Mechanical wounding improves salt tolerance by maintaining root ion homeostasis in a desert shrub.

Soil salinization, especially in arid environments, is a leading cause of land degradation and desertification. Excessive salt in the soil is detrimental to plants. Plants have developed various sophisticated regulatory mechanisms that allow them to withstand adverse environments. Through cross-adaptation, plants improve their resistance to an adverse condition after experiencing a different kind of adversity. Our analysis of Ammopiptanthus nanus, a desert shrub, showed that mechanical wounding activates the biosynthesis of jasmonic acid (JA) and abscisic acid (ABA), enhancing plasma membrane H+-ATPase activity to establish an electrochemical gradient that promotes Na+ extrusion via Na+/H+ antiporters. Mechanical wounding reduces K+ loss under salt stress, improving the K/Na and maintaining root ion balance. Meanwhile, mechanical damage enhances the activity of antioxidant enzymes and the content of osmotic substances, working together with cellular ions to alleviate water loss and growth inhibition under salt stress. This study provides new insights and approaches for enhancing salt tolerance and stress adaptation in plants by elucidating the signaling mechanisms of cross-adaptation.

What keeps the style under tension? Experimental tests to understand the biomechanics of the explosive style movement in Marantaceae.

Pollination in Marantaceae is mediated by an explosive style movement. Before release, style tension is held by the hooded staminode. When a pollinator touches the trigger appendage of the hooded staminode the latter deforms and the style rapidly curls upwards. This movement has been interpreted as a turgor movement by some authors, but recent studies clearly indicate that setup, hold and release of tension are purely mechanical processes. However, in view of the high diversity of hooded staminodes, the question arises what keeps the tension in species with very thin staminodes. To test the holding mechanisms, we conducted mechanical and physico-chemical release experiments in four species with robust and four species with thin hooded staminodes in their natural tropical environment. We found almost the same response of all species to mechanical treatments, but species-specific reactions to different physico-chemical conditions. This indicates that style release follows the same mechanical principles in all species, but that the sensitivity of the explosive movement depends on material properties like tissue thickness and turgescence. As to the holding mechanisms, we found different degrees of floral synorganization. The hood of the hooded staminode formerly interpreted as an important holding structure does not play a noteworthy role. Instead, the basal plate of the hooded staminode antagonises the pressure of the style head against the holding point of the hooded staminode in species with robust hooded staminodes and well-developed basal plates. In some species with a thin hooded staminode, the latter is closely attached to the style and most likely stabilises tension by adhesive forces. In another species, a morphologically analogous structure adopts the function of the basal plate. We conclude that the holding mechanism of the style tension diversified during the evolution of Marantaceae whereas the release mechanism itself has been conserved throughout the family.

Wound-Induced Temporal Reprogramming of Gene Expression during Agarwood Formation in Aquilaria sinensis.

Agarwood is a resinous heartwood of Aquilaria sinensis that is formed in response to mechanical wounding. However, the transcriptional response of A. sinensis to mechanical wounding during the agarwood formation process is still unclear. Here, three five-year-old A. sinensis trees were mechanically damaged by a chisel, and time-series transcriptomic analysis of xylem tissues in the treated area (TA) was performed at 15 (TA1), 70 (TA2) and 180 days after treatment (TA3). Samples from untreated areas at the corresponding time points (UA1, UA2, UA3, respectively) were collected as controls. A total of 1862 (TA1 vs. UA1), 961 (TA2 vs. UA2), 1370 (TA3 vs. UA3), 3305 (TA2 vs. TA1), 2625 (TA3 vs. TA1), 2899 (TA3 vs. TA2), 782 (UA2 vs. UA1), 4443 (UA3 vs. UA1) and 4031 (UA3 vs. UA2) genes were differentially expressed (DEGs). Functional enrichment analysis showed that DEGs were significantly enriched for secondary metabolic processes, signal transduction and transcriptional regulation processes. Most of the genes involved in lignin biosynthesis were more abundant in the TA groups, which included phenylalanine ammonia-lyase, 4-coumarate CoA ligase, cinnamate 4-hydroxylase, caffeoyl-CoA O-methyltransferase and cinnamoyl-CoA reductase. DEGs involved in sesquiterpene biosynthesis were also identified. Hydroxymethylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, phosphomevalonate kinase and terpene synthase genes were significantly increased in the TA groups, promoting sesquiterpene biosynthesis in the wounded xylem tissues. The TF-gene transcriptomic networks suggested that MYB DNA-binding, NAM, WRKY, HLH and AP2 TFs co-expressed with genes related to lignin and sesquiterpene synthesis, indicating their critical regulatory roles in the biosynthesis of these compounds. Overall, our study reveals a dynamic transcriptional response of A. sinensis to mechanical wounding, provides a resource for identifying candidate genes for molecular breeding of agarwood quality, and sheds light on the molecular mechanisms of agarwood formation in A. sinensis.

