Phospholipases C and D and Their Role in Biotic and Abiotic Stresses.

Plants, as sessile organisms, have adapted a fine sensing system to monitor environmental changes, therefore allowing the regulation of their responses. As the interaction between plants and environmental changes begins at the surface, these changes are detected by components in the plasma membrane, where a molecule receptor generates a lipid signaling cascade via enzymes, such as phospholipases (PLs). Phospholipids are the key structural components of plasma membranes and signaling cascades. They exist in a wide range of species and in different proportions, with conversion processes that involve hydrophilic enzymes, such as phospholipase-C (PLC), phospholipase-D (PLD), and phospholipase-A (PLA). Hence, it is suggested that PLC and PLD are highly conserved, compared to their homologous genes, and have formed clusters during their adaptive history. Additionally, they generate responses to different functions in accordance with their protein structure, which should be reflected in specific signal transduction responses to environmental stress conditions, including innate immune responses. This review summarizes the phospholipid systems associated with signaling pathways and the innate immune response.

Link between Lipid Second Messengers and Osmotic Stress in Plants.

Plants are subject to different types of stress, which consequently affect their growth and development. They have developed mechanisms for recognizing and processing an extracellular signal. Second messengers are transient molecules that modulate the physiological responses in plant cells under stress conditions. In this sense, it has been shown in various plant models that membrane lipids are substrates for the generation of second lipid messengers such as phosphoinositide, phosphatidic acid, sphingolipids, and lysophospholipids. In recent years, research on lipid second messengers has been moving toward using genetic and molecular approaches to reveal the molecular setting in which these molecules act in response to osmotic stress. In this sense, these studies have established that second messengers can transiently recruit target proteins to the membrane and, therefore, affect protein conformation, activity, and gene expression. This review summarizes recent advances in responses related to the link between lipid second messengers and osmotic stress in plant cells.

Phospholipid signaling pathway in Capsicum chinense suspension cells as a key response to consortium infection.

BACKGROUND: Mexico is considered the diversification center for chili species, but these crops are susceptible to infection by pathogens such as Colletotrichum spp., which causes anthracnose disease and postharvest decay in general. Studies have been carried out with isolated strains of Colletotrichum in Capsicum plants; however, under growing conditions, microorganisms generally interact with others, resulting in an increase or decrease of their ability to infect the roots of C. chinense seedlings and thus, cause disease. RESULTS: Morphological changes were evident 24 h after inoculation (hai) with the microbial consortium, which consisted primarily of C. ignotum. High levels of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA) were found around 6 hai. These metabolic changes could be correlated with high transcription levels of diacylglycerol-kinase (CchDGK1 and CchDG31) at 3, 6 and 12 hai and also to pathogen gene markers, such as CchPR1 and CchPR5. CONCLUSIONS: Our data constitute the first evidence for the phospholipids signalling events, specifically DGPP and PA participation in the phospholipase C/DGK (PI-PLC/DGK) pathway, in the response of Capsicum to the consortium, offering new insights on chilis' defense responses to damping-off diseases.

Phospholipid Signaling Is a Component of the Salicylic Acid Response in Plant Cell Suspension Cultures.

Salicylic acid (SA) is an important signaling molecule involved in plant defense. While many proteins play essential roles in SA signaling, increasing evidence shows that responses to SA appear to involve and require lipid signals. The phospholipid-generated signal transduction involves a family of enzymes that catalyze the hydrolysis or phosphorylation of phospholipids in membranes to generate signaling molecules, which are important in the plant cellular response. In this review, we focus first, the role of SA as a mitigator in biotic/abiotic stress. Later, we describe the experimental evidence supporting the phospholipid-SA connection in plant cells, emphasizing the roles of the secondary lipid messengers (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA)) and related enzymes (phospholipase D (PLD) and phospholipase C (PLC)). By placing these recent finding in context of phospholipids and SA in plant cells, we highlight the role of phospholipids as modulators in the early steps of SA triggered transduction in plant cells.

