Sphingolipid Long-Chain Base Signaling in Compatible and Non-Compatible Plant-Pathogen Interactions in Arabidopsis.

The chemical diversity of sphingolipids in plants allows the assignment of specific roles to special molecular species. These roles include NaCl receptors for glycosylinositolphosphoceramides or second messengers for long-chain bases (LCBs), free or in their acylated forms. Such signaling function has been associated with plant immunity, with an apparent connection to mitogen-activated protein kinase 6 (MPK6) and reactive oxygen species (ROS). This work used in planta assays with mutants and fumonisin B1 (FB1) to generate varying levels of endogenous sphingolipids. This was complemented with in planta pathogenicity tests using virulent and avirulent Pseudomonas syringae strains. Our results indicate that the surge of specific free LCBs and ceramides induced by FB1 or an avirulent strain trigger a biphasic ROS production. The first transient phase is partially produced by NADPH oxidase, and the second is sustained and is related to programmed cell death. MPK6 acts downstream of LCB buildup and upstream of late ROS and is required to selectively inhibit the growth of the avirulent but not the virulent strain. Altogether, these results provide evidence that a LCB- MPK6- ROS signaling pathway contributes differentially to the two forms of immunity described in plants, upregulating the defense scheme of a non-compatible interaction.

Plasma and vacuolar membrane sphingolipidomes: composition and insights on the role of main molecular species.

Lipid structures affect membrane biophysical properties such as thickness, stability, permeability, curvature, fluidity, asymmetry, and interdigitation, contributing to membrane function. Sphingolipids are abundant in plant endomembranes and plasma membranes (PMs) and comprise four classes: ceramides, hydroxyceramides, glucosylceramides, and glycosylinositolphosphoceramides (GIPCs). They constitute an array of chemical structures whose distribution in plant membranes is unknown. With the aim of describing the hydrophobic portion of sphingolipids, 18 preparations from microsomal (MIC), vacuolar (VM), PM, and detergent-resistant membranes (DRM) were isolated from Arabidopsis (Arabidopsis thaliana) leaves. Sphingolipid species, encompassing pairing of long-chain bases and fatty acids, were identified and quantified in these membranes. Sphingolipid concentrations were compared using univariate and multivariate analysis to assess sphingolipid diversity, abundance, and predominance across membranes. The four sphingolipid classes were present at different levels in each membrane: VM was enriched in glucosylceramides, hydroxyceramides, and GIPCs; PM in GIPCs, in agreement with their key role in signal recognition and sensing; and DRM in GIPCs, as reported by their function in nanodomain formation. While a total of 84 sphingolipid species was identified in MIC, VM, PM, and DRM, only 34 were selectively distributed in the four membrane types. Conversely, every membrane contained a different number of predominant species (11 in VM, 6 in PM, and 17 in DRM). This study reveals that MIC, VM, PM, and DRM contain the same set of sphingolipid species but every membrane source contains its own specific assortment based on the proportion of sphingolipid classes and on the predominance of individual species.

Effects of MPK3 and MPK6 kinases on the chloroplast architecture and function induced by cold acclimation in Arabidopsis.

Exposure to low, non-freezing temperatures develops freezing tolerance in many plant species. Such process is called cold acclimation. Molecular changes undergone during cold acclimation are orchestrated by signalling networks including MAP kinases. Structure and function of chloroplasts are affected by low temperatures. The aim of this work was to study how the MAP kinases MPK3 and MPK6 are involved in the chloroplast performance upon a long period of cold acclimation. We used Arabidopsis thaliana wild type and mpk3 and mpk6 mutants. Adult plants were acclimated during 7 days at 4 °C and then measurements of PSII performance and chloroplast ultrastructure were carried out. Only the mpk6 acclimated plants showed a high freezing sensitivity. No differences in the PSII function were observed in the plants from the three genotypes exposed to non-acclimated or acclimated conditions. The acclimation of wild-type plants produced severe alterations in the ultrastructure of chloroplast and thylakoids, which was more accentuated in the mpk plants. However, only the mpk6 mutant was unable to internalize the damaged chloroplasts into the vacuole. These results indicate that cold acclimation induces alterations in the chloroplast architecture leading to preserve an optimal performance of PSII. MPK3 and MPK6 are necessary to regulate these morphological changes, but besides, MPK6 is needed to the vacuolization of the damaged chloroplasts, suggesting a role in the chloroplast recycling during cold acclimation. The latter could be quite relevant, since it could explain why this mutant is the only one showing an extremely low freezing tolerance.