Metabolomic responses to the mechanical wounding of Catharanthus roseus' upper leaves.

Purpose: Plant secondary metabolites are used to treat various human diseases. However, it is difficult to produce a large number of specific metabolites, which largely limits their medicinal applications. Many methods, such as drought and nutrient application, have been used to induce the biosynthetic production of secondary metabolites. Among these secondary metabolite-inducing methods, mechanical wounding maintains the composition of secondary metabolites with little potential risk. However, the effects of mechanical stress have not been fully investigated, and thus this method remains widely unused. Methods: In this study, we used metabolomics to investigate the metabolites produced in the upper and lower leaves of Catharanthus roseus in response to mechanical wounding. Results: In the upper leaves, 13 different secondary metabolites (three terpenoid indole alkaloids and 10 phenolic compounds) were screened using an orthogonal partial least squares discriminant analysis (OPLS-DA) score plot. The mechanical wounding of different plant parts affected the production of secondary metabolites. Specifically, when lower leaves were mechanically wounded, the upper leaves became a strong source of resources. Conversely, when upper leaves were injured, the upper leaves themselves became a resource sink. Changes in the source-sink relationship reflected a new balance between resource tradeoff and the upregulation or downregulation of certain metabolic pathways. Conclusion: Our findings suggest that mechanical wounding to specific plant parts is a novel approach to increase the biosynthetic production of specific secondary metabolites. These results indicate the need for a reevaluation of production practices for secondary metabolites from select commercial plants.

RNA-Sequencing Reveals the Involvement of Sesquiterpene Biosynthesis Genes and Transcription Factors during an Early Response to Mechanical Wounding of Aquilaria sinensis.

Plants respond to wounding by reprogramming the expression of genes involved in secondary metabolism. Aquilaria trees produce many bioactive secondary metabolites in response to wounding, but the regulatory mechanism of agarwood formation in the early response to mechanical wounding has remained unclear. To gain insights into the process of transcriptome changes and to determine the regulatory networks of Aquilaria sinensis to an early response (15 days) to mechanical wounding, we collected A. sinensis samples from the untreated (Asc1) and treated (Asf1) xylem tissues and performed RNA sequencing (RNA-seq). This generated 49,102,523 (Asc1) and 45,180,981 (Asf1) clean reads, which corresponded to 18,927 (Asc1) and 19,258 (Asf1) genes, respectively. A total of 1596 differentially expressed genes (DEGs) were detected in Asf1 vs. Asc1 (|log2 (fold change)| ≥ 1, Padj ≤ 0.05), of which 1088 were up-regulated and 508 genes were down-regulated. GO and KEGG enrichment analysis of DEGs showed that flavonoid biosynthesis, phenylpropanoid biosynthesis, and sesquiterpenoid and triterpenoid biosynthesis pathways might play important roles in wound-induced agarwood formation. Based on the transcription factor (TF)-gene regulatory network analysis, we inferred that the bHLH TF family could regulate all DEGs encoding for farnesyl diphosphate synthase, sesquiterpene synthase, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS), which contribute to the biosynthesis and accumulation of agarwood sesquiterpenes. This study provides insight into the molecular mechanism regulating agarwood formation in A. sinensis, and will be helpful in selecting candidate genes for improving the yield and quality of agarwood.