Heat stress during development affects immunocompetence in workers, queens and drones of Africanized honey bees (Apis mellifera L.) (Hymenoptera: Apidae).

Though social insects generally seem to have a reduced individual immunoresponse compared to solitary species, the impact of heat stress on that response has not been studied. In the honey bee, the effect of heat stress on reproductives (queens and males/drones) may also vary compared to workers, but this is currently unknown. Here, we quantified the activity of an enzyme linked to the immune response in insects and known to be affected by heat stress in solitary species: phenoloxidase (PO), in workers, queens and drones of Africanized honey bees (AHBs) experimentally subjected to elevated temperatures during the pupal stage. Additionally, we evaluated this marker in individuals experimentally infected with the entomopathogenic fungus Metarhizium anisopliae. Differences in PO activity were found between sexes and castes, with PO activity generally higher in workers and lower in reproductives. Such differences are associated with the likelihood of exposure to infection and the role of different individuals in the colony. Contrary to our expectation, heat stress did not cause an increase in PO activity equally in all classes of individual. Heat stress during the pupal stage significantly decreased the PO activity of AHB queens, but not that of workers or drones, which more frequently engage in extranidal activity. Experimental infection with Metarhizium anisopliae reduced PO activity in queens and workers, but increased it in drones. Notably, heat stressed workers lived significantly shorter after infection despite exhibiting greater PO activity than queens or drones. We suggest that this discrepancy may be related to trade-offs among immune response cascades in honey bees such as between heat shock proteins and defensin peptides used in microbial defence. Our results provide evidence for complex relationships among humoral immune responses in AHBs and suggest that heat stress could result in a reduced life expectancy of individuals.

Biochemical characterization of phospholipases C from Coffea arabica in response to aluminium stress.

Signal transduction in plants determines their successful adaptation to diverse stress factors. Our group employed suspension cells to study the phosphoinositide pathway, which is triggered by aluminium stress. We investigated about members of the PI-specific phospholipase C (PLC) family and evaluated their transcription profiles in Coffea arabica (Ca) suspension cells after 14days of culture when treated or not with 100µM AlCl3. The four CaPLC1-4 members showed changes in their transcript abundance upon AlCl3 treatment. The expression profiles of CaPLC1/2 exhibited a rapid and transitory increase in abundance. In contrast, CaPLC3 and CaPLC4 showed that transcript levels were up-regulated in short times (at 30s), while only CaPLC4 kept high levels and CaPLC3 was reduced to basal after 3h of treatment. CaPLC proteins were heterologously expressed, and CaPLC2 and CaPLC4 were tested for in vitro activity in the presence or absence of AlCl3 and compared to Arabidopsis PLC2 (AtPLC2). A crude extract was isolated from coffee cells. CaPLC2 showed a similar inhibition (30%) as in AtPLC2 and in the crude extract, while in CaPLC4, the activity was enhanced by AlCl3. Additionally, we visualized the yellow fluorescent protein PH domain of human PLCδ1 (YFP-PHPLCδ1) subcellular localization in cells that were treated or not with AlCl3. In non-treated cells, we observed a polar fluorescence signal towards the fused membrane. However, when cells were treated with AlCl3, these signals were disrupted. Finally, this is the first time that PLC activity has been shown to be stimulated in vitro by AlCl3.

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

Production of Plant Secondary Metabolites: Examples, Tips and Suggestions for Biotechnologists.

Plants are sessile organisms and, in order to defend themselves against exogenous (a)biotic constraints, they synthesize an array of secondary metabolites which have important physiological and ecological effects. Plant secondary metabolites can be classified into four major classes: terpenoids, phenolic compounds, alkaloids and sulphur-containing compounds. These phytochemicals can be antimicrobial, act as attractants/repellents, or as deterrents against herbivores. The synthesis of such a rich variety of phytochemicals is also observed in undifferentiated plant cells under laboratory conditions and can be further induced with elicitors or by feeding precursors. In this review, we discuss the recent literature on the production of representatives of three plant secondary metabolite classes: artemisinin (a sesquiterpene), lignans (phenolic compounds) and caffeine (an alkaloid). Their respective production in well-known plants, i.e., Artemisia, Coffea arabica L., as well as neglected species, like the fibre-producing plant Urtica dioica L., will be surveyed. The production of artemisinin and caffeine in heterologous hosts will also be discussed. Additionally, metabolic engineering strategies to increase the bioactivity and stability of plant secondary metabolites will be surveyed, by focusing on glycosyltransferases (GTs). We end our review by proposing strategies to enhance the production of plant secondary metabolites in cell cultures by inducing cell wall modifications with chemicals/drugs, or with altered concentrations of the micronutrient boron and the quasi-essential element silicon.