MPK6 Kinase Regulates Plasma Membrane H+-ATPase Activity in Cold Acclimation.

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.

The Protective Effect of Trichoderma asperellum on Tomato Plants against Fusarium oxysporum and Botrytis cinerea Diseases Involves Inhibition of Reactive Oxygen Species Production.

Trichoderma species are fungi widely employed as plant-growth-promoting agents and for biological control. Several commercial and laboratory-made solid formulations for mass production of Trichoderma have been reported. In this study, we evaluated a solid kaolin-based formulation to promote the absortion/retention of Trichoderma asperellum in the substrate for growing tomato plants. The unique implementation of this solid formulation resulted in an increased growth of the tomato plants, both in roots and shoots after 40 days of its application. Plants were challenged with two fungal pathogens, Fusarium oxysporum and Botrytis cinerea, and pretreatment with T. asperellum resulted in less severe wilting and stunting symptoms than non-treated plants. Treatment with T. asperellum formulation inhibited Reactive Oxygen Species (ROS) production in response to the pathogens in comparison to plants that were only challenged with both pathogens. These results suggest that decrease in ROS levels contribute to the protective effects exerted by T. asperellum in tomato.

Isolation of detergent-resistant membranes (DRMs) from Escherichia coli.

Lipid rafts or membrane microdomains have been proposed to compartmentalize cellular processes by spatially organizing diverse molecules/proteins in eukaryotic cells. Such membrane microdomains were recently reported to also exist in a few bacterial species. In this work, we report the development of a procedure for membrane microdomain isolation from Escherichia coli plasma membranes as well as a method to purify the latter. The method here reported could easily be adapted to other gram-negative bacteria, wherein the isolation of this kind of sub-membrane preparation imposes special difficulties. The analysis of isolated membrane microdomains might provide important information on the nature and function of these bacterial structures and permit their comparison with the ones of eukaryotic cells.

Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

Early carbon mobilization and radicle protrusion in maize germination.

Considerable amounts of information is available on the complex carbohydrates that are mobilized and utilized by the seed to support early seedling development. These events occur after radicle has protruded from the seed. However, scarce information is available on the role of the endogenous soluble carbohydrates from the embryo in the first hours of germination. The present work analysed how the soluble carbohydrate reserves in isolated maize embryos are mobilized during 6-24 h of water imbibition, an interval that exclusively embraces the first two phases of the germination process. It was found that sucrose constitutes a very significant reserve in the scutellum and that it is efficiently consumed during the time in which the adjacent embryo axis is engaged in an active metabolism. Sucrose transporter was immunolocalized in the scutellum and in vascular elements. In parallel, a cell-wall invertase activity, which hydrolyses sucrose, developed in the embryo axis, which favoured higher glucose uptake. Sucrose and hexose transporters were active in the embryo tissues, together with the plasma membrane H(+)-ATPase, which was localized in all embryo regions involved in both nutrient transport and active cell elongation to support radicle extension. It is proposed that, during the initial maize germination phases, a net flow of sucrose takes place from the scutellum towards the embryo axis and regions that undergo elongation. During radicle extension, sucrose and hexose transporters, as well as H(+)-ATPase, become the fundamental proteins that orchestrate the transport of nutrients required for successful germination.

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis.

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.