The mechanisms and prediction of non-structural carbohydrates accretion and depletion after mechanical wounding in slash pine (Pinus elliottii) using near-infrared reflectance spectroscopy.

BACKGROUND: The allocation of non-structural carbohydrates (NSCs) plays a critical role in the physiology and metabolism of tree growth and survival defense. However, little is known about the allocation of NSC after continuous mechanical wounding of pine by resin tapping during tree growth. RESULTS: Here, we examine the NSC allocation in plant tissues after 3 year lasting resin tapping, and also investigate the use of near-infrared reflectance (NIR) spectroscopy to quantify the NSC, starch and free sugar (e.g., sucrose, glucose, and fructose) concentrations in different plant tissues of slash pine. Spectral measurements on pine needle, branch, trunk phloem, and root were obtained before starch and free sugar concentrations were measured in the laboratory. The variation of NSC, starch and free sugars in different plant tissues after resin tapping was analyzed. Partial least squares regression was applied to calibrate prediction models, models were simulated 100 times for model performance and error estimation. More NSC, starch and free sugars were stored in winter than summer both in tapped and control trees. The position of resin tapping significantly influenced the NSCs allocation in plant tissues: more NSCs were transformed into free sugars for defensive resin synthesis close to the tapping wound rather than induced distal systemic responses. Models for predicting NSC and free sugars of plant tissues showed promising results for the whole tree for fructose (R2CV = 0.72), glucose (R2CV = 0.67), NSCs (R2CV = 0.66) and starch (R2CV = 0.58) estimates based on NIR models. Models for individual plant tissues also showed reasonable predictive ability: the best model for NSCs and starch prediction was found in root. The significance multivariate correlation algorithm for variable selection significantly reduced the number of variables. Important variables were identified, including features at 1021-1290 nm, 1480, 1748, 1941, 2020, 2123 and 2355 nm, which are highly related to NSC, starch, fructose, glucose and sucrose. CONCLUSIONS: NIR spectroscopy provided a rapid and cost-effective method to monitor NSC, starch and free sugar concentrations after continuous resin tapping. It can be used for studying the trade-off between growth and production of defensive metabolites.

The Arabinogalactan Protein Family of Centaurium erythraea Rafn.

Centaurium erythraea (centaury) is a medicinal plant with exceptional developmental plasticity in vitro and vigorous, often spontaneous, regeneration via shoot organogenesis and somatic embryogenesis, during which arabinogalactan proteins (AGPs) play an important role. AGPs are highly glycosylated proteins belonging to the super family of O-glycosylated plant cell surface hydroxyproline-rich glycoproteins (HRGPs). HRGPs/AGPs are intrinsically disordered and not well conserved, making their homology-based mining ineffective. We have applied a recently developed pipeline for HRGP/AGP mining, ragp, which is based on machine learning prediction of proline hydroxylation, to identify HRGP sequences in centaury transcriptome and to classify them into motif and amino acid bias (MAAB) classes. AGP sequences with low AG glycomotif representation were also identified. Six members of each of the three AGP subclasses, fasciclin-like AGPs, receptor kinase-like AGPs and AG peptides, were selected for phylogenetic and expression analyses. The expression of these 18 genes was recorded over 48 h following leaf mechanical wounding, as well as in 16 tissue samples representing plants from nature, plants cultivated in vitro, and developmental stages during shoot organogenesis and somatic embryogenesis. None of the selected genes were upregulated during both wounding recovery and regeneration. Possible functions of AGPs with the most interesting expression profiles are discussed.