Relationship between aluminum stress and caffeine biosynthesis in suspension cells of Coffea arabica L.

Toxicity by aluminum is a growth-limiting factor in plants cultivated in acidic soils. This metal also promotes signal transduction pathways leading to the biosynthesis of defense compounds, including secondary metabolites. In this study, we observed that Coffea arabica L. cells that were kept in the dark did not produce detectable levels of caffeine. However, irradiation with light and supplementation of the culture medium with theobromine were the best conditions for cell maintenance to investigate the role of aluminum in caffeine biosynthesis. The addition of theobromine to the cells did not cause any changes to cell growth and was useful for the bioconversion of theobromine to caffeine. During a short-term AlCl3-treatment (500µM) of C. arabica cells kept under light irradiation, increases in the caffeine levels in samples that were recovered from both the cells and culture media were evident. This augmentation coincided with increases in the enzyme activity of caffeine synthase (CS) and the transcript level of the gene encoding this enzyme (CS). Together, these results suggest that actions by Al and theobromine on the same pathway lead to the induction of caffeine biosynthesis.

Protoplasts: a friendly tool to study aluminum toxicity and coffee cell viability.

OBJECTIVE: Aluminum toxicity is a major limiting factor with regard to crop production and quality in most acidic soils around the world. We propose the use of C. arabica L. protoplasts to evaluate the toxic effects of aluminum, the nuclear localization of aluminum and propensity of aluminum to cause DNA damage. RESULTS: After protoplasts were exposed to aluminum (Al) for varying periods of time (0, 5, 10, 20 and 30 min), we detected a reduction in protoplast viability. Additionally, we observed a rapid decline in the ability of protoplasts to synthesize DNA following exposure to Al for 30 min. Furthermore, DNA damage was observed after 10 min of treatment with Al. CONCLUSIONS: Protoplasts can be used to evaluate the effects of Al upon entry into the cell, which affects the structure of the nucleus. These results indicate that protoplasts provide a useful model for the study Al toxicity at the cellular level.

Tissue integration and inflammatory reaction in full-thickness abdominal wall repair using an innovative composite mesh.

PURPOSE: When composite meshes are used in abdominal wall repair, seroma formation may persist and delay the desired integration leading to recurrence. This study compares tissue integration and inflammatory response in abdominal wall repair with composites with different absorbable synthetic barriers. METHODS: Full-thickness defects created in the abdominal wall of rabbits were repaired using polypropylene prosthesis or the following composites: Physiomesh™ (Phy); Ventralight™ (Vent) and "new composite mesh" (Ncm) not yet used clinically in humans. The collected seroma was evaluated for IFN-γ/IL-4 by ELISA. Tissue integration, anti- (IL-13/TGFß-1/IL-10/IL-4) and pro-inflammatory (TNF-α/IL-6/IFN-γ/VEGF) cytokine mRNA expression and TGFß/VEGF immunolabeling were evaluated at 14 and 90 days post-implant. RESULTS: Seroma was observed in 10 of 12 Phy/Vent and 4 of 12 Ncm. Wound fluid IFN-γ showed a time-dependent significant increase in Vent and tendency to decrease in Ncm, while all composites exhibited IL-4 upward trend. Prostheses were fully infiltrated by an organized connective tissue at end time although the area had shown prior seroma. A stable mesothelium was developed, except in adhesion areas. Vent/Phy displayed a significant increase in TNF-α/IFN-γ-mRNA over time. Significant decrease in VEGF mRNA was observed in Phy/Ncm, while a significant increase of TGFß-1 mRNA was evident in all composites over time. Ncm exhibited the highest TGFß protein expression area at short term and the greatest percentage of VEGF positive vessels at end time. CONCLUSION: Ncm could be an appropriate candidate to improve clinical outcome showing the lower development of seroma and optimal tissue integration with minimal pro-inflammatory cytokine response over time and consistent pro-wound healing cytokine expression.

