for plant wounding responses [2+ and ROS, versatile secondary messenger, were generated that plants use to sense and transform environmental stimuli into an adaptive intracellular response [2+ concentration, and these spatiotemporal variations have been shown to yield Ca2+ signatures [2, H2O2, and OH−. ROS which has been demonstrated to act as a self-propagating long-distance and fast wound signal [m, Ca2+, and ROS in plant response to herbivory and provide an overview of what is currently known about the role of ion channels in plant-herbivore interactions.There has been considerable research on identifying the factors that are involved in long-distance signaling. Plants can appraise their unaffected parts by extensive network of intracellular regulators, V and ROS ,41. The  and ROS . The proesponses . Finallyresponse . Insect gnatures ,46,47,48d signal . Throughm is an electrical potential of the cell membrane that is maintained via the balance of ion fluxes across the plasma membrane. Vm indicates whether a cell is excited or not. It is responsible for generating action potentials in tissues, muscles, and nerves in animals and plays a crucial role in diverse biological functions such as biological sensing, hearing, cell cycle, proliferation, contractility, and circadian rhythm, to name a few [m to regulate plant cellular functions such as maintaining turgor pressure, osmotic balance, and stomatal closure. There is no net flux of ions through the membrane when in equilibrium, called the resting membrane potential. Changes in the resting membrane potential will occur due to an unbalanced movement of ions, thus leading to Vm being more positive (depolarization) or more negative (hyperpolarization). In general, plants maintain a negative resting membrane potential in the order of −110 to −150 mV [m leads to plant defense responses. The way plants sense insect cues and initiate defense responses has been a point of interest for many years. One hypothesis that has evolved by studying cellular responses following herbivory suggests that the first event following herbivory generates the fluctuation in Vm [Spodoptera littoralis) alter Vm in lima bean (Phaseolus lunatus L.) at increasing distances of 5, 30, and 60 mm from the bite zone. Vm depolarization was observed within the first 15 min of feeding by S. littoralis in the palisade cells. The effect of S. littoralis regurgitant and its components were also tested on Vm in P. lunatus leaf and the results showed that Vm alterations were independent of regurgitate concentration. In addition, they also examined changes in Vm in response to the application of various H2O2 concentrations to mechanically damaged and herbivore-wounded P. lunatus leaves. H2O2 treatment induced a robust Vm that was significantly greater in herbivory-wounded plants than in mechanically injured leaves [The Vme a few . Unlike  −150 mV ,52. It hon in Vm . Maffei on in Vm  has alsod leaves .m alterations in wild-type and plasmodesmata mutated A. thaliana pdko3 lines; plasmodesmata are channels within the plant cell that allow chemicals to pass through, establishing a pathway for cell-to-cell communication. A strong Vm depolarization occurred in wild-type A. thaliana plants within 7 to 8 min after herbivory, but the pdko3 mutant did not exhibit Vm depolarization in response to herbivory or application of OS from S. littoralis. However, Ca2+ elevation was observed in both wild types as well as in pdko3 mutant. This observation ruled out the possibility of Ca2+ channels being involved in Vm depolarization. To dissect the dependence of Vm depolarization on potassium (K+) channels, the K+ channel activity was measured using fluorescent indicator FluxORTM. A significant increase in K+ channel activity was observed in wild-type plants, whereas a complete loss of K+ channel activity was observed in pdko3 plants. This finding also suggests that K+ channels are involved in Vm depolarization and supports the hypothesis that plant cells respond to OS by a Vm-mediated signal transduction pathway.Bricchi et al.  studied m has been known to be induced by the binding of specific components from herbivore OS with the receptors present at the plasma membrane [Ginkgo biloba), a living fossil plant, responds to S. littoralis