Molecular Mechanism Underlying Mechanical Wounding-Induced Flavonoid Accumulation in Dalbergia odorifera T. Chen, an Endangered Tree That Produces Chinese Rosewood.

Dalbergia odorifera, a critically endangered tree species, produces heartwood containing a vast variety of flavonoids. This heartwood, also known as Chinese rosewood, has high economic and medicinal value, but its formation takes several decades. In this study, we showed that discolored wood induced by pruning displays similar color, structure, and flavonoids content to those of natural heartwood, suggesting that wounding is an efficient method for inducing flavonoid production in D. odorifera. Transcriptome analysis was performed to investigate the mechanism underlying wounding-induced flavonoids production in D. odorifera heartwood. Wounding upregulated the expression of 90 unigenes, which covered 19 gene families of the phenylpropanoid and flavonoid pathways, including PAL, C4H, 4CL, CHS, CHI, 6DCS, F3'5'H, F3H, FMO, GT, PMAT, CHOMT, IFS, HI4'OMT, HID, IOMT, I2'H, IFR, and I3'H. Furthermore, 47 upregulated unigenes were mapped to the biosynthesis pathways for five signal molecules (ET, JA, ABA, ROS, and SA). Exogenous application of these signal molecules resulted in the accumulation of flavonoids in cell suspensions of D. odorifera, supporting their role in wounding-induced flavonoid production. Insights from this study will help develop new methods for rapidly inducing the formation of heartwood with enhanced medicinal value.

SpitWorm, a Herbivorous Robot: Mechanical Leaf Wounding with Simultaneous Application of Salivary Components.

Induction of jasmonate-mediated plant defense against insect herbivory is initiated by a combination of both mechanical wounding and chemical factors. In order to study both effects independently on plant defense induction, SpitWorm, a computer-controlled device which mimics the damage pattern of feeding insect larvae on leaves and, in addition, can apply oral secretions (OS) or other solutions to the 'biting site' during 'feeding,' was developed and evaluated. The amount of OS left by a Spodoptera littoralis larva during feeding on Phaseolus lunatus (lima bean) leaves was estimated by combining larval foregut volume, biting rate, and quantification of a fluorescent dye injected into the larvae's foregut prior to feeding. For providing OS amounts by SpitWorm equivalent to larval feeding, dilution and delivery rate were optimized. The effectiveness of SpitWorm was tested by comparing volatile organic compounds (VOC) emissions of P. lunatus leaves treated with either SpitWorm, MecWorm, or S. littoralis larvae. Identification and quantification of emitted VOCs revealed that SpitWorm induced a volatile bouquet that is qualitatively and quantitatively similar to herbivory. Additionally, RT-qPCR of four jasmonic acid responsive genes showed that SpitWorm, in contrast to MecWorm, induces the same regulation pattern as insect feeding. Thus, SpitWorm mimics insect herbivory almost identically to real larvae feeding.

Double benefits of mechanical wounding in enhancing chilling tolerance and lodging resistance in wheat plants.

Chilling and lodging are major threats to wheat production. However, strategies that can be used to effectively mitigate the adverse effects of these threats are still far from clear. Mechanical wounding is a traditional agronomic measure, whereas information about the role it plays in wheat chilling and lodging is scant. The aim of the present study was to investigate mechanisms underlying the protective roles of mechanical wounding in alleviating damage from chilling at jointing stage and enhancing lodging resistance after anthesis of winter wheat (Triticum aestivum L.). Our data show that net photosynthesis rate, maximum photochemical efficiency of photosystem II, activity of the antioxidant enzymes and osmolytes were significantly increased in the latest fully expanded leaves of wounded plants under chilling. Wounding also reduced hydrogen peroxide accumulation, electrolyte leakage and water loss in wounded plants. Moreover, mechanical wounding significantly reduced the length but increased the diameter and wall thickness of the basal second internode of the main stem. Quantitative and histochemical analysis further indicated that wounding increased lignin accumulation and activity of enzymes involved in lignin synthesis, which resulted in increased mechanical strength and the lodging resistance index in the main stem. We conclude from our data that mechanical wounding confers both cold tolerance by alleviating the damage caused by chilling at jointing stage and lodging resistance after anthesis of wheat plants.

