Edible Medicinal Guava Fruit (Psidium guajava L.) Are a Source of Anti-Biofilm Compounds against Pseudomonas aeruginosa.

Psidium guajava is one of the most common edible medicinal plants frequently used in Malagasy traditional medicine to treat gastrointestinal infections. In order to evaluate their probable antibacterial activities, three organic extracts (successive extractions by hexane, dichloromethane, and ethanol) of ripe guava fruits were assessed for their bactericidal and anti-virulence properties against P. aeruginosa PAO1. Although these three extracts have shown no direct antibacterial activity (MIC of 1000 µg/mL) and, at the non-bactericidal concentration of 100 µg/mL, no impact on the production of major P. aeruginosa PAO1 virulence factors (pyocyanin and rhamnolipids), the hexane and dichloromethane extracts showed significant anti-biofilm properties and the dichloromethane extract disrupted the P. aeruginosa PAO1 swarming motility. Bioguided fractionation of the dichloromethane extract led to the isolation and identification of lycopene and ß-sitosterol-ß-D-glucoside as major anti-biofilm compounds. Interestingly, both compounds disrupt P. aeruginosa PAO1 biofilm formation and maintenance with IC50 of 1383 µM and 131 µM, respectively. More interestingly, both compounds displayed a synergistic effect with tobramycin with a two-fold increase in its effectiveness in killing biofilm-encapsulated P. aeruginosa PAO1. The present study validates the traditional uses of this edible medicinal plant, indicating the therapeutic effectiveness of guava fruits plausibly through the presence of these tri- and tetraterpenoids, which deserve to be tested against pathogens generally implicated in diarrhea.

Ratiometric Fluorescent Safranin-O Staining Allows the Quantification of Lignin Contents In Muro.

In some specific vascular plant tissues, lignin can impregnate the entire cell wall to make it more rigid and hydrophobic. Different techniques have been developed in the past years to make possible the quantification of this polyphenolic polymer at the organ or tissue level, but difficulties of access to the cellular level remain. Here we describe an approach based on ratiometric emission measurements using safranin-O and the development of a macro adapted for the FIJI software, which makes it possible to quantify lignin in three different layers of the cell wall on images captured on a fluorescent confocal microscope.

Evidence for poplar PtaPLATZ18 in the regulation of plant growth and vascular tissues development.

Introduction: Plant A/T-rich protein and zinc-binding protein (PLATZ) are plant-specific transcription factors playing a role in plant development and stress response. To assess the role of PLATZs in vascular system development and wood formation in poplar, a functional study for PtaPLATZ18, whose expression was associated with the xylem, was carried out. Methods: Poplar dominant repressor lines for PtaPLATZ18 were produced by overexpressing a PtaPLATZ18-SRDX fusion. The phenotype of three independent transgenic lines was evaluated at morphological, biochemical, and molecular levels and compared to the wild type. Results: The PtaPLATZ18-SRDX lines showed increased plant height resulting from higher internode length. Besides, a higher secondary xylem thickness was also evidenced in these dominant repression lines as compared to the wild type suggesting an activation of cambial activity. A higher amount of lignin was evidenced within wood tissue as compared to the wild type, indicating an alteration in cell wall composition within xylem cell types. This latter phenotype was linked to an increased expression of genes involved in lignin biosynthesis and polymerization. Discussion: The phenotype observed in the PtaPLATZ18-SRDX lines argues that this transcription factor targets key regulators of plant growth and vascular tissues development.

The Xanthophyll Carotenoid Lutein Reduces the Invasive Potential of Pseudomonas aeruginosa and Increases Its Susceptibility to Tobramycin.

Recently, the xanthophyll carotenoid lutein has been qualified as a potential quorum sensing (QS) and biofilm inhibitor against Pseudomonas aeruginosa. To address the potential of this xanthophyll compound as a relevant antivirulence agent, we investigated in depth its impact on the invasion capabilities and aggressiveness of P. aeruginosa PAO1, which rely on the bacterial ability to build and maintain protective barriers, use different types of motilities and release myriad virulence factors, leading to host cell and tissue damages. Our data, obtained on the PAO1 strain, indicate that all-trans lutein (Lut; 22 µM) disrupts biofilm formation and disorganizes established biofilm structure without affecting bacterial viability, while improving the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Furthermore, this xanthophyll affects PAO1 twitching and swarming motilities while reducing the production of the extracellular virulence factors pyocyanin, elastase and rhamnolipids as well as the expression of the QS-regulated lasB and rhlA genes without inhibiting the QS-independent aceA gene. Interestingly, the expression of the QS regulators rhlR/I and lasR/I is significantly reduced as well as that of the global virulence factor regulator vfr, which is suggested to be a major target of Lut. Finally, an oxidative metabolite of Lut, 3'-dehydrolutein, induces a similar inhibition phenotype. Taken together, lutein-type compounds represent potential agents to control the invasive ability and antibiotic resistance of P. aeruginosa.