Bacterial adhesion to biological versus polymer prosthetic materials used in abdominal wall defect repair: do these meshes show any differences in vitro?

PURPOSE: Although clinical data suggest the similar performance of collagen-based biological prosthetic materials to some polymer materials, the use of a biomesh for abdominal hernia repair in a setting of infection is controversial. This in vitro study compares the adhesion of two Staphylococcus strains to polymer and biological meshes. METHODS: Sterile fragments of Optilene(®) (Op), Surgipro™ (Surg), Preclude(®) (Precl), TIGR(®) (TIGR), Bio-A(®) (BioA), Permacol™ (Perm), Surgisis(®) (SIS), and Tutomesh(®) (Tuto) were inoculated with 10(6) CFU of S. aureus (Sa) or S. epidermidis (Se) (n = 18 per strain per mesh). The first five meshes are polymer materials while Perm, SIS and Tuto are biomeshes. After 24/48 h of incubation, bacterial adhesion was examined by sonication, scanning electron microscopy (SEM) and light microscopy. RESULTS: Sa and Se showed a high affinity for the absorbable meshes (TIGR, BioA, Perm, SIS, Tuto) (p < 0.001). Precl yielded the lowest bacterial loads (p < 0.001). Surg, Precl and BioA underwent no substantial change over time, while Op (p < 0.001) and TIGR (p < 0.05) showed decreasing bacterial loads during incubation. The Sa-contaminated biomeshes behaved similarly while biomeshes inoculated with Se returned higher bacterial yields at 48 h, especially SIS (p < 0.001). SEM and light microscopy observations revealed planktonic bacteria and biofilms on the polymer surface and bacterial niches in biomesh pores. CONCLUSIONS: Within 48 h of contamination, the absorbable polymer and biological meshes exhibited high bacterial loads. Given their lower affinity for both bacterial strains, the conventional non-absorbable polymer materials could be better candidates for use in contaminated surgical fields.

Salicylic-acid elicited phospholipase D responses in Capsicum chinense cell cultures.

The plant response to different stress types can occur through stimulus recognition and the subsequent signal transduction through second messengers that send information to the regulation of metabolism and the expression of defense genes. The phospholipidic signaling pathway forms part of the plant response to several phytoregulators, such as salicylic acid (SA), which has been widely used to stimulate secondary metabolite production in cell cultures. In this work, we studied the effects of SA treatment on [(32)-P]Pi phospholipid turnover and phospholipase D (PLD) activity using cultured Capsicum chinense cells. In cultured cells, the PIP2 turnover showed changes after SA treatment, while the most abundant phospholipids (PLs), such as phosphatidylcholine (PC), did not show changes during the temporal course. SA treatment significantly increased phosphatidic acid (PA) turnover over time compared to control cells. The PA accumulation in cells treated with 1-butanol showed a decrease in messengers; at the same time, there was a 1.5-fold increase in phosphatidylbutanol. These results suggest that the participation of the PLD pathway is a source of PA production, and the activation of this mechanism may be important in the cell responses to SA treatment.

Extraperitoneal and intraperitoneal behavior of several biological meshes currently used to repair abdominal wall defects.