The formation and accumulation of protein-networks by physical interactions in the rapid occlusion of laticifer cells in rubber tree undergoing successive mechanical wounding.

BACKGROUND: Although the wound response of plants has been extensively studied, little is known of the rapid occlusion of wounded cell itself. The laticifer in rubber tree is a specific type of tissue for natural rubber biosynthesis and storage. In natural rubber production, tapping is used to harvest the latex which flows out from the severed laticifer in the bark. Therefore, study of the rapid wound-occlusion of severed laticifer cells is important for understanding the rubber tree being protected from the continuously mechanical wounding. RESULTS: Using cytological and biochemical techniques, we revealed a biochemical mechanism for the rapid occlusion of severed laticifer cells. A protein-network appeared rapidly after tapping and accumulated gradually along with the latex loss at the severed site of laticifer cells. Triple immunofluorescence histochemical localization showed that the primary components of the protein-network were chitinase, ß-1,3-glucanase and hevein together with pro-hevein (ProH) and its carboxyl-terminal part. Molecular sieve chromatography showed that the physical interactions among these proteins occurred under the condition of neutral pH. The interaction of ß-1,3-glucanase respectively with hevein, chitinase and ProH was testified by surface plasmon resonance (SPR). The interaction between actin and ß-1,3-glucanase out of the protein inclusions of lutoids was revealed by pull-down. This interaction was pharmacologically verified by cytochalasin B-caused significant prolongation of the duration of latex flow in the field. CONCLUSIONS: The formation of protein-network by interactions of the proteins with anti-pathogen activity released from lutoids and accumulation of protein-network by binding to the cytoskeleton are crucial for the rapid occlusion of laticifer cells in rubber tree. The protein-network at the wounded site of laticifer cells provides not only a physical barrier but also a biochemical barrier to protect the wounded laticifer cells from pathogen invasion.

Unloading of intercellular tension induces the directional translocation of PKCα.

The migration of endothelial cells (ECs) is closely associated with a Ca2+ -dependent protein, protein kinase Cα (PKCα). The disruption of intercellular adhesion by single-cell wounding has been shown to induce the directional translocation of PKCα. We hypothesized that this translocation of PKCα is induced by mechanical stress, such as unloading of intercellular tension, or by intercellular communication, such as gap junction-mediated and paracrine signaling. In the current study, we found that the disruption of intercellular adhesion induced the directional translocation of PKCα even when gap junction-mediated and paracrine signaling were inhibited. Conversely, it did not occur when the mechanosensitive channel was inhibited. In addition, the strain field of substrate attributable to the disruption of intercellular adhesion tended to be larger at the areas corresponding with PKCα translocation. Recently, we found that a direct mechanical stimulus induced the accumulation of PKCα at the stimulus area, involving Ca 2+ influx from extracellular space. These results indicated that the unloading of intercellular tension induced directional translocation of PKCα, which required Ca 2+ influx from extracellular space. The results of this study indicate the involvement of PKCα in the Ca 2+ signaling pathway in response to mechanical stress in ECs.

