Plant Plasma Membrane Research Articles

Verticillium dahliae CFEM proteins manipulate host immunity and differentially contribute to virulence

BackgroundVerticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown.ResultsNine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type.ConclusionsIn the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.

Antipermeability Strategy to Achieve Extremely High Specificity and Ultralong Imaging of Diverse Cell Membranes Based on Restriction-Induced Emission of AIEgens

Long-term in situ cell membrane-targeted bioimaging is of great significance for studying specific biological processes and functions, but currently developed membrane probes are rarely simultaneously used to image the plasma membrane of animal and plant cells, and these probes lack sufficiently high long-term targeting ability. Herein, we proposed an antipermeability strategy to achieve highly specific and long-term imaging of plasma membranes of both human and plant cells using the steric hindrance effect and restriction-induced emission of AIE-active probes based on an updated membrane model. A certain degree of rigidity of plasma membrane containing a large ratio of rigid cholesterol molecules in the updated membrane model provides a promising opportunity to design antipermeable probes by introducing a rigid steric hindrance group in the probe. The designed antipermeable probes can anchor inside plasma membrane for a long term relying on the combination of the steric hindrance effect and the electrostatic and hydrophobic interactions between the probe and the membrane, as well as light up the membrane via the restriction-induced emission mechanism. The excellent performance in imaging completeness and specificity for both human cells and plant cells clearly shows that these designed probes possess outstanding antipermeability to achieve long-term specific imaging of membrane. These probes also show some advanced features such as ultrafast staining, wash-free merit, favorable biocompatibility, good photostability, and effective resistance to viscosity and pH alteration. This work also provides a valuable design principle for membrane probes of plant cells that the designed probes require a suitable molecular size favoring the penetration of small pores of cell walls.

Plant exocytosis: Weaving distinct pathways to the plant plasma membrane

During plant growth and development newly synthesized proteins and other cargo molecules must be selectively targeted to a variety of cellular compartments. Perhaps the most important targeting involves the trafficking of proteins to the plasma membrane, where they control interactions with the environment and neighboring cells. During this process, proteins entering the secretory pathway are imported into the endoplasmic reticulum where they are folded and processed. They are then sorted and delivered to the Golgi complex, where they are further processed and modified.

A critical review on natural compounds interacting with the plant plasma membrane H+ -ATPase and their potential as biologicals in agriculture.

The plant plasma membrane (PM) H+ -ATPase is an essential enzyme controlling plant growth and development. It is an important factor in response to abiotic and biotic stresses and is subject to tight regulation. We are in demand for new sustainable natural growth regulators and as a key enzyme for regulation of transport into the plant cell the PM H+ -ATPase is a potential target for these. In this review, we have evaluated the known non-protein natural compounds with regulatory effects on the PM H+ -ATPase, focusing on their mechanism of action and their potential as biologicals/growth regulators in plant production of future sustainable agriculture.

Modulation of plant plasma membrane structure by exogenous fatty acid hydroperoxide is a potential perception mechanism for their eliciting activity.

Oxylipins are lipid-derived molecules that are ubiquitous in eukaryotes and whose functions in plant physiology have been widely reported. They appear to play a major role in plant immunity by orchestrating reactive oxygen species (ROS) and hormone-dependent signalling pathways. The present work focuses on the specific case of fatty acid hydroperoxides (HPOs). Although some studies report their potential use as exogenous biocontrol agents for plant protection, evaluation of their efficiency in planta is lacking and no information is available about their mechanism of action. In this study, the potential of 13(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid (13-HPOD) and 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13-HPOT), as plant defence elicitors and the underlying mechanism of action is investigated. Arabidopsis thaliana leaf resistance to Botrytis cinerea was observed after root application with HPOs. They also activate early immunity-related defence responses, like ROS. As previous studies have demonstrated their ability to interact with plant plasma membranes (PPM), we have further investigated the effects of HPOs on biomimetic PPM structure using complementary biophysics tools. Results show that HPO insertion into PPM impacts its global structure without solubilizing it. The relationship between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events.