Isolation of detergent-resistant membranes from plant photosynthetic and non-photosynthetic tissues.

Microdomains, or lipid rafts, are transient membrane regions enriched in sphingolipids and sterols that have only recently, but intensively, been studied in plants. In this work, we report a detailed, easy-to-follow, and fast procedure to isolate detergent-resistant membranes (DRMs) from purified plasma membranes (PMs) that was used to obtain DRMs from Phaseolus vulgaris and Nicotiana tabacum leaves and germinating Zea mays embryos. Characterized according to yield, ultrastructure, and sterol composition, these DRM preparations showed similarities to analogous preparations from other eukaryotic cells. Isolation of DRMs from germinating maize embryos reveals the presence of microdomains at very early developmental stages of plants.

Plasma Membrane Fluidity: An Environment Thermal Detector in Plants.

The lipid matrix in cell membranes is a dynamic, bidimensional array of amphipathic molecules exhibiting mesomorphism, which contributes to the membrane fluidity changes in response to temperature fluctuation. As sessile organisms, plants must rapidly and accurately respond to environmental thermal variations. However, mechanisms underlying temperature perception in plants are poorly understood. We studied the thermal plasticity of membrane fluidity using three fluorescent probes across a temperature range of -5 to 41 °C in isolated microsomal fraction (MF), vacuolar membrane (VM), and plasma membrane (PM) vesicles from Arabidopsis plants. Results showed that PM were highly fluid and exhibited more phase transitions and hysteresis, while VM and MF lacked such attributes. These findings suggest that PM is an important cell hub with the capacity to rapidly undergo fluidity modifications in response to small changes of temperatures in ranges spanning those experienced in natural habitats. PM fluidity behaves as an ideal temperature detector: it is always present, covers the whole cell, responds quickly and with sensitivity to temperature variations, functions with a cell free-energy cost, and it is physically connected with potential thermal signal transducers to elicit a cell response. It is an optimal alternative for temperature detection selected for the plant kingdom.

Arabidopsis thaliana eIF4E1 and eIF(iso)4E Participate in Cold Response and Promote Translation of Some Stress-Related mRNAs.

Plant defense and adaptation to adverse environmental conditions rely on gene expression control, such as mRNA transcription, processing, stability, and translation. Sudden temperature changes are common in the era of global warming; thus, understanding plant acclimation responses at the molecular level becomes imperative. mRNA translation initiation regulation has a pivotal role in achieving the synthesis of the appropriate battery of proteins needed to cope with temperature stress. In this study, we analyzed the role of translation initiation factors belonging to the eIF4E family in Arabidopsis acclimation to cold temperatures and freezing tolerance. Using knockout (KO) and overexpressing mutants of AteIF4E1 or AteIF(iso)4E, we found that AteIF4E1 but not AteIF(iso)4E overexpressing lines displayed enhanced tolerance to freezing without previous acclimation at 4°C. However, KO mutant lines, eif(iso)4e-1 and eif4e1-KO, were more sensitive to the stress. Cold acclimation in wild-type plants was accompanied by increased levels of eIF4E1 and eIF(iso)4E transcript levels, polysomes (P) enrichment, and shifts of these factors from translationally non-active to active fractions. Transcripts, previously found as candidates for eIF(iso)4E or eIF4E1 selective translation, changed their distribution in both P and total RNA in the presence of cold. Some of these transcripts changed their polysomal distribution in the mutant and one eIF4E1 overexpressing line. According to this, we propose a role of eIF4E1 and eIF(iso)4E in cold acclimation and freezing tolerance by regulating the expression of stress-related genes.

Organized Disassembly of Photosynthesis During Programmed Cell Death Mediated By Long Chain Bases.