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Insect herbivore feeding causes mechanical damage to plants, which can activate plant defense responses. Whether symbiosis with beneficial microorganisms can enhance the responses of plants to mechanical damage is of importance for plant anti-herbivore resistance. In this study, defense responses of tomato (Lycopersicon esculentum) plants to mechanical wounding was investigated after the tomato roots being infected by arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae. The results showed that in response to leaf mechanical wounding, the activities of phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and catalase (CAT) in the leaves of tomato pre-inoculated with AMF (FD), as well as transcript levels of genes encoding phenylalanine ammonia lyase (PAL) and ß-1,3-glucanase (PR2) in the leaves and roots were significantly higher in relative to sole mechanical wounding (D), sole mycorrhizal inoculation (F), and control without mechanical wounding and mycorrhizal inoculation (CK). Although the activities of protective enzyme and transcript levels of the two defense-related genes were induced in the plants of sole mechanical wounding (D) and sole mycorrhizal inoculation (F), the induction was faster and stronger in the plants with leaf mechanical wounding and mycorrhizal pre-inoculation (FD). Our findings indicated that arbuscular mycorrhizal colonization could prime quicker and stronger defense responses of tomato plants to mechanical damage.

The Role of Hydrogen Peroxide in Mediating the Mechanical Wounding-Induced Freezing Tolerance in Wheat.

Systemic wound response (SWR), a well-characterized systemic signaling response, plays crucial roles in plant defense responses. Progress in understanding of the SWR in abiotic stress has also been aided by the researchers. However, the function of SWR in freezing stress remains elusive. In this study, we showed that local mild mechanical wounding enhanced freezing tolerance in newly occurred systemic leaves of wheat plants (Triticum aestivum L.). Wounding significantly increased the maximal photochemical efficiency of photosystem II, net photosynthetic rate, and the activities of the antioxidant enzymes under freezing stress. Wounding also alleviated freezing-induced chlorophyll decomposition, electrolyte leakage, water lose, and membrane peroxidation. In addition, wounding-induced freezing stress mitigation was closely associated with the ratio between reduced glutathione (GSH) and oxidized glutathione (GSSG), and the ratio between ascorbate (AsA) and dehydroascorbate (DHA), as well as the contents of total soluble sugars and free amino acids. Importantly, pharmacological study showed that wounding-induced freezing tolerance was substantially arrested by pretreatment of wheat leaves with the scavenger of hydrogen peroxide (H2O2) or the inhibitor of NADPH oxidase (RBOH). These results support the hypothesis that local mechanical wounding-induced SWR in newly occurred leaves is largely attributed to RBOH-dependent H2O2 production, which may subsequently induce freezing tolerance in wheat plants. This mechanism may have a potential application to reduce the yield losses of wheat under late spring freezing conditions. Highlights: In our previous research, we found that local mechanical wounding could induce freezing tolerance in the upper systemic leaves of wheat plants. Surprisingly, in this paper, we further demonstrated that local mechanical wounding could also increase freezing resistance in newly occurred leaves of wheat plants. RBOH mediated H2O2 and ascorbate-glutathione cycle participate in this systemic wound response.

Mechanical stress rapidly induces E-resveratrol and E-piceatannol biosynthesis in grape canes stored as a freshly-pruned byproduct.

Grape canes represent a promising source of bioactive phytochemicals. However the stabilization of the raw material after pruning remains challenging. We recently reported the induction of stilbenoid metabolism after winter pruning including a strong accumulation of E-resveratrol and E-piceatannol during the first six weeks of storage. In the present study, the effect of mechanical wounding on freshly-pruned canes was tested to increase the induction of stilbenoid metabolism. Cutting the grape canes in short segments immediately after pruning triggered a transient expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes, followed by a rapid accumulation of E-resveratrol and E-piceatannol. The degree of stilbenoid induction was related to the intensity of mechanical wounding. Data suggest that a global defense response is triggered involving jasmonate signaling, PR proteins and stilbenoid metabolism. Mechanical wounding of freshly-pruned canes drastically shortens the time required to reach maximal stilbenoid accumulation from 6 to 2weeks.

Mechanical wounding promotes local and long distance response in the halophyte Cakile maritima through the involvement of the ROS and RNS metabolism.