herbivory by inducing Vm depolarization, which was evident up to 6 h. Another study using A. thaliana also showed that the extent of Vm depolarization was the same for S. littoralis, green peach aphid (Myzus persicae), and the plant pathogenic bacteria Pseudomonas syringe, but the timing of the occurrence of Vm depolarization was different for each of these biotrophs. Moreover, the magnitude of early defense response depends upon the amount of tissue damage by the biotroph. Vm depolarization was rapid upon the attack of chewing herbivore, S. littoralis (30 min to 2 h), as it caused substantial tissue loss, since it consumed large amounts of leaf tissue. On the other hand, less damage was observed by a phloem feeder, M. persicae (4 to 6 h), that delayed the plant defense response since phloem feeders with sucking mouthparts feed on vascular tissues without visible tissue damage as observed with chewing herbivores [2+ and ROS generation are directly tied to Vm when herbivores interact with plants, and Vm is essential for plant defense responses.The fluctuation in Vmembrane . These cmembrane . A studymembrane  showed trbivores . It is a2+ is a ubiquitous signaling molecule in plants. It functions as a secondary messenger in cellular pathways that regulate plant growth and development, cell polarity, cytoskeleton organization, ion transport across membranes, stomatal regulation, root growth, fertilization, nutrient signaling, and plant immunity [2+ signature,” linked with distinct fluctuations in Ca2+ concentration in the cytosol and sometimes in a particular intracellular compartment. Therefore, Ca2+ fluxes, especially oscillations between calcium stores and the cytosol, are important for cell signaling [Caimmunity . Consequignaling ,61,62.2+ concentration is maintained at or below 100 nM; however, the majority of Ca2+ is stored in the apoplast, vacuole, endoplasmic reticulum (ER), and Golgi apparatus. The apoplast serves as the first Ca2+ reservoir of a cell that can store 0.33 mM free resting Ca2+ and the first area that responds to stimuli, while the vacuole serves as the largest Ca2+ pool of a cell that can store up to 0.2–5 mM free resting Ca2+ [In plants, the cytosolic Caing Ca2+ ,63.2+ signature plays an important role in long-distance signal transduction during herbivore attack through which HAEs such as OS, oviposition, and frass is sensed by the cell membrane, and then, a Ca2+ is rapidly propagated in the cytosol and travels throughout the plant to induce defense responses. The shaping of this “Ca2+ signature” during plant-herbivore interactions is achieved through the amplification and integration of Ca2+ signals. The amplification step is mediated via specific ion channels or transporter proteins and enhances Ca2+ fluxes at sites of herbivore attack, whereas the integration step is mediated via Ca2+ sensor proteins, which allow efficient transmission of Ca2+ signals from one cell to another in a tissue or organ. Herbivory induces Ca2+ entry from the apoplast to the cytosol via plasma membrane Ca2+ channels which stimulates Ca2+ signals in the cytosol leading to the amplification of Ca2+ signals. The localized Ca2+ signals from the cytosol are distributed throughout the whole plant. In this way, amplification, and integration of Ca2+ signals constitute two important ways by which “Ca2+ signature” contributes as a signaling molecule during plant-herbivore interactions [The Caractions .2+ signal requires selective Ca2+ sensor proteins that respond to changes in cytosolic Ca2+ levels and encipher the frequency, amplitude, and signal localization of Ca2+ signatures. It is estimated that A. thaliana contains around 250 Ca2+ sensor proteins [2+ dependent protein kinases (CPKs) and the Ca2+ and calmodulin-dependent protein kinase (CCPK) [2+ binding and cause conformational changes in their structure [The amplification of intracellular Caproteins . These cproteins ; (2) theproteins ; and (3)e (CCPK) . All of tructure .Arabidopsis genome has seven calmodulin genes encoding four different isoforms  [A. thaliana signal responsive (AtSR1) proteins [Atsr1 mutants of A. thaliana were sensitive to attack by dark winged fungus gnats (Bradysia impatiens), suggesting the role of CaM as an important sensor in the early stages of the