BACKGROUND: This study compares the behavior of several cross- and noncrosslinked biomeshes (Permacol®, CollaMend®, Surgisis®, Tutomesh®, and Strattice®) currently used for abdominal wall repair when implanted intraperitoneally and extraperitoneally. Material and Methods. Intraperitoneal (IP) implants were fixed on the parietal peritoneum and partial abdominal wall defects (EP) were repaired using each of the biomeshes, in the rabbit abdominal wall. After 90 days of implant, the biomeshes were examined to assess biomesh degradation, collagen I and III expression (Sirius red staining) and the host macrophage response (immunohistochemistry). Results. Following implant, the thinner noncrosslinked biomeshes Tutomesh and Surgisis, were almost fully degraded in both models. In contrast, Strattice behavior was similar to crosslinked biomeshes, showing negligible degree of degradation. This mesh also showed high expression of collagen I, similar to the crosslinked. The noncrosslinked materials elicited lower macrophage counts, significantly so for Strattice. In IP and EP models, Permacol showed similarly high macrophages while counts were lower for CollaMend and Surgisis in the EP model. Conclusions. The intra or extraperitoneal implant of the different meshes did not affect host tissue incorporation or mesh degradation. The crosslinked biomeshes induced a more intense macrophage response regardless of their IP or EP location.

Intraperitoneal behaviour of a new composite mesh (Parietex™ Composite Ventral Patch) designed for umbilical or epigastric hernia repair.

INTRODUCTION: The most common treatment option for ventral and umbilical hernias is the implant of a prosthetic mesh. This study compares the behaviour of a new mesh, Parietex™ Composite Ventral Patch (Ptx), with two commercially available meshes, Ventralex™ ST Hernia Patch and Proceed™ Ventral Patch. MATERIALS AND METHODS: The following meshes were tested in a umbilical-hernia repair model using 54 rabbits: Ventralex™ ST Hernia Patch (Vent) (Bard Davol Inc., USA); Proceed™ Ventral Patch (PVP) (Ethicon, USA) and Ptx (Covidien, Sofradim, France) (n = 18 each). At 3, 7 and 14 days postimplantation, peritoneal behaviour and adhesion formation were assessed by sequential laparoscopy. Adhesions were scored for consistency and quantified by image analysis. The animals were euthanized at 2 (n = 27) and 6 weeks (n = 27) postsurgery. Mesothelial cover of meshes and tissue ingrowth were determined by scanning and light microscopy. RESULTS: Seroma was observed in 1/18 Vent, 7/18 PVP and 4/18 Ptx, mainly between the implant and subcutaneous tissue. Firm omental adhesions between the mesh and parietal peritoneum were noted in 2/9 Vent, 6/9 PVP and 3/9 Ptx at 2 weeks and in 3/9 Vent, 5/9 PVP and 1/9 Ptx at 6 weeks. Three (out of 9) encapsulated PVP implants showed "tissue-integrated" adhesions affecting the intestinal loops. No differences between implants were detected in the surface area occupied by adhesions at 2 weeks, though at 6 weeks, percentages were significantly higher (p < 0.01; Mann-Whitney U test) for PVP compared to Ptx or Vent. At this time point, Ptx and Vent showed good host tissue incorporation and optimal mesothelialization. CONCLUSIONS: The PVP implants showed greater adhesion formation than the other materials. Postimplantation behaviour was comparable for Ptx and Vent including scarce adhesion formation and optimal mesothelialization. Regarding tissue integration, Ptx showed greater long-term collagenization of the neoformed tissue.

Short- and long-term biomechanical and morphological study of new suture types in abdominal wall closure.

