Olive Mill Wastes: A Source of Bioactive Molecules for Plant Growth and Protection against Pathogens.

Olive oil production generates high amounts of liquid and solid wastes. For a long time, such complex matrices were considered only as an environmental issue, due to their polluting properties. On the other hand, olive mill wastes (OMWs) exert a positive effect on plant growth when applied to soil due to the high content of organic matter and mineral nutrients. Moreover, OMWs also exhibit antimicrobial activity and protective properties against plant pathogens possibly due to the presence of bioactive molecules including phenols and polysaccharides. This review covers the recent advances made in the identification, isolation, and characterization of OMW-derived bioactive molecules able to influence important plant processes such as plant growth and defend against pathogens. Such studies are relevant from different points of view. First, basic research in plant biology may benefit from the isolation and characterization of new biomolecules to be potentially applied in crop growth and protection against diseases. Moreover, the valorization of waste materials is necessary for the development of a circular economy, which is foreseen to drive the future development of a more sustainable agriculture.

AtPME17 is a functional Arabidopsis thaliana pectin methylesterase regulated by its PRO region that triggers PME activity in the resistance to Botrytis cinerea.

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid-ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.

Cell wall features transferred from common into durum wheat to improve Fusarium Head Blight resistance.

Durum wheat is naturally more susceptible to Fusarium graminerum infection in comparison to common wheat. The improvement of durum wheat resistance against F. graminearum is a challenge due to the lack of resistance sources in its gene pool. FHB-resistance factors were introduced in durum wheat by generating recombinant inbred lines (RILs), obtained by crossing the hexaploid resistant accession 02-5B-318 with the susceptible durum wheat cv. Saragolla. In this work we explored the possible contribution of cell wall (CW) in RILs with improved FHB resistance. We thoroughly studied CW components, mycotoxins content and the expression of related genes in different RILs selected for their extremely high and low resistance to FHB. Differences were found in resistant and susceptible lines in the degree of pectin methylesterification and in deoxynivalenol (DON) accumulation after fungal infection. Genes involved in biochemical modification of CW structure (WheatPme-1, Glu-1) and mycotoxins accumulation (ns-LTP-1) were analyzed as putative candidates for FHB resistance. Our results indicate that durum wheat plants with cell wall structure and gene response acquired from common wheat displayed an increased resistance to FHB.

Pectic enzymes as potential enhancers of ascorbic acid production through the D-galacturonate pathway in Solanaceae.

The increase of L-Ascorbic Acid (AsA) content in tomato (Solanum lycopersicum) is a common goal in breeding programs due to its beneficial effect on human health. To shed light into the regulation of fruit AsA content, we exploited a Solanum pennellii introgression line (IL12-4-SL) harbouring one quantitative trait locus that increases the content of total AsA in the fruit. Biochemical and transcriptomic analyses were carried out in fruits of IL12-4-SL in comparison with the cultivated line M82 at different stages of ripening. AsA content was studied in relation with pectin methylesterase (PME) activity and the degree of pectin methylesterification (DME). Our results indicated that the increase of AsA content in IL12-4-SL fruits was related with pectin de-methylesterification/degradation. Specific PME, polygalacturonase (PG) and UDP-D-glucuronic-acid-4-epimerase (UGlcAE) isoforms were proposed as components of the D-galacturonate pathway leading to AsA biosynthesis. The relationship between AsA content and PME activity was also exploited in PMEI tobacco plants expressing a specific PME inhibitor (PMEI). Here we report that tobacco PMEI plants, altered in PME activity and degree of pectin methylesterification, showed a reduction in low methylesterified pectic domains and exhibited a reduced AsA content. Overall, our results provide novel biochemical and genetic traits for increasing antioxidant content by marker-assisted selection in the Solanaceae family.

Three Pectin Methylesterase Inhibitors Protect Cell Wall Integrity for Arabidopsis Immunity to Botrytis.