insect-plant attack [2+ binding and act as Ca2+ relays/sensors [A. thaliana upon S. littoralis OS treatment, implying a function in early defense plant signaling [2+ binding domain and a serine/threonine kinase domain into a single protein that performs the fundamental function of converting Ca2+ signals to phosphorylation events [A. thaliana contains 34 CPK family genes that play a role in plant defense responses. CPK 3 and CPK 13 both participate in signaling after Ca2+ influx upon S. littoralis attack through regulation of plant defensin gene (PDF1.2) by phosphorylation of the transcription factor, HsfB2a [cpk3 and cpk13 mutants had much lower transcript levels of the plant defensin gene PDF1.2 in comparison to wild-type plants.CaM functions as a sensor relay protein since it lacks an enzymatic function. The nd CaM7) . CaM/CaMnd CaM7) . A. thalproteins , also knproteins , have bet attack . Along w/sensors . CML and/sensors . CML42 gignaling . CPKs han events ,77. A. t, HsfB2a . The cpk2+ signaling has gained momentum with the advance in Ca2+ imaging techniques. Therefore, it is important to discuss different plant Ca2+ imaging methods, which are widely used in the context of plant-herbivore interactions to observe and record cytosolic Ca2+ concentration in herbivore-infested plants. These techniques include the use of Ca2+ sensing fluorescent dyes and genetically encoded Ca2+ indicators. Various fluorescent Ca2+ sensing dyes, such as Fluo-3, Calcium Orange, etc., have been used to investigate the dynamics of cytosolic Ca2+ signals in plant- herbivore interaction [2+ indicator Ca2+ orange was utilized to identify changes in cytosolic Ca2+ concentrations in P. lunatus following S. littoralis herbivory. The changes in Ca2+ concentration were compared in response to a single wounding (MD) event, continual mechanical damage caused by a robotic worm , and herbivory. After 30 min, a considerable increase in Ca2+ fluorescence was observed due to herbivory in the wounding zone, which persisted for 4 h, but in MD and MW plants, just a faint fluorescence was noticed [2+ sensing dyes have some limitations, including toxicity, fragility, low fluorescence signals, and they cannot be imaged in living plants without permeabilization. To overcome these limitations, researchers have initiated research on the use of genetically encoded Ca2+ indicators. The most widely used Ca2+ imaging method includes genetically encoded Ca2+ indicators, such as GCaMP, Yellow Cameleon (YC) Ca2+-sensors. The Ca2+ sensors were developed from GFP by combining them with calmodulin. These Ca2+ sensors can be expressed in the whole plant and are functional throughout the entire plant. Therefore, it can be used to monitor cytosolic Ca2+ in plants subjected to various herbivore attack conditions [P. rapae caterpillars induced cytosolic Ca2+ responses in the leaves of A. thaliana can be monitored with GCaMP3. This study reported that the increases in cytosolic Ca2+ concentration were associated with ion influx through plasma membrane Ca2+ channels such as GLR3.3/GLR3.6. Another example is Verrillo et al. [2+ induction could be monitored with YC3.60, a YC-based Ca2+ sensor, following application of S. littoralis OS on mechanically damaged A. thaliana leaves. By using these tools, it is now possible to study the dynamics of Ca2+ signaling in plant-herbivore interactions at single-leaf, whole-plant, and whole-plant-insect herbivore attack conditions.In recent years, the research on Caeraction ,79,80,81 noticed . Even thnditions ,82,83,84nditions  showed t2+ level is controlled by the influx of Ca2+ ions from extracellular through apoplastic and vacuolar membranes. Therefore, plant ion channels play an important role in regulating plant development and the perception of many stimuli, including herbivory.Intracellular Ca6 ions per second. Ion channels can control ion fluxes in their target compartment and, thus, modify cellular homeostasis, and are vital in osmoregulation, development, signaling, mobility, and uptake of nutrients by the root and long-distance communication [+ channel, Stelar K+ outward rectifier (SKOR) [+ channels such as the shaker family , tandem pore, and two-pore K+ channels , are responsible for permeation of K+ ion across the plasma membrane