UGT72, a Major Glycosyltransferase Family for Flavonoid and Monolignol Homeostasis in Plants.

Plants have developed the capacity to produce a diversified range of specialized metabolites. The glycosylation of those metabolites potentially decreases their toxicity while increasing their stability and their solubility, modifying their transport and their storage. The UGT, forming the largest glycosyltransferase superfamily in plants, combine enzymes that glycosylate mainly hormones and phenylpropanoids by using UDP-sugar as a sugar donor. Particularly, members of the UGT72 family have been shown to glycosylate the monolignols and the flavonoids, thereby being involved in their homeostasis. First, we explore primitive UGTs in algae and liverworts that are related to the angiosperm UGT72 family and their role in flavonoid homeostasis. Second, we describe the role of several UGT72s glycosylating monolignols, some of which have been associated with lignification. In addition, the role of other UGT72 members that glycosylate flavonoids and are involved in the development and/or stress response is depicted. Finally, the importance to explore the subcellular localization of UGTs to study their roles in planta is discussed.

Leaf necrosis resulting from downregulation of poplar glycosyltransferase UGT72A2.

Reactive species (RS) causing oxidative stress are unavoidable by-products of various plant metabolic processes, such as photosynthesis, respiration or photorespiration. In leaves, flavonoids scavenge RS produced during photosynthesis and protect plant cells against deleterious oxidative damages. Their biosynthesis and accumulation are therefore under tight regulation at the cellular level. Glycosylation has emerged as an essential biochemical reaction in the homeostasis of various specialized metabolites such as flavonoids. This article provides a functional characterization of the Populus tremula x P. alba (poplar) UGT72A2 coding for a UDP-glycosyltransferase that is localized in the chloroplasts. Compared with the wild type, transgenic poplar lines with decreased expression of UGT72A2 are characterized by reduced growth and oxidative damages in leaves, as evidenced by necrosis, higher content of glutathione and lipid peroxidation products as well as diminished soluble peroxidase activity and NADPH to NADP+ ratio under standard growing conditions. They furthermore display lower pools of phenolics, anthocyanins and total flavonoids but higher proanthocyanidins content. Promoter analysis revealed the presence of cis-elements involved in photomorphogenesis, chloroplast biogenesis and flavonoid biosynthesis. The UGT72A2 is regulated by the poplar MYB119, a transcription factor known to regulate the flavonoid biosynthesis pathway. Phylogenetic analysis and molecular docking suggest that UGT72A2 could glycosylate flavonoids; however, the actual substrate(s) was not consistently evidenced with either in vitro assays nor analyses of glycosylated products in leaves of transgenic poplar overexpressing or downregulated for UGT72A2. This article provides elements highlighting the importance of flavonoid glycosylation regarding protection against oxidative stress in poplar leaves and raises new questions about the link between this biochemical reaction and regulation of the redox homeostasis system.

You Want it Sweeter: How Glycosylation Affects Plant Response to Oxidative Stress.

Oxidative stress is a cellular threat which puts at risk the productivity of most of crops valorized by humankind in terms of food, feed, biomaterial, or bioenergy. It is therefore of crucial importance to understand the mechanisms by which plants mitigate the deleterious effects of oxidizing agents. Glycosylation of antioxidant molecules and phytohormones modifies their chemical properties as well as their cellular and histological repartition. This review emphasizes the mechanisms and the outcomes of this conjugation reaction on plant ability to face growing conditions favoring oxidative stress, in mirror with the activity of deglycosylating enzymes. Pioneer evidence bridging flavonoid, glycosylation, and redox homeostasis paved the way for numerous functional analyses of UDP-glycosyltransferases (UGTs), such as the identification of their substrates and their role to circumvent oxidative stress resulting from various environmental challenges. (De)glycosylation appears as a simple chemical reaction regulating the biosynthesis and/or the activity of a myriad of specialized metabolites partaking in response to pathogen and abiotic stresses. This outcome underlies the possibility to valorize UGTs potential to upgrade plant adaptation and fitness in a rising context of sub-optimal growing conditions subsequent to climate change.