Mechanical wounding in plants, which are capable of generating defense responses possibly associated with nitro-oxidative stress, can be caused by (a)biotic factors such as rain, wind, herbivores and insects. Sea rocket (Cakile maritima L.), a halophyte plant belonging to the mustard family Brassicaceae, is commonly found on sandy coasts throughout Europe. Using 7-day-old Cakile maritima L. seedlings, mechanical wounding was induced in hypocotyls by pinching with a striped-tip forceps; after 3 h, several biochemical parameters were analyzed in both the damaged and unwounded organs (green cotyledons and roots). We thus determined NO production, H2O2 content, lipid oxidation as well as protein nitration patterns; we also identified several antioxidant enzymes including catalase, superoxide dismutase (SOD) isozymes, peroxidases, ascorbate-glutathione cycle enzymes and NADP-dehydrogenases. All these parameters were differentially modulated in the damaged (hypocotyls) and unwounded organs, which clearly indicated an induction of CuZnSOD V in the three organs, an increase in protein nitration in green cotyledons and an induction of NADP-isocitrate dehydrogenase activity in roots. On the whole, our results indicate that the wounding of hypocotyls, which showed an active ROS metabolism and oxidative stress, causes long-distance signals that also trigger responses in unwounded tissues with a more active RNS metabolism. These data therefore confirm the existence of local and long-distance responses which counteract negative effects and provide appropriate responses, enabling the wounded seedlings to survive.

Mechanical Barriers Restrict Invasion of Herpes Simplex Virus 1 into Human Oral Mucosa.

Oral mucosa is one of the main target tissues of the human pathogen herpes simplex virus 1 (HSV-1). How the virus overcomes the protective epithelial barriers and penetrates the tissue to reach its receptors and initiate infection is still unclear. Here, we established an ex vivo infection assay with human oral mucosa that allows viral entry studies in a natural target tissue. The focus was on the susceptibility of keratinocytes in the epithelium and the characterization of cellular receptors that mediate viral entry. Upon ex vivo infection of gingiva or vestibular mucosa, we observed that intact human mucosa samples were protected from viral invasion. In contrast, the basal layer of the oral epithelium was efficiently invaded once the connective tissue and the basement membrane were removed. Later during infection, HSV-1 spread from basal keratinocytes to upper layers, demonstrating the susceptibility of the stratified squamous epithelium to HSV-1. The analysis of potential receptors revealed nectin-1 on most mucosal keratinocytes, whereas herpesvirus entry mediator (HVEM) was found only on a subpopulation of cells, suggesting that nectin-1 acts as primary receptor for HSV-1 in human oral mucosa. To mimic the supposed entry route of HSV-1 via microlesions in vivo, we mechanically wounded the mucosa prior to infection. While we observed a limited number of infected keratinocytes in some wounded mucosa samples, other samples showed no infected cells. Thus, we conclude that mechanical wounding of mucosa is insufficient for the virus to efficiently overcome epithelial barriers and to make entry-mediating receptors accessible.IMPORTANCE To invade the target tissue of its human host during primary infection, herpes simplex virus (HSV) must overcome the epithelial barriers of mucosa, skin, or cornea. For most viruses, the mechanisms underlying the invasion into the target tissues of their host organism are still open. Here, we established an ex vivo infection model of human oral mucosa to explore how HSV can enter its target tissue. Our results demonstrate that intact mucosa samples and even compromised tissue allow only very limited access of HSV to keratinocytes. Detailed understanding of barrier functions is an essential precondition to unravel how HSV bypasses the barriers and approaches its receptors in tissue and why it is beneficial for the virus to use a cell-cell adhesion molecule, such as nectin-1, as a receptor.

Nitric Oxide and Hydrogen Peroxide Mediate Wounding-Induced Freezing Tolerance through Modifications in Photosystem and Antioxidant System in Wheat.