Infection by necrotrophs is a complex process that starts with the breakdown of the cell wall (CW) matrix initiated by CW-degrading enzymes and results in an extensive tissue maceration. Plants exploit induced defense mechanisms based on biochemical modification of the CW components to protect themselves from enzymatic degradation. The pectin matrix is the main CW target of Botrytis cinerea, and pectin methylesterification status is strongly altered in response to infection. The methylesterification of pectin is controlled mainly by pectin methylesterases (PMEs), whose activity is posttranscriptionally regulated by endogenous protein inhibitors (PMEIs). Here, AtPMEI10, AtPMEI11, and AtPMEI12 are identified as functional PMEIs induced in Arabidopsis (Arabidopsis thaliana) during B. cinerea infection. AtPMEI expression is strictly regulated by jasmonic acid and ethylene signaling, while only AtPMEI11 expression is controlled by PME-related damage-associated molecular patterns, such as oligogalacturonides and methanol. The decrease of pectin methylesterification during infection is higher and the immunity to B. cinerea is compromised in pmei10, pmei11, and pmei12 mutants with respect to the control plants. A higher stimulation of the fungal oxalic acid biosynthetic pathway also can contribute to the higher susceptibility of pmei mutants. The lack of PMEI expression does not affect hemicellulose strengthening, callose deposition, and the synthesis of structural defense proteins, proposed as CW-remodeling mechanisms exploited by Arabidopsis to resist CW degradation upon B. cinerea infection. We show that PME activity and pectin methylesterification are dynamically modulated by PMEIs during B. cinerea infection. Our findings point to AtPMEI10, AtPMEI11, and AtPMEI12 as mediators of CW integrity maintenance in plant immunity.

Pyramiding PvPGIP2 and TAXI-III But Not PvPGIP2 and PMEI Enhances Resistance Against Fusarium graminearum.

Plant protein inhibitors counteract the activity of cell wall-degrading enzymes (CWDEs) secreted by pathogens to breach the plant cell-wall barrier. Transgenic plants expressing a single protein inhibitor restrict pathogen infections. However, since pathogens secrete a number of CWDEs at the onset of infection, we combined more inhibitors in a single wheat genotype to reinforce further the cell-wall barrier. We combined polygalacturonase (PG) inhibiting protein (PGIP) and pectin methyl esterase inhibitor (PMEI), both controlling the activity of PG, one of the first CWDEs secreted during infection. We also pyramided PGIP and TAXI-III, a xylanase inhibitor that controls the activity of xylanases, key factors for the degradation of xylan, a main component of cereal cell wall. We demonstrated that the pyramiding of PGIP and PMEI did not contribute to any further improvement of disease resistance. However, the presence of both pectinase inhibitors ensured a broader spectrum of disease resistance. Conversely, the PGIP and TAXI-III combination contributed to further improvement of Fusarium head blight (FHB) resistance, probably because these inhibitors target the activity of different types of CWDEs, i.e., PGs and xylanases. Worth mentioning, the reduction of FHB symptoms is accompanied by a reduction of deoxynivalenol accumulation with a foreseen great benefit to human and animal health.

Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens.

The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens.

The Grapevine VvPMEI1 Gene Encodes a Novel Functional Pectin Methylesterase Inhibitor Associated to Grape Berry Development.

Pectin is secreted in a highly methylesterified form and partially de-methylesterified in the cell wall by pectin methylesterases (PMEs). PME activity is expressed during plant growth, development and stress responses. PME activity is controlled at the post-transcriptional level by proteins named PME inhibitors (PMEIs). We have identified, expressed and characterized VvPMEI1, a functional PME inhibitor of Vitis vinifera. VvPMEI1 typically affects the activity of plant PMEs and is inactive against microbial PMEs. The kinetics of PMEI-PME interaction, studied by surface plasmon resonance, indicates that the inhibitor strongly interacts with PME at apoplastic pH while the stability of the complex is reduced by increasing the pH. The analysis of VvPMEI1 expression in different grapevine tissues and during grape fruit development suggests that this inhibitor controls PME activity mainly during the earlier phase of berry development. A proteomic analysis performed at this stage indicates a PME isoform as possible target of VvPMEI1.

Cell wall traits as potential resources to improve resistance of durum wheat against Fusarium graminearum.