UDP-GLYCOSYLTRANSFERASE 72E3 Plays a Role in Lignification of Secondary Cell Walls in Arabidopsis.

Lignin is present in plant secondary cell walls and is among the most abundant biological polymers on Earth. In this work we investigated the potential role of the UGT72E gene family in regulating lignification in Arabidopsis. Chemical determination of floral stem lignin contents in ugt72e1, ugt72e2, and ugt72e3 mutants revealed no significant differences compared to WT plants. In contrast, the use of a novel safranin O ratiometric imaging technique indicated a significant increase in the cell wall lignin content of both interfascicular fibers and xylem from young regions of ugt72e3 mutant floral stems. These results were globally confirmed in interfascicular fibers by Raman microspectroscopy. Subsequent investigation using a bioorthogonal triple labelling strategy suggested that the augmentation in lignification was associated with an increased capacity of mutant cell walls to incorporate H-, G-, and S-monolignol reporters. Expression analysis showed that this increase was associated with an up-regulation of LAC17 and PRX71, which play a key role in lignin polymerization. Altogether, these results suggest that UGT72E3 can influence the kinetics of lignin deposition by regulating monolignol flow to the cell wall as well as the potential of this compartment to incorporate monomers into the growing lignin polymer.

Characterization of the UDP-glycosyltransferase UGT72 Family in Poplar and Identification of Genes Involved in the Glycosylation of Monolignols.

Monolignols are the building blocks for lignin polymerization in the apoplastic domain. Monolignol biosynthesis, transport, storage, glycosylation, and deglycosylation are the main biological processes partaking in their homeostasis. In Arabidopsis thaliana, members of the uridine diphosphate-dependent glucosyltransferases UGT72E and UGT72B subfamilies have been demonstrated to glycosylate monolignols. Here, the poplar UGT72 family, which is clustered into four groups, was characterized: Group 1 UGT72AZ1 and UGT72AZ2, homologs of Arabidopsis UGT72E1-3, as well as group 4 UGT72B37 and UGT72B39, homologs of Arabidopsis UGT72B1-3, glycosylate monolignols. In addition, promoter-GUS analyses indicated that poplar UGT72 members are expressed within vascular tissues. At the subcellular level, poplar UGT72s belonging to group 1 and group 4 were found to be associated with the nucleus and the endoplasmic reticulum. However, UGT72A2, belonging to group 2, was localized in bodies associated with chloroplasts, as well as possibly in chloroplasts. These results show a partial conservation of substrate recognition between Arabidopsis and poplar homologs, as well as divergent functions between different groups of the UGT72 family, for which the substrates remain unknown.

Alterations in the phenylpropanoid pathway affect poplar ability for ectomycorrhizal colonisation and susceptibility to root-knot nematodes.

This study investigates the impact of the alteration of the monolignol biosynthesis pathway on the establishment of the in vitro interaction of poplar roots either with a mutualistic ectomycorrhizal fungus or with a pathogenic root-knot nematode. Overall, the five studied transgenic lines downregulated for caffeoyl-CoA O-methyltransferase (CCoAOMT), caffeic acid O-methyltransferase (COMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD) or both COMT and CAD displayed a lower mycorrhizal colonisation percentage, indicating a lower ability for establishing mutualistic interaction than the wild-type. The susceptibility to root-knot nematode infection was variable in the five lines, and the CAD-deficient line was found to be less susceptible than the wild-type. We discuss these phenotypic differences in the light of the large shifts in the metabolic profile and gene expression pattern occurring between roots of the CAD-deficient line and wild-type. A role of genes related to trehalose metabolism, phytohormones, and cell wall construction in the different mycorrhizal symbiosis efficiency and nematode sensitivity between these two lines is suggested. Overall, these results show that the alteration of plant metabolism caused by the repression of a single gene within phenylpropanoid pathway results in significant alterations, at the root level, in the response towards mutualistic and pathogenic associates. These changes may constrain plant fitness and biomass production, which are of economic importance for perennial industrial crops such as poplar.