and tonoplast membrane. Anion channels include slowly activating anion channels , aluminum-activated malate transporters , chloride channels/transporters , and detoxification efflux carrier . Ligand-gated channels include cyclic nucleotide-gated channel  and glutamate receptor-like channels  [2+-transporting ion channels have been shown to play a role in plant-herbivore interactions, including cyclic nucleotide-gated channels (CNGC19) [Ion channels are macromolecular pores in the membrane that regulate the influx and efflux of ions across the membrane at a rate of 10nication ,86. The r (SKOR) . The lasansport)   , stomataansport) ,91, pollansport) , salt toansport) , and plaansport) , to name(CNGC19) ,96, glut(CNGC19) ,42,97, t(CNGC19) ,84,98, a(CNGC19) ,100.2+ channels, first discovered in retinal photoreceptors and olfactory neurons [A. thaliana, which consists of 20 family members [The cyclic nucleotide-gated channels (CNGCs) are ligand-gated Ca neurons . They pl neurons . These i neurons ,103,104  neurons ,106, the neurons , and sal neurons . These c members .C-termini of the channel  channels are ionotropic glutamate receptor homologs in mammals (iGluRs). The iGluRs have been extensively studied for their central nervous system and have been known to play a vital role in synaptic transmission, learning, and memory [Arabidopsis consists of 20 GLR genes; each subunit hosts a N-terminal domain, two extracellular ligand-binding sites , and transmembrane domains (S1–S4), including a pore region (P) and the C-terminal domain [d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and Kainate receptors. Plant GluRs share a high degree of similarity with the NMDA receptors that range from 16 to 63% in the ligand-binding domains and the transmembrane domains [Glutamate receptor-like (GLR) is a non-selective ion channel responsible for permeating Cad memory ,124. It d memory . In pland memory , gravitrd memory , pollen d memory ,129, immd memory ,132,133,d memory . Arabidol domain   var xanthi while the application of lanthanum and Ca2+ chelator, BAPTA, inhibited glutamate-induced Ca2+ increase. This observation suggests that the plant GLR has a role in the modulation of Ca2+ influx that ensures plant defense responses against insect herbivores.Electrophysiological studies have shown the involvement of GLRs in inducing a Carbivores ,140. A s2+ waves from wounded to unwounded sections of the plant. When S. littoralis larvae were allowed to feed on A. thaliana wild-type plants, wound-induced surface potential alterations were detected. However, wounding reduced the surface potential alterations in the four GLR mutants GLR3.1, GLR3.2, GLR3.3, and GLR 3.6. [Pieris rapae) caterpillars in A. thaliana leaf expressing genetically encoded Ca2+ sensor GCaMP3. The cytosolic Ca2+ elevation and subsequent defense gene expression were observed after the application of glutamate and not with other amino acids such as sorbitol. Furthermore, the Ca2+ signals were completely abolished in the GLR3.3/GLR3.6 double mutant in A. thaliana, suggesting that GLR3.3 and GLR3.6 are essential for transmitting Ca2+ signals induced by wounding and herbivory. Another recent study by Shao et al. [2+ elevation and surface wave potential (SWP) in A. thaliana expressing calcium sensor GCaMP6. Additionally, the application of glutamate to the wound site in the root induced an increase in Ca2+ and SWP in all leaves. Interestingly, in the GLR3.3/GLR3.6 double mutant, this wound and glutamate-induced rise in root to shoot Ca2+ was attenuated. This finding suggests that GLR3.3 and GLR3.6 are involved in propagating systemic Ca2+ signaling from leaf to leaf and root to shoot. These results provide evidence for the role of plant GLRs in the modulation of Ca2+ signaling during plant defense responses against insect herbivores.GLR3.3 has been implicated in the transmission of signals in the form of CaGLR 3.6. . This suGLR 3.6.  