BACKGROUND: Fusarium graminearum, one of the causal agents of Fusarium Head Blight (FHB, scab), leads to severe losses in grain yield and quality due to the production of mycotoxins which are harmful to human and livestock. Different traits for FHB resistance in wheat were identified for common wheat (Triticum aestivum L.) while the sources of FHB resistance in durum wheat (Triticum turgidum ssp. Durum), one of the cereals most susceptible to F. graminearum infection, have not been found. New lines of evidence indicate that content and composition of cell wall polymers affect the susceptibility of the wall to degrading enzymes produced by pathogens during infection and can play a role in the outcome of host-pathogen interactions. The objective of our research is to identify potential cell wall biochemical traits linked to Fusariosis resistance to be transferred from a resistant common wheat to a susceptible durum wheat line. RESULTS: A detailed analysis of cell wall composition in spikes isolated from a highly resistant common wheat accession "02-5B-318", a breeding line derived from the FHB-resistant Chinese cv. Sumai-3 and a high susceptible durum wheat cv. Saragolla was performed. Significant differences in lignin monolignols composition, arabinoxylan (AX) substitutions and pectin methylesterification were found between resistant and susceptible plants. We isolated and characterized a pectin methylesterase gene WheatPME1, which we found being down regulated in the FHB-resistant line and induced by fungal infection in the susceptible wheat. CONCLUSIONS: Our results indicate cell wall traits differing between the FHB sensitive and resistant wheat genotypes, possibly related to FHB-resistance, and identify the line 02-5B-318R as a potential resource of such traits. Evidence suggests that WheatPME1 is involved in wheat response to F. graminearum.

How do pectin methylesterases and their inhibitors affect the spreading of tobamovirus?

After replication in the cytoplasm, viruses spread from the infected cell into the neighboring cells through plasmodesmata, membranous channels embedded by the cell wall. As obligate parasites, viruses have acquired the ability to utilize host factors that unwillingly cooperate for the viral infection process. For example, the viral movement proteins (MP) interacts with the host pectin methylesterase (PME) and both proteins cooperate to sustain the viral spread. However, how and where PMEs interact with MPs and how the PME/MP complexes favor the viral translocation is not well understood. Recently, we demonstrated that the overexpression of PME inhibitors (PMEIs) in tobacco and Arabidopsis plants limits the movement of Tobacco mosaic virus and Turnip vein clearing virus and reduces plant susceptibility to these viruses. Here we discuss how overexpression of PMEI may reduce tobamovirus spreading.

Plant cell wall dynamics and wall-related susceptibility in plant-pathogen interactions.

The cell wall is a dynamic structure that often determines the outcome of the interactions between plants and pathogens. It is a barrier that pathogens need to breach to colonize the plant tissue. While fungal necrotrophs extensively destroy the integrity of the cell wall through the combined action of degrading enzymes, biotrophic fungi require a more localized and controlled degradation of the cell wall in order to keep the host cells alive and utilize their feeding structures. Also bacteria and nematodes need to degrade the plant cell wall at a certain stage of their infection process, to obtain nutrients for their growth. Plants have developed a system for sensing pathogens and monitoring the cell wall integrity, upon which they activate defense responses that lead to a dynamic cell wall remodeling required to prevent the disease. Pathogens, on the other hand, may exploit the host cell wall metabolism to support the infection. We review here the strategies utilized by both plants and pathogens to prevail in the cell wall battleground.

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis.

Plant infection by a virus is a complex process influenced by virus-encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well-characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI-2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.

Analysis of pectin mutants and natural accessions of Arabidopsis highlights the impact of de-methyl-esterified homogalacturonan on tissue saccharification.