A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification.

One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.

Molecular Changes Concomitant with Vascular System Development in Mature Galls Induced by Root-Knot Nematodes in the Model Tree Host Populus tremula × P. alba.

One of the most striking features occurring in the root-knot nematode Meloidogyne incognita induced galls is the reorganization of the vascular tissues. During the interaction of the model tree species Populus and M. incognita, a pronounced xylem proliferation was previously described in mature galls. To better characterise changes in expression of genes possibly involved in the induction and the formation of the de novo developed vascular tissues occurring in poplar galls, a comparative transcript profiling of 21-day-old galls versus uninfected root of poplar was performed. Genes coding for transcription factors associated with procambium maintenance and vascular differentiation were shown to be differentially regulated, together with genes partaking in phytohormones biosynthesis and signalling. Specific signatures of transcripts associated to primary cell wall biosynthesis and remodelling, as well as secondary cell wall formation (cellulose, xylan and lignin) were revealed in the galls. Ultimately, we show that molecules derived from the monolignol and salicylic acid pathways and related to secondary cell wall deposition accumulate in mature galls.

A Molecular Blueprint of Lignin Repression.

Although lignin is essential to ensure the correct growth and development of land plants, it may be an obstacle to the production of lignocellulosics-based biofuels, and reduces the nutritional quality of crops used for human consumption or livestock feed. The need to tailor the lignocellulosic biomass for more efficient biofuel production or for improved plant digestibility has fostered considerable advances in our understanding of the lignin biosynthetic pathway and its regulation. Most of the described regulators are transcriptional activators of lignin biosynthesis, but considerably less attention has been devoted to the repressors of this pathway. We provide a comprehensive overview of the molecular factors that negatively impact on the lignification process at both the transcriptional and post-transcriptional levels.

Escherichia colimazEF Toxin-Antitoxin System as a Tool to Target Cell Ablation in Plants.

BACKGROUND/AIMS: The Escherichia coli MazF is an endoribonuclease that cleaves mRNA at ACA sequences, thereby triggering inhibition of protein synthesis. The aim of this study is to evaluate the efficiency of the mazEF toxin-antitoxin system in plants to develop biotechnological tools for targeted cell ablation. METHODS: A double transformation strategy, combining expression of the mazE antitoxin gene under the control of the CaMV 35S promoter, reported to drive expression in all plant cells except within the tapetum, together with the expression of the mazF gene under the control of the TA29 tapetum-specific promoter in transgenic tobacco, was applied. RESULTS: No transgenic TA29-mazF line could be regenerated, suggesting that the TA29 promoter is not strictly tapetum specific and that MazF is toxic for plant cells. The regenerated 35S-mazE/TA29-mazF double-transformed lines gave a unique phenotype where the tapetal cell layer was necrosed resulting in the absence of pollen. CONCLUSION: These results show that the E. colimazEF system can be used to induce death of specific plant cell types and can provide a new tool to plant cell ablation.

Poplar-Root Knot Nematode Interaction: A Model for Perennial Woody Species.

Plant root-knot nematode (RKN) interaction studies are performed on several host plant models. Though RKN interact with trees, no perennial woody model has been explored so far. Here, we show that poplar (Populus tremula × P. alba) grown in vitro is susceptible to Meloidogyne incognita, allowing this nematode to penetrate, to induce feeding sites, and to successfully complete its life cycle. Quantitative reverse transcription-polymerase chain reaction analysis was performed to study changes in poplar gene expression in galls compared with noninfected roots. Three genes (expansin A, histone 3.1, and asparagine synthase), selected as gall development marker genes, followed, during poplar-nematode interaction, a similar expression pattern to what was described for other plant hosts. Downregulation of four genes implicated in the monolignol biosynthesis pathway was evidenced in galls, suggesting a shift in the phenolic profile within galls developed on poplar roots. Raman microspectroscopy demonstrated that cell walls of giant cells were not lignified but mainly composed of pectin and cellulose. The data presented here suggest that RKN exercise conserved strategies to reproduce and to invade perennial plant species and that poplar is a suitable model host to study specific traits of tree-nematode interactions.

PtaRHE1, a Populus tremula × Populus alba RING-H2 protein of the ATL family, has a regulatory role in secondary phloem fibre development.