In plants, pathogen triggered programmed cell death (PCD) is frequently mediated by polar lipid molecules referred as long chain bases (LCBs) or ceramides. PCD interceded by LCBs is a well-organized process where several cell organelles play important roles. In fact, light-dependent reactions in the chloroplast have been proposed as major players during PCD, however, the functional aspects of the chloroplast during PCD are largely unknown. For this reason, we investigated events that lead to disassembly of the chloroplast during PCD mediated by LCBs. To do so, LCB elevation was induced with Pseudomonas syringae pv. tomato (a non-host pathogen) or Fumonisin B1 in Phaseolus vulgaris. Then, we performed biochemical tests to detect PCD triggering events (phytosphingosine rises, MPK activation and H2O2 generation) followed by chloroplast structural and functional tests. Observations of the chloroplast, via optical phenotyping methods combined with microscopy, indicated that the loss of photosynthetic linear electron transport coincides with the organized ultrastructure disassembly. In addition, structural changes occurred in parallel with accumulation of H2O2 inside the chloroplast. These features revealed the collapse of chloroplast integrity and function as a mechanism leading to the irreversible execution of the PCD promoted by LCBs.

Sphingolipid Effects on the Plasma Membrane Produced by Addition of Fumonisin B1 to Maize Embryos.

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides that modifies the membrane properties from animal cells and inhibits complex sphingolipids synthesis through the inhibition of ceramide synthase. The aim of this work was to determine the effect of Fumonisin B1 on the plant plasma membrane when the mycotoxin was added to germinating maize embryos. Fumonisin B1 addition to the embryos diminished plasma membrane fluidity, increased electrolyte leakage, caused a 7-fold increase of sphinganine and a small decrease in glucosylceramide in the plasma membrane, without affecting phytosphingosine levels or fatty acid composition. A 20%-30% inhibition of the plasma membrane H+-ATPase activity was observed when embryos were germinated in the presence of the mycotoxin. Such inhibition was only associated to the decrease in glucosylceramide and the addition of exogenous ceramide to the embryos relieved the inhibition of Fumonisin B1. These results indicate that exposure of the maize embryos for 24 h to Fumonisin B1 allowed the mycotoxin to target ceramide synthase at the endoplasmic reticulum, eliciting an imbalance of endogenous sphingolipids. The latter disrupted membrane properties and inhibited the plasma membrane H+-ATPase activity. Altogether, these results illustrate the mode of action of the pathogen and a plant defense strategy.

Proteomic analysis of Escherichia coli detergent-resistant membranes (DRM).

Membrane microdomains or lipid rafts compartmentalize cellular processes by laterally organizing membrane components. Such sub-membrane structures were mainly described in eukaryotic cells, but, recently, also in bacteria. Here, the protein content of lipid rafts in Escherichia coli was explored by mass spectrometry analyses of Detergent Resistant Membranes (DRM). We report that at least three of the four E. coli flotillin homologous proteins were found to reside in DRM, along with 77 more proteins. Moreover, the proteomic data were validated by subcellular localization, using immunoblot assays and fluorescence microscopy of selected proteins. Our results confirm the existence of lipid raft-like microdomains in the inner membrane of E. coli and represent the first comprehensive profiling of proteins in these bacterial membrane platforms.

Sphingolipid Distribution, Content and Gene Expression during Olive-Fruit Development and Ripening.

Plant sphingolipids are involved in the building of the matrix of cell membranes and in signaling pathways of physiological processes and environmental responses. However, information regarding their role in fruit development and ripening, a plant-specific process, is unknown. The present study seeks to determine whether and, if so, how sphingolipids are involved in fleshy-fruit development and ripening in an oil-crop species such as olive (Olea europaea L. cv. Picual). Here, in the plasma-membranes of live protoplasts, we used fluorescence to examine various specific lipophilic stains in sphingolipid-enriched regions and investigated the composition of the sphingolipid long-chain bases (LCBs) as well as the expression patterns of sphingolipid-related genes, OeSPT, OeSPHK, OeACER, and OeGlcCerase, during olive-fruit development and ripening. The results demonstrate increased sphingolipid content and vesicle trafficking in olive-fruit protoplasts at the onset of ripening. Moreover, the concentration of LCB [t18:1(8Z), t18:1 (8E), t18:0, d18:2 (4E/8Z), d18:2 (4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E)] increases during fruit development to reach a maximum at the onset of ripening, although these molecular species decreased during fruit ripening. On the other hand, OeSPT, OeSPHK, and OeGlcCerase were expressed differentially during fruit development and ripening, whereas OeACER gene expression was detected only at the fully ripe stage. The results provide novel data about sphingolipid distribution, content, and biosynthesis/turnover gene transcripts during fleshy-fruit ripening, indicating that all are highly regulated in a developmental manner.