Turgor pressure change in stomatal guard cells arises from interactions between water influx and mechanical responses of their cell walls

The ability of plants to absorb CO2 for photosynthesis and transport water from root to shoot depends on the reversible swelling of guard cells that open stomatal pores in the epidermis. Despite decades of experimental and theoretical work, the biomechanical drivers of stomatal opening and closure are still not clearly defined. We combined mechanical principles with a growing body of knowledge concerning water flux across the plant cell membrane and the biomechanical properties of plant cell walls to quantitatively test the long-standing hypothesis that increasing turgor pressure resulting from water uptake drives guard cell expansion during stomatal opening. To test the alternative hypothesis that water influx is the main motive force underlying guard cell expansion, we developed a system dynamics model accounting for water influx. This approach connects stomatal kinetics to whole plant physiology by including values for water flux arising from water status in the plant .

Quantifying the Organization and Dynamics of the Plant Plasma Membrane Across Scales Using Light Microscopy.

The plant cell surface continuum is composed of the cell wall, plasma membrane, and cytoskeleton. Plasmodesmata are specialized channels in the cell wall allowing intercellular communication and resource distribution. Proteins within these organelles play fundamental roles in development, perception of the external environment, and resource acquisition. Therefore, an understanding of protein dynamics and organization within the membrane and plasmodesmata is of fundamental importance to understanding both how plants develop as well as perceive the myriad of external stimuli they experience and initiate appropriate downstream responses. In this chapter, I will describe protocols for quantifying the dynamics and organization of the plasma membrane and plasmodesmata proteins across scales. The protocols described below allow researchers to determine bulk protein mobility within the membrane using fluorescence recovery after photobleaching (FRAP), imaging, and quantification of nanodomain size (with Airyscan confocal microscopy) and determining the dynamics of these nanodomains at the single particle level using total internal reflection (TIRF)single particle imaging.

Short-term effects of combined freeze–thaw and saline–alkali stresses on the physiological response in highland barley (Hordeum vulgare).

Highland barley (Hordeum vulgare L.), as the dominant crop on the Qinghai-Tibetan Plateau, is a typical representative of plants adapted to extreme environmental conditions. However, the harsh environment, severe salinisation and frequent freezing and thawing in the Qinghai-Tibetan Plateau are main limiting factor for crop growth in this region. The physiological response of highland barley to salinisation and freeze-thaw stresses was studied in this paper. Under the combined stresses of 60mmol/LNaCl·60mmol/LNaHCO3 and freeze-thaw cycles (10, -5, and 10°C), the changes in the relative moisture content, relative electrical conductivity, soluble protein, malondialdehyde (MDA) and photosynthetic indices Pn and E in seedling leaves of eight groups of treatments (CK, S, A, S-A, CK (FT), S (FT), A (FT), and S-A (FT)) were analysed. Results showed that a single stress did not cause a change in the MDA content. All of the combined stresses in S-A, CK (FT), S (FT), A (FT), and S-A (FT) treatments increased the MDA content of barley seedlings, and the MDA content of S-A (FT) reached 28.438 at T2 (-5°C) μmol/g. During the freeze-thaw cycle, the cell membrane of seedlings was damaged more seriously by alkali stress, which showed a significant increase in relative conductivity. The relative moisture content value of seedlings was more than 100% because the seedlings could absorb more moisture due to mechanical injury. The protein content of osmoregulatory substances in highland barley seedlings increased with increasing stress, indicating resistance to stress. Moreover, the effect of freeze-thaw stress on photosynthesis was more significant. The changes in indices proved that an appropriate amount of salt stress could improve the resistance of the plant cell membrane. Alkali stress had a significant effect on the growth of highland barley seedlings. Freezing and thawing can aggravate the damage of saline-alkali stress to highland barley seedlings, resulting in changes in the biological membrane permeability and photosynthesis of seedlings. The fluctuation of osmoregulation substance content confirmed that highland barley seedlings had a certain degree of stress resistance. Freeze-thaw cycles will aggravate the damage of land salinisation to highland barley seedlings. To better reduce the impact and loss of land salinisation and freeze-thaw disasters on agriculture in the Qinghai-Tibetan Plateau, priority should be given to solving freeze-thaw stress in the process of grain production.

Physiological Response of the Resurrection Fern Subjected to Environmental Stress as Shown by Plant Cell Membrane Integrity

Resurrection fern has a unique ability to maintain cell wall integrity when the plant cell is desiccated. It uses proteins such as late embryogenesis proteins and heat shock proteins to maintain their cellular functions. The purpose of this experiment is to determine the effects of environmental stressors on the physiological response of the resurrection fern (Pleopeltis polypodioides). The physiological response of resurrection fern plants was subjected to various temperatures (-50°C, 0°C, 25°C, and 50°C) for 7 days. Results indicated that there was a significant difference between hydrated and desiccated ferns based on the temperature. Additionally, electrolyte leakage measurements confirmed cell damage following exposure to temperature extremes of -50°C and 50°C.