REALLY INTERESTING NEW GENE (RING) proteins play important roles in the regulation of many processes by recognizing target proteins for ubiquitination. Previously, we have shown that the expression of PtaRHE1, encoding a Populus tremula × Populus alba RING-H2 protein with E3 ubiquitin ligase activity, is associated with tissues undergoing secondary growth. To further elucidate the role of PtaRHE1 in vascular tissues, we have undertaken a reverse genetic analysis in poplar. Within stem secondary vascular tissues, PtaRHE1 and its corresponding protein are expressed predominantly in the phloem. The downregulation of PtaRHE1 in poplar by artificial miRNA triggers alterations in phloem fibre patterning, characterized by an increased portion of secondary phloem fibres that have a reduced cell wall thickness and a change in lignin composition, with lower levels of syringyl units as compared with wild-type plants. Following an RNA-seq analysis, a biological network involving hormone stress signalling, as well as developmental processes, could be delineated. Several candidate genes possibly associated with the altered phloem fibre phenotype observed in amiRPtaRHE1 poplar were identified. Altogether, our data suggest a regulatory role for PtaRHE1 in secondary phloem fibre development.

Analysis of genome sequences from plant pathogenic Rhodococcus reveals genetic novelties in virulence loci.

Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.

Does PtaRHE1, a poplar RING-H2 protein, play a role in water conduction through ABA signaling?

RING (REALLY INTERESTING NEW GENE) proteins with E3 ligase activity are largely represented in plants. They have been shown to play important roles in the regulation of many biological processes by recognizing target proteins for ubiquitination. PtaRHE1, encoding a poplar RING-H2 domain-containing protein with E3 ligase activity has been previously shown to be expressed during the establishment of secondary vascular system in poplar. In the present report, we demonstrate that the expression of PtaRHE1 and the accumulation of its corresponding protein are modulated by the relative atmospheric and soil humidity and by abscisic acid. Overall, the integrated data are discussed within a working model highlighting a plausible function of PtaRHE1 in the signaling and/or in the regulation of water status in poplar.

Bioplastics science from a policy vantage point.

Society is fundamentally ambivalent to the use of plastics. On the one hand, plastics are uniquely flexible materials that have seen them occupy a huge range of functions, from simple packing materials to complex engineering components. On the other hand, their durability has raised concerns about their end-of-life disposal. When that disposal route is landfill, their invulnerability to microbial decomposition, combined with relatively low density and high bulk, means that plastics will occupy increasing amounts of landfill space in a world where available suitable landfill sites is shrinking. The search for biodegradable plastics and their introduction to the marketplace would appear to be a suitable amelioration strategy for such a problem. And yet the uptake of biodegradable plastics has been slow. The term biodegradable itself has entered public controversy, with accidental and intended misuse of the term; the intended misuse has led to accusations and instances of 'greenwashing'. For this and other reasons standards for biodegradability and compostability testing of plastics have been sought. An environmental dilemma with more far-reaching implications is climate change. The need for rapid and deep greenhouse gas (GHG) emissions cuts is one of the drivers for the resurgence of industrial biotechnology generally, and the search for bio-based plastics more specifically. Bio-based has come to mean plastics based on renewable resources, but this need not necessarily imply biodegradability. If the primary purpose is GHG emissions savings, then once again plastics durability can be a virtue, if the end-of-life solution can be energy recovery during incineration or recycling. The pattern of production is shifting from the true biodegradable plastics to the bio-based plastics, and that trend is likely to persist into the future. This paper looks at aspects of the science of biodegradable and bio-based plastics from the perspective of policy advisers and makers. It is often said that the bioplastics suffer from a lack of a favourable policy regime when compared to the wide-ranging set of policy instruments that are available on both the supply and demand side of biofuels production. Some possible policy measures are discussed.

Insight into plant annexin function: from shoot to root signaling.

The multifunctionality of plant annexins and their importance for coordinating development and responses to biotic and abiotic environment have been largely reviewed. We recently described a tobacco annexin, named Ntann12, which is mainly localized in the nucleus of root cells when the plant is grown under light conditions. We also found that auxin and polar auxin transport are essential for Ntann12 accumulation in root cells. Under dark condition, Ntann12 is no longer detected in the root system. In the present addendum, light, regulating auxin signaling, is evidenced as an essential determinant for the synchronization of growth and development between the shoot and the root during light/dark cycle. A speculative model for Ntann12 is described and discussed with regards to relevant literature data.