Localization of Sphingolipid Enriched Plasma Membrane Regions and Long-Chain Base Composition during Mature-Fruit Abscission in Olive.

Sphingolipids, found in membranes of eukaryotic cells, have been demonstrated to carry out functions in various processes in plant cells. However, the roles of these lipids in fruit abscission remain to be determined in plants. Biochemical and fluorescence microscopy imaging approach has been adopted to investigate the accumulation and distribution of sphingolipids during mature-fruit abscission in olive (Olea europaea L. cv. Picual). Here, a lipid-content analysis in live protoplasts of the olive abscission zone (AZ) was made with fluorescent dyes and lipid analogs, particularly plasma membrane sphingolipid-enriched domains, and their dynamics were investigated in relation to the timing of mature-fruit abscission. In olive AZ cells, the measured proportion of both polar lipids and sphingolipids increased as well as endocytosis was stimulated during mature-fruit abscission. Likewise, mature-fruit abscission resulted in quantitative and qualitative changes in sphingolipid long-chain bases (LCBs) in the olive AZ. The total LCB increase was due essentially to the increase of t18:1(8E) LCBs, suggesting that C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation were quantitatively the most important sphingolipids in olive AZ during abscission. However, our results also showed a specific association between the dihydroxylated LCB sphinganine (d18:0) and the mature-fruit abscission. These results indicate a clear correlation between the sphingolipid composition and mature-fruit abscission. Moreover, measurements of endogenous sterol levels in the olive AZ revealed that it accumulated sitosterol and campesterol with a concomitant decrease in cycloartenol during abscission. In addition, underlying the distinct sterol composition of AZ during abscission, genes for key biosynthetic enzymes for sterol synthesis, for obtusifoliol 14α-demethylase (CYP51) and C-24 sterol methyltransferase2 (SMT2), were up-regulated during mature-fruit abscission, in parallel to the increase in sitosterol content. The differences found in AZ lipid content and the relationships established between LCB and sterol composition, offer new insights about sphingolipids and sterols in abscission.

Long-chain bases, phosphatidic acid, MAPKs, and reactive oxygen species as nodal signal transducers in stress responses in Arabidopsis.

Due to their sessile condition, plants have developed sensitive, fast, and effective ways to contend with environmental changes. These mechanisms operate as informational wires conforming extensive and intricate networks that are connected in several points. The responses are designed as pathways orchestrated by molecules that are transducers of protein and non-protein nature. Their chemical nature imposes selective features such as specificity, formation rate, and generation site to the informational routes. Enzymes such as mitogen-activated protein kinases and non-protein, smaller molecules, such as long-chain bases, phosphatidic acid, and reactive oxygen species are recurrent transducers in the pleiotropic responses to biotic and abiotic stresses in plants. In this review, we considered these four components as nodal points of converging signaling pathways that start from very diverse stimuli and evoke very different responses. These pleiotropic effects may be explained by the potentiality that every one of these four mediators can be expressed from different sources, cellular location, temporality, or magnitude. Here, we review recent advances in our understanding of the interplay of these four specific signaling components in Arabidopsis cells, with an emphasis on drought, cold and pathogen stresses.

Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species.

It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical-chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes.

hrp genes of Erwinia chrysanthemi 3937 are important virulence factors.

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.