The antioxidant response of Hedera helix leaves to seasonal temperature variations

Seasonal variations in the environment (e.g. of temperature and light intensity) can lead to the excess production of reactive oxygen species and oxidative stress, inducing changes in the permeability of the plant cell membrane and the structure and function of cellular molecules. To address these deleterious effects, plants activate various non-enzymatic and enzymatic antioxidants. This study seeks to detect the influence of seasonal variation in Hedera helix (ivy) leaves, collected in Banj brdo (Banja Luka, Republika Srpska, Bosnia and Herzegovina) between December 2017 and November 2018, on oxidative (hydrogen peroxide and malondialdehyde) and antioxidant (superoxide dismutase, catalase, Class III peroxidases, and phenolic compounds) parameters. During the winter-early spring months (a temperature range of 0.7 to 5.4?C), we detected an increase in the values of all the oxidative and antioxidant parameters, whereas during the spring, summer, and autumn months (a temperature range of 15 to 25?C), the values of most of these parameters fell. However, a peak in the parameter values was detected during June and July 2018, which might be attributable to the influence of the changes in both light intensity and temperature and to the effects of intensive shoot growth. Our results highlight the importance of the antioxidant protection system of H. helix for its acclimation to seasonal variations in the environment, especially temperature.

Extraction of Galactolipids from Waste By-Products: The Feasibility of Green Chemistry Methods

Galactolipids are a class of lipids present, inter alia, in the plastid membranes of plant cells. Apart from their biological significance, they are recognized as an important group of bioactive agents, especially in the treatment of osteoarthritis. The aim of this research was to evaluate the usefulness of the green chemistry approach in the extraction of these compounds. Waste products of food processing were selected as a raw material to improve the sustainability of the process even further, and their galactolipid content was investigated using an LC-MS analysis. The rosehip pomace, which was recognized as the most promising amongst materials used in this study, was subjected to supercritical fluid extraction (SFE) and ultrasound-assisted extraction (UAE). It transpired that SFE using pure CO2 was not an effective method for the extraction of galactolipids, although the use of ethanol as a cosolvent favored the separation. The results of UAE were also very promising—the improvement of the extraction yield up to 74% was observed. The green chemistry approaches used for galactolipid isolation were compared with a conventional processing method and proved to be an interesting alternative.

Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport.

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.

Deciphering the role of plant plasma membrane lipids in response to invasion patterns: how could biology and biophysics help?

Plants have to constantly face pathogen attacks. To cope with diseases, they have to detect the invading pathogen as early as possible via the sensing of conserved motifs called invasion patterns. The first step of perception occurs at the plasma membrane. While many invasion patterns are perceived by specific proteinaceous immune receptors, several studies have highlighted the influence of the lipid composition and dynamics of the plasma membrane in the sensing of invasion patterns. In this review, we summarize current knowledge on how some microbial invasion patterns could interact with the lipids of the plasma membrane, leading to a plant immune response. Depending on the invasion pattern, different mechanisms are involved. This review outlines the potential of combining biological with biophysical approaches to decipher how plasma membrane lipids are involved in the perception of microbial invasion patterns.

Regulation of Cytosolic pH: The Contributions of Plant Plasma Membrane H+-ATPases and Multiple Transporters.

Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H+ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H+ transport activity, namely, H+-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H+ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H+-ATPases mediate H+ extrusion, whereas most members of other six proteins mediate H+ influx, thus contributing to cytosolic pH homeostasis by directly modulating H+ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H+-coupled substrate influx, whereas other intra-family members facilitate H+-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H+-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H+ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H+ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.

An atypical Phytophthora sojae RxLR effector manipulates host vesicle trafficking to promote infection.

In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway.

Potentiation of plant defense by bacterial outer membrane vesicles is mediated by membrane nanodomains.