showed to et al.  demonstr2+ channels [A. thaliana by genome sequencing [N-terminal head and a C-terminal annexin core [2+ binding activity occurs in type II and III binding sites of annexin proteins [N-terminal region than their animal counterparts [N-terminal region that interacts with other proteins.Annexins are the phospholipid-binding proteins and are considered novel mechanosensitive Cachannels ,142. In channels . They archannels ,144. Eigquencing . Plant axin core   herbivory. The study found that annexin 1 was responsible for inducing cytosolic free Ca2+ elevation upon wounding and simulated herbivory in A. thaliana. ANN-1 knock-out and ANN-1 overexpressing lines were employed in this work to evaluate their role in herbivory-mediated Ca2+ signaling. The result showed that in the ANN-1 deletion line, the increase in cytosolic Ca2+ upon herbivory by S. littoralis was impaired, and the larvae gained more weight than those fed on wild-type plants. On the other hand, weight increase was significantly lower in larvae that fed on the ANN-1 overexpressed line compared to the wild type. Additionally, jasmonate accumulation and defense responses were diminished in ANN-1 systemic leaves, demonstrating that ANN-1 is involved in systemic cytosolic Ca2+-dependent jasmonate induction. This finding suggests that ANN-1 modulates plant defenses against herbivore damage through the Ca2+-dependent jasmonate signaling pathway and is required for systemic rather than local defense activation in plants attacked by herbivorous insects.A recent study by Malabarba et al.  reportedA. thaliana (AtTPC1), with 733 amino acids identical to the rat TPC1 sequence [Two pore channels (TPCs) are organellar cation channels that are widely expressed in animals and plants. In animals, they are localized in the endolysosomal membrane, while in plants they reside in the tonoplast of plant vacuoles ,153,154.sequence .2+ and Na+ channels and feature two shaker-like units with six transmembrane domains (S1–S6), each joined by a cytosolic linker containing two Ca2+-binding EF-hands (EF1 and EF2). , the plasma membrane Ca2+ permeable ion channels glutamate receptor-like 3.3 and 3.6 (GLR3.3 and GLR3.6), which are critical regulators of extracellular Ca2+ import into the cytoplasm of plant cells. In addition, this study also revealed that the increase in cytosolic Ca2+ induced TPC1 mediated vacuolar Ca2+ release in response to aphid feeding, suggesting that the TPC1 channel operates in conjunction with the plasma membrane Ca2+ permeable ion channels GLR3.3 and GLR3.6 in mediating cytosolic Ca2+ increase during insect herbivory [Plant and animal TPCs are similar in sequence to voltage-gated Cand EF2). . Voltage opening . The SV 2+ to K+ ,157. Ca2 release ,158. The release  has showt et al.  employederbivory .+-ATPases) are the primary pumps responsible for the generation of a proton gradient across cellular membranes. This electrogenic transporter uses energy from ATP hydrolysis to drive the translocation of protons against their concentration gradient from the cytosol to the external aqueous environment [+-ATPase is located in the plasma membrane (PM) of plant cells. It has been demonstrated that the activation and suppression of the H+-ATPase activity in the plant plasma membrane modulate Vm, resulting in the alteration of PM ion channels and transporters functions [+-ATPase is a single 100 kDa polypeptide and a member of the large family of phosphorylation (P)-type ATPases. It is composed of six transmembrane helices (M1–M6) and a cytoplasmic domain containing phosphorylation (P), nucleotide-binding (N), and actuator (A) domains involved in ATP hydrolysis. The PM H+-ATPase has been implicated in various physiological processes, including cell development, intracellular pH regulation, food uptake, stomatal opening, salt tolerance, and cellular expansion [The proton-pumping ATPases  is involved in the formation of slow wave potentials (SWPs), which are required for long-distance electrical transmission during herbivore-induced plant defense. Fusicoccin, a PM H+-ATPase activator, prolonged the SWP repolarization phase in leaves distal to wounds. The repolarization phase was significantly prolonged in reduced function aha1 mutants, whereas the duration of SWP repolarization was reduced in the presence of a gain-of-function mutant ost2-2D. Additionally, S littoralis larvae performed better on aha1-7 mutants than on wild-type plants. Overall, these observations suggest that the PM H+-ATPase is required for the regulation of the Vm and electrical signal propagation between different parts of a plant during