BACKGROUND: Plant biomass is a potentially important renewable source of energy and industrial products. The natural recalcitrance of the cell walls to enzymatic degradation (saccharification), which plants have evolved to defend themselves from biotic stresses, represents a major bottleneck for the industrial bioconversion of lignocellulosic biomasses. The identification of factors that influence the cell wall recalcitrance to saccharification may help to overcome the existing limitations that hamper the utilization of biomass. RESULTS: Here we have investigated in Arabidopsis thaliana the impact of homogalacturonan (HG) content and structure on tissue saccharification. We characterized mutants affected in genes encoding proteins involved in HG biosynthesis (quasimodo2-1; qua2-1) and methylesterification (pectin methylesterase 3; pme3). We also analyzed the natural variation of Arabidopsis through the characterization of a nested core collection of 24 accessions generated to maximize genetic variability. We found a negative correlation between the level of de-methyl-esterified HG (HGA) and cellulose degradability. CONCLUSIONS: We propose to use the level of HGA domains as a biochemical marker of the cell wall recalcitrance to saccharification. This may be utilized for selecting, on a large scale, natural variants or mutants with improved bioconversion features.

Cell wall integrity: targeted post-synthetic modifications to reveal its role in plant growth and defense against pathogens.

The plant cell wall, a dynamic network of polysaccharides and glycoproteins of significant compositional and structural complexity, functions in plant growth, development and stress responses. In recent years, the existence of plant cell wall integrity (CWI) maintenance mechanisms has been demonstrated, but little is known about the signaling pathways involved, or their components. Examination of key mutants has shed light on the relationships between cell wall remodeling and plant cell responses, indicating a central role for the regulatory network that monitors and controls cell wall performance and integrity. In this review, we present a short overview of cell wall composition and discuss post-synthetic cell wall modification as a valuable approach for studying CWI perception and signaling pathways.

Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens.

The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens.

Methyl esterification of pectin plays a role during plant-pathogen interactions and affects plant resistance to diseases.

The cell wall is a complex structure mainly composed by a cellulose-hemicellulose network embedded in a cohesive pectin matrix. Pectin is synthesized in a highly methyl esterified form and is de-esterified in muro by pectin methyl esterases (PMEs). The degree and pattern of methyl esterification affect the cell wall structure and properties with consequences on both the physiological processes of the plants and their resistance to pathogens. PME activity displays a crucial role in the outcome of the plant-pathogen interactions by making pectin more susceptible to the action of the enzymes produced by the pathogens. This review focuses on the impact of pectin methyl esterification in plant-pathogen interactions and on the dynamic role of its alteration during pathogenesis.

A functional pectin methylesterase inhibitor protein (SolyPMEI) is expressed during tomato fruit ripening and interacts with PME-1.

A pectin methylesterase inhibitor (SolyPMEI) from tomato has been identified and characterised by a functional genomics approach. SolyPMEI is a cell wall protein sharing high similarity with Actinidia deliciosa PMEI (AdPMEI), the best characterised inhibitor from kiwi. It typically affects the activity of plant pectin methylesterases (PMEs) and is inactive against a microbial PME. SolyPMEI transcripts were mainly expressed in flower, pollen and ripe fruit where the protein accumulated at breaker and turning stages of ripening. The expression of SolyPMEI correlated during ripening with that of PME-1, the major fruit specific PME isoform. The interaction of SolyPMEI with PME-1 was demonstrated in ripe fruit by gel filtration and by immunoaffinity chromatography. The analysis of the zonal distribution of PME activity and the co-localization of SolyPMEI with high esterified pectins suggest that SolyPMEI regulates the spatial patterning of distribution of esterified pectins in fruit.

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Pectin methylesterase is induced in Arabidopsis upon infection and is necessary for a successful colonization by necrotrophic pathogens.

The ability of bacterial or fungal necrotrophs to produce enzymes capable of degrading pectin is often related to a successful initiation of the infective process. Pectin is synthesized in a highly methylesterified form and is subsequently de-esterified in muro by pectin methylesterase. De-esterification makes pectin more susceptible to the degradation by pectic enzymes such as endopolygalacturonases (endoPG) and pectate lyases secreted by necrotrophic pathogens during the first stages of infection. We show that, upon infection, Pectobacterium carotovorum and Botrytis cinerea induce in Arabidopsis a rapid expression of AtPME3 that acts as a susceptibility factor and is required for the initial colonization of the host tissue.