Mechanical wounding is a common stress caused by herbivores or manual and natural manipulations, whereas its roles in acclimation response to a wide spectrum of abiotic stresses remain unclear. The present work showed that local mechanical wounding enhanced freezing tolerance in untreated systemic leaves of wheat plants (Triticum aestivum L.), and meanwhile the signal molecules hydrogen peroxide (H2O2) and nitric oxide (NO) were accumulated systemically. Pharmacological study showed that wounding-induced NO synthesis was substantially arrested by pretreatment with scavengers of reactive oxygen species and an inhibitor of NADPH oxidase (respiratory burst oxidase homolog, RBOH). On the contrary, wounding-induced H2O2 accumulation was not sensitive to NO synthetic inhibitors or scavenger, indicating that H2O2 acts upstream of NO in wounding signal transduction pathways. Cytochemical and vascular tissues localizations approved that RBOH-dependent H2O2 acts as long-distance signal in wounding response. Transcriptome analysis revealed that 279 genes were up-regulated in plants treated with wounding and freezing, but not in plants treated with freezing alone. Importantly, freezing- and wounding-induced genes were significantly enriched in the categories of "photosynthesis" and "signaling." These results strongly supported that primary mechanical wounding can induce freezing tolerance in wheat through the systemic accumulation of NO and H2O2, and further modifications in photosystem and antioxidant system.

The Hippeastrum hybridum PepR1 gene (HpPepR1) encodes a functional guanylyl cyclase and is involved in early response to fungal infection.

It is generally known that cyclic GMP widespread in prokaryotic and eukaryotic cells, is involved in essential cellular processes and stress signal transduction. However, in contrast to animals the knowledge about plant guanylyl cyclases (GCs) which catalyze the formation of cGMP from GTP is still quite obscure. Recent studies of plant GCs are focused on identification and functional analysis of a new family of membrane proteins called "moonlighting kinases with GC activity" with guanylyl cyclase catalytic center encapsulated within intracellular kinase domain. Here we report identification and characterization of plasma membrane receptor of peptide signaling molecules - HpPepR1 in Hippeastrum hybridum. Both bioinformatic analysis of amimo acid sequence and in vitro studies revealed that the protein can act as guanylyl cyclase. The predicted amino acid sequence contains highly conserved 14 aa-long search motif in the catalytic center of GCs from lower and higher eukaryotes. Here, we provide experimental evidence to show that the intracellular domain of HpPepR1 can generate cGMP in vitro. Moreover, it was shown that the accumulation of HpPepR1 transcript was sharply increased after Peyronellaea curtisii (=Phoma narcissi) fungal infection, whereas mechanical wounding has no influence on expression profile of studied gene. These results may indicate the participation of cGMP-dependent pathway in rapid, alarm plant reactions induced by pathogen infection.

Lipid, phenol and carotenoid changes in 'Bianca' grapevine leaves after mechanical wounding: a case study.

Metabolic changes can occur in plants in response to abiotic stress. Extensive use of leaf discs (mechanical wounding) in studies regarding the effect on the biochemical response of the grapevine to different types of biotic stress makes it necessary to understand metabolic perturbation after injury. In this study, we investigate how mechanical wounding can affect the metabolism of grapevine leaf tissue using Bianca variety as case study. Two sizes of leaf discs (1.1 and 2.8 cm in diameter) were excised from leaves, and phenol, lipid and carotenoid perturbation were investigated 0, 6, 12, 24, 48, 96 and 120 h post cutting. In our study, we found an accumulation of molecules belonging to stilbenoid and stilbene classes such as trans-resveratrol, trans-piceide, Z-miyabenol C, E-cis-miyabenol C and ampelopsin D + quadrangularin A after abiotic stress. The increase in fatty acids such as linoleic acid, linolenic acid and oleic + cis-vaccenic acid during the first 12 h after injury, followed by a return to basal level, allowed us to surmise their role in response to abiotic stress, in particular to mechanical wounding in grapevine leaves. Different-sized discs caused a different response to the tissue, with a higher accumulation in 1.1-cm-diameter discs, especially of phenol compounds. The results of this work can be used to better understand metabolic changes due to biotic stress, having previous knowledge about the perturbation caused by abiotic stress.