insect herbivory.Plant PM H feeding . A study feeding  demonstri et al.  has reve2−•−), hydrogen peroxide (H2O2•), hydroxyl radical (HO•), peroxynitrite (ONOO), and singlet oxygen (1O2) [2O2. In plants, respiratory burst oxidase homologs (RBOHs) were found to be the key enzymes that catalyze the formation of ROS, which is a key step in plant protection against herbivores [N. attenuate where tobacco hornworm (Manduca sexta) OS enhanced NaRBOHD (N. attenuata NADPH oxidase homolog) on damaged leaves. ROS accumulation was diminished in M. sexta OS treated NaRBOHD-silenced N. attenuata plants without affecting OS-induced gene expression of defense-related genes [Reactive oxygen species (ROS) are highly reactive molecules generated by redox reactions. They are part of several biological processes, such as photorespiration, oxidative phosphorylation, the electron transport chain (ETC), as well as a plant defense against pathogens and herbivores. ROS is produced in the mitochondria, chloroplast, and peroxisomes. There are several forms of ROS like superoxide anion (Oen (1O2) . ROS is rbivores ,171,172.rbivores . The siged genes .A. thaliana [Diuraphis noxia). A strong burst of H2O2, as well as NADPH oxidase activity, was observed in resistant plants 3 h after infestation in comparison to susceptible plants. Treatments of plants with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, suppressed the H2O2 production. Elevation in H2O2 levels (47%) was observed by treating resistant wheat plants with a mixture of glucose and glucose oxidase [2O2 plays a role in the defense response against D. noxia infestation.The production of ROS is an inevitable by-product of metabolism in many cell types. Previously, it was assumed that ROS are toxic molecules that cause cellular damage to macromolecules . Still, thaliana . Plants thaliana . ROS prothaliana ,180. The oxidase , suggestPterisvittata) response to herbivory by S. littoralis. The P. vittata plants responded to the attack of S. littoralis by activating peroxidases which produced H2O2. The concentration of H2O2 in leaves was lower in mechanically wounded young leaves than herbivory wounded leaves, suggesting that P. vittata can distinguish between mechanical and herbivory wounding by modulating the amount of ROS production. A study by Shinya et al. [Mythimna loreyi), caused a strong intracellular ROS generation on rice cells, and a similar effect was obtained upon application of synthetically prepared N-linolenoyl-L-Glu, the most abundant FAC present in OS of M. loreyi, indicating that FAC from M. loreyi OS promoted ROS production in rice cells.Studies have shown that ROS serve as early defense signaling molecules in response to herbivore-induced wounding and secretions such as OS and oviposition. Imbiscuso et al.  investiga et al.  demonstrM. sexta oral secretions (OS) induced ROS generation in isolated tomato protoplasts. Interestingly, our study showed that the application of tomato plant-fed (PF) M. sexta OS enhanced ROS generation while artificial diet-fed (DF) OS could not induce ROS in tomato protoplasts, suggesting that the oral secretions of M. sexta play an indispensable role in inducing ROS generation in tomato protoplasts. Our study also showed that the M. sexta PF-OS induced ROS increase was diminished in the presence of a Ca2+ chelator, BAPTA-AM, suggesting that there is a link between Ca2+ and ROS signaling. Several lines of evidence have indicated the existence of a positive feedback mechanism between ROS and Ca2+ production. In a heterologous expression system, treatment with ionomycin, an ionophore that leads to Ca2+ influx into cells, resulting in activation of RHD2 NADPH oxidase (root hair defective 2 reduced nicotinamide adenine dinucleotide phosphate) in root tips of A. thaliana confirming Ca2+ triggered RHD2 NADPH oxidase activity. These observations suggest that Ca2+ acts upstream of ROS production [Recently, our group Gandhi et al.  demonstroduction .2+ binding [2+ dependent modulation [2+ and ROS. A. thaliana mutants lacking certain NADPH oxidases (AtRBOHD and AtRBOHF) do not close their stomata and produce ROS, Ca2+, and Ca2+ channel activation when they are exposed to ABA. Supplementation of H2O2 to guard cells rescues the mutant phenotype, implying that