Outer membrane vesicles (OMVs) are released from the outer membranes of Gram-negative bacteria during infection and modulate host immunity during host-pathogen interactions. The mechanisms by which OMVs are perceived by plants and affect host immunity are unclear. Here, we used the pathogen Xanthomonas campestris pv. campestris to demonstrate that OMV-plant interactions at the Arabidopsis thaliana plasma membrane (PM) modulate various host processes, including endocytosis, innate immune responses, and suppression of pathogenesis by phytobacteria. The lipid phase of OMVs is highly ordered and OMVs directly insert into the Arabidopsis PM, thereby enhancing the plant PM's lipid order; this also resulted in strengthened plant defenses. Strikingly, the integration of OMVs into the plant PM is host nanodomain- and remorin-dependent. Using coarse-grained simulations of molecular dynamics, we demonstrated that OMV integration into the plant PM depends on the membrane lipid order. Our computational simulations further showed that the saturation level of the OMV lipids could fine-tune the enhancement of host lipid order. Our work unraveled the mechanisms underlying the ability of OMVs produced by a plant pathogen to insert into the host PM, alter host membrane properties, and modulate plant immune responses.

Calcium Sensor SlCBL4 Associates with SlCIPK24 Protein Kinase and Mediates Salt Tolerance in Solanum lycopersicum.

Soil salinity is one of the major environmental stresses that restrict the growth and development of tomato (Solanum lycopersicum L.) worldwide. In Arabidopsis, the calcium signaling pathway mediated by calcineurin B-like protein 4 (CBL4) and CBL-interacting protein kinase 24 (CIPK24) plays a critical role in salt stress response. In this study, we identified and isolated two tomato genes similar to the Arabidopsis genes, designated as SlCBL4 and SlCIPK24, respectively. Bimolecular fluorescence complementation (BiFC) and pull-down assays indicated that SlCBL4 can physically interact with SlCIPK24 at the plasma membrane of plant cells in a Ca2+-dependent manner. Overexpression of SlCBL4 or superactive SlCIPK24 mutant (SlCIPK24M) conferred salt tolerance to transgenic tomato (cv. Moneymaker) plants. In particular, the SlCIPK24M-overexpression lines displayed dramatically enhanced tolerance to high salinity. It is notable that the transgenic plants retained higher contents of Na+ and K+ in the roots compared to the wild-type tomato under salt stress. Taken together, our findings clearly suggest that SlCBL4 and SlCIPK24 are functional orthologs of the Arabidopsis counterpart genes, which can be used or engineered to produce salt-tolerant tomato plants.

Plant Sterol Clustering Correlates with Membrane Microdomains as Revealed by Optical and Computational Microscopy.

Local inhomogeneities in lipid composition play a crucial role in the regulation of signal transduction and membrane traffic. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used a new push–pull pyrene probe and fluorescence lifetime imaging microscopy (FLIM) combined with all-atom multiscale molecular dynamics simulations to provide a detailed view on the interaction between phospholipids and phytosterol and the effect of modulating cellular phytosterols on membrane-associated microdomains and phase separation formation. Our understanding of the organization principles of biomembranes is limited mainly by the challenge to measure distributions and interactions of lipids and proteins within the complex environment of living cells. Comparing phospholipids/phytosterol compositions typical of liquid-disordered (Ld) and liquid-ordered (Lo) domains, we furthermore show that phytosterols play crucial roles in membrane homeostasis. The simulation work highlights how state-of-the-art modeling alleviates some of the prior concerns and how unrefuted discoveries can be made through a computational microscope. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of plant cells and suggest that they are key compounds for the formation of plant PM microdomains and the lipid-ordered phase.

Correlative Light-Environmental Scanning Electron Microscopy of Plasma Membrane Efflux Carriers of Plant Hormone Auxin.

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

Foliar Application or Seed Priming of Cholic Acid-Glycine Conjugates can Mitigate/Prevent the Rice Bacterial Leaf Blight Disease via Activating Plant Defense Genes.

Xanthomonas Oryzae pv. oryzae (Xoo) causes bacterial blight and Rhizoctonia solani (R. solani) causes sheath blight in rice accounting for >75% of crop losses. Therefore, there is an urgent need to develop strategies for the mitigation of these pathogen infections. In this study, we report the antimicrobial efficacy of Cholic Acid-Glycine Conjugates (CAGCs) against Xoo and R. solani. We show that CAGC C6 is a broad-spectrum antimicrobial and is also able to degrade biofilms. The application of C6 did not hamper plant growth and showed minimal effect on the plant cell membranes. Exogenous application of C6 on pre-infection or post-infection of Xoo on rice susceptible genotype Taichung native (TN1) can mitigate the bacterial load and improve resistance through upregulation of plant defense genes. We further demonstrate that C6 can induce plant defense responses when seeds were primed with C6 CAGC. Therefore, this study demonstrates the potential of CAGCs as effective antimicrobials for crop protection that can be further explored for field applications.