To perform an abdominal-wall closure, a continuous suture is the preferred method. The suture materials that are most commonly employed in abdominal surgery are polypropylene and polydioxanone. However, in recent times, new products have been marketed, such as non-absorbable polyurethane with elastic properties (Assuplus(®), Assut Europe, Italy) and absorbable barbed polydioxanone (Filbloc(®), Assut Europe, Italy). The purpose of this study was to compare the ability of those against the standard polypropylene (Surgipro(TM), Covidien, USA) and polydioxanone (Assufil(®), Assut Europe, Italy) to mimic the biomechanical behavior of the abdominal wall closure. Comparison of the sutures was made first with the materials alone and later in a laparotomy closure of a rabbit abdomen, used as an animal model. The biomechanical analysis consisted of uniaxial tensile tests of threads and sutured samples of the animal abdomen. In the latter case, results were analyzed at short- (21days) and long- (180days) term intervals after the surgery. The morphology studies and collagen expression of the samples were also investigated. The results determined that polydioxanone and polypropylene sutures showed a linear elastic behavior, with barbed polydioxanone as the most compliant suture and polyurethane as the stiffest. The sutured samples showed a statistically significant loss of resistance, measured as the load needed to perform a certain stretch, when compared with the corresponding control tissue. Analysis of the stress-stretch curves showed that elastic polyurethane was the only suture able to reproduce the mechanical behavior of healthy tissue in the short term, while the rest of the sutures remained less stiff. This coincides with the expression of type I collagen observed in this group at this point in the study. In the long term, there was no difference among the sutures, and none was able to mimic control behavior.

Differential effects of aluminum on in vitro primary root growth, nutrient content and phospholipase C activity in coffee seedlings (Coffea arabica).

Coffea arabica is a woody species that grows in acid soils, where aluminum is available and may affect growth and productivity. To determine the effect of aluminum on primary root growth of C. arabica cv. Typica, seedlings were exposed over 30 days to different concentrations of AlCl3 (0, 100, 300 and 500 µM) in vitro. The aluminum effect on primary root growth was dose-dependent: low aluminum concentrations (100 and 300 µM) stimulated primary root growth (6.98 ± 0.15 and 6.45 ± 0.17 cm, respectively) compared to the control (0 µM; 5.24 ± 0.17 cm), while high concentrations (500 µM) induced damage to the root tips and inhibition of primary root growth (2.96 ± 0.28 cm). Aluminum (100 µM) also increased the K and Ca contents around 33% and 35% in the coffee roots. It is possible that aluminum toxicity resides in its association with cell nuclei in the meristematic region of the root. Additionally, after 30 days of treatment with aluminum, two different effects could be observed on phospholipase C (PLC) activity. In shoots, aluminum concentrations ≥ 300 µM inhibited more than 50% of PLC activity. In contrast, in roots a contrasting behavior was determined: low (100 µM) and toxic concentrations (500 µM) increased the activity of PLC (100%). These results suggest the possible involvement of the phosphoinositide signal transduction pathway, with the phospholipase C enzyme participating in the beneficial and toxic effects of aluminum in plants.

Long term behavior of biological prostheses used as abdominal wall substitutes.

BACKGROUND: Despite their degradation in the host organism, the benefits of collagen bioprostheses remain unclear. This study addresses the absorption and long-term host tissue incorporation of several collagen biomeshes. MATERIAL AND METHODS: Partial ventral hernial defects created in the abdominal wall of rabbits were repaired using the crosslinked meshes Permacol® or CollaMend®, or the non-crosslinked Surgisis®, Tutomesh® or Strattice®. After 90 and 180 days of implant, morphological studies and morphometric analysis of the thickness of the meshes were performed. Immunofluorescence confocal microscopy combined with differential interference contrast (DIC) imaging was used to distinguish newly formed collagen from that comprising the mesh. The macrophage response was examined by immunohistochemistry. RESULTS: At 90 days, the thinner non-crosslinked biomeshes Tutomesh and Surgisis were more fully degraded with much of their collagen replaced with loose connective tissue. By 180 days, both implants had been practically fully absorbed. In contrast, in Strattice only the outermost third was infiltrated by neoformed tissue. On both surfaces of the crosslinked meshes, a fibrous capsule with host cells lining its perimeter was observed at both time points, though at 180 days these cells had penetrated the mesh interior. At both implant times, Strattice showed the higher expression of collagen type I while collagen III expression was similar for all the meshes. The non-crosslinked materials elicited lower macrophage counts at both time points, significantly so for Strattice. The macrophage response decreased over time for all the meshes but Surgisis. CONCLUSIONS: Strattice, the thicker, more compacted non-crosslinked mesh showed the best balance between tissue incorporation and absorption while eliciting a minimal foreign-body reaction in the long-term.