Ca2+ entry proceeds downstream of ROS generation in ABA signaling [Compelling evidence indicates that ROS production by RBOHD is dependent on the Ca binding ,187. RBOdulation . Abscisiignaling ,190.A. thaliana, the production of H2O2 was observed in leaves 72 h after oviposition by cabbage moth (Pieris brassicae) and was recognized by the formation of a reddish-brown precipitate. This result indicates that oviposition can trigger a localized response that resembles the hypersensitive response induced by pathogens [P. brassicae induced generation of H2O2, salicylic acid and defense gene expression in A. thaliana. This study also revealed phosphatidylcholines (PCs) released from eggs is the key signaling molecule that activates gene expression and triggers various defenses in the plants.In athogens . A recenathogens  showed t2DCFDA). Genetically encoded ROS sensors “Ro-GFP” can monitor the cellular redox status at a high spatiotemporal resolution [2DCFDA are easier to use and can measure ROS in real-time with high sensitivity [2DCFDA has gained attention for its potential to measure the ROS levels in real-time on whole plants and as well as plant protoplasts [P. syringae pv. tomato DC 3000 on ROS signaling in H2DCFDA dye sprayed A. thaliana by using whole plant-live imaging. This study suggests that the combination of live-cell imaging and the use of H2DCFDA enables real-time monitoring of ROS in plants in response to various stress and pathogen treatments. This study also utilized an RBOHD (rbohD) knockout, and upon treatment with different stimuli, less ROS generation was observed. In contrast, another cytosolic ascorbate peroxidase 1 (apx) knockout produced more local as well as systemic ROS upon wounding or light stress treatments implying that this mutant had less ROS quenching capacity.While ROS relevance in plant-herbivore interaction is gaining momentum, the detection and characterization of ROS are still a significant bottleneck in this field. The early detection and quantification of ROS can be carried out by either utilizing genetically encoded fluorescent ROS sensors such as redox-sensitive green fluorescence protein (Ro-GFP), or synthetic fluorescent probes, such as 3,3′-diaminobenzidine (DAB) and 2′,7′-dichlorofluorescein diacetate (Hsolution ,198,199.solution  measuredsitivity . DAB hassitivity ,203,204.toplasts ,205,206.2+ and ROS generation, gene expression, and, ultimately, the host plant defense response against herbivores. Transforming plants with biosensors such as GCaMP-Ca2+ and Ro-GFP-ROS sensors can help in the early identification of the plant defense responses. HAEs such as OS, frass, and oviposition could be used to develop strategies for early detection of the impending herbivory. So far, only a handful of Ca2+ permeable channels have been identified that plays a role in plant-herbivore interactions. Further studies are needed to unravel other ion channels that may be contributing to the modulation of Vm, Ca2+, and ROS, the downstream signaling cascade, and, more importantly, the role of these ion channels in triggering a rapid defense response. A deeper understanding of these early signaling events will eventually help us to minimize herbivory by developing pest management strategies based on plant-herbivore monitoring systems. Such knowledge can be instrumental in the design of plants with improved resistance against herbivores. As such, in the future, it will be important to develop effective small-molecule modulators that can inhibit or enhance the early signaling events in plant-herbivore interactions. Such an approach would not only facilitate research on early plant signaling events but also help in developing novel strategies for the development of herbivore-resistant crops.Recent years have witnessed immense progress in identifying the early defense signaling components in plant defense against herbivores, but studies on the molecular identification and characterization of these components are still a work in progress. However, with the advent of state-of-the-art imaging techniques, physiological and biochemical assays, and genomics may help us to understand the early defense signaling events by coordinating the plasma membrane potential changes, ion channels modulation, intracellular Ca