Plant Plasma Membrane Research Articles

Covalent labeling of the Arabidopsis plasma membrane H+-ATPase reveals 3D conformational changes involving the C-terminal regulatory domain.

The plasma membrane proton pump is the primary energy transducing, electrogenic ion pump of the plasma membrane in plants and fungi. Compared to its fungal counterpart, the plant plasma membrane proton pump's regulatory C-terminal domain (CTD) contains an additional regulatory segment that links multiple sensory pathways regulating plant cell length through phosphorylation and recruitment of regulatory 14-3-3 proteins. However, a complete structural model of a plant proton pump is lacking. Here, we performed covalent labeling with mass spectrometric analysis (CL-MS) on the Arabidopsis pump AHA2 to identify potential interactions between the CTD and the catalytic domains. Our results suggest that autoinhibition in the plant enzyme is much more structurally complex than in the fungal enzyme.

Plant PI4P is required for bacteria to translocate type-3 effectors.

Type-3 effectors (T3E) of phytopathogenic Gram-negative bacteria fulfill a virulent role, causing disease, or an avirulent role, inducing immunity, following their translocation into plant cells. This study aimed to validate the hypothesis that bacterial T3E translocation requires lipidic compounds in plant cell membranes. Based on genetic, molecular, and biochemical assays, we determined that phosphatidylinositol 4-phosphate (PI4P) associated with plant cell membranes is essential for the translocation of T3E by bacterial pathogens. Replicate experimental data revealed that PI4P cooperates with the type-3 translocase HrpF to facilitate the translocation of effectors TAL and Xop from Xanthomonas oryzae and Hop from Pseudomonas syringae into the cells of Oryza sativa and Nicotiana benthamiana, respectively. Genetic and molecular analyses confirmed that, once translocated into plant cells, the distinct effectors induce disease or immunity. Combined genetic and pharmacological analyses revealed that when PI4P content is suppressed via genetic or pharmacological measures, the T3 effector translocation is considerably suppressed, resulting in serious inhibition of bacterial infection. Overall, these findings demonstrate that cooperative functioning of HrpF-PI4P is conserved in bacterial effectors and plants.

Transcriptome Analysis Reveals the Crucial Role of Phenylalanine Ammonia-Lyase in Low Temperature Response in Ammopiptanthus mongolicus.

Background: Ammopiptanthus mongolicus is a rare temperate evergreen shrub with high tolerance to low temperature, and understanding the related gene expression regulatory network can help advance research on the mechanisms of plant tolerance to abiotic stress. Methods: Here, time-course transcriptome analysis was applied to investigate the gene expression network in A. mongolicus under low temperature stress. Results: A total of 12,606 differentially expressed genes (DEGs) were identified at four time-points during low temperature stress treatment, and multiple pathways, such as plant hormones, secondary metabolism, and cell membranes, were significantly enriched in the DEGs. Trend analysis found that the expression level of genes in cluster 19 continued to upregulate under low temperatures, and the genes in cluster 19 were significantly enriched in plant hormone signaling and secondary metabolic pathways. Based on the transcriptome data, the expression profiles of the genes in abscisic acid, salicylic acid, and flavonoid metabolic pathways were analyzed. It was found that biosynthesis of abscisic acid and flavonoids may play crucial roles in the response to low temperature stress. Furthermore, members of the phenylalanine ammonia-lyase (PAL) family in A. mongolicus were systematically identified and their structures and evolution were characterized. Analysis of cis-acting elements showed that the PAL genes in A. mongolicus were closely related to abiotic stress response. Expression pattern analysis showed that PAL genes responded to various environmental stresses, such as low temperature, supporting their involvement in the low temperature response in A. mongolicus. Conclusions: Our study provides important data for understanding the mechanisms of tolerance to low temperatures in A. mongolicus.

A fungal phospholipase C involved in the degradation of plant glycosylinositol phosphorylceramides during Arabidopsis/Botrytis interaction

This study investigates the presence and significance of phosphorylated oligosaccharides that accumulate during the interaction between Arabidopsis thaliana and Botrytis cinerea, a necrotrophic fungus that poses a major threat to crops worldwide. While previous research has extensively characterized cell wall-derived molecules during fungal infection, the role of plasma membrane-derived ones remains unclear. Here, we reveal the discovery of inositol phosphate glycans (IPGs) released during infection, originating from plant sphingolipids, specifically glycosylinositol phosphorylceramides (GIPC). Advanced chromatography, mass spectrometry techniques and molecular biology were employed to identify these IPGs, and determine their origins. In addition to the well-characterized role of B. cinerea in releasing cell wall-degrading enzymes, this research suggests that B. cinerea’s enzymatic machinery may also target the degradation of the plant plasma membrane. As a consequence of this, IPGs identical to those generated by the host plant are released, most likely due to activity of a putative phospholipase C that acts on GIPC plasma membrane lipids. These insights could pave the way for developing new strategies to enhance crop resistance by focusing on membrane integrity in addition to cell wall fortification.

Associations of Arbuscular Mycorrhizal Fungi (AMF) for Enhancements in Soil Fertility and Promotion of Plant Growth: A Review

Arbuscular Mycorrhizal Fungi are used for soil fertility enhancements and stimulating plant growth in which they association with other organisms like terrestrial plants. Mycorrhizas create an association between fungi and the roots of plants. Therefore, the review was made to point out important fungal species involved in fungal plant interaction and their major roles in agriculture as well as ecosystem. 80% of plants form associations with mycorrhizal fungi. The fungal are used to use their different organs like chain, arbuscular, vesicle, supportive cells and spore to interact with the other plant/ plat’s organ. The mycorrhizal fungi can be categorized into two principal classifications based on their anatomical interactions with the roots of host plants. Arbuscular Mycorrhizal and Ectomycorrhizal fungi utilize two distinct strategies for nutrient acquisition. The main categories of vesicular arbuscular mycorrhizal associations are linear or coiling and of ectomycorrhizal associations are epidermal or cortical. The rhizospheric and endophytic microbes promote plant growth as inoculated with crop. AM fungi as an obligate symbiont share a distinct feature called arbuscules as a site of nutrient exchanges between host and fungi. Arbuscules developed between cell wall and plasma membrane of root cortical cells and differentiated from plant plasma membrane by periarbuscular membrane. Arbuscular mycorrhizal fungi (AMF) play an indispensable role in augmenting plant nutrient acquisition, enhancing plant resilience and tolerance to various environmental stresses, improving soil fertility and structure, and providing numerous beneficial effects. AMF engage in interactions with other soil microorganisms, such as plant growth-promoting rhizobacteria, resulting in a synergistic effect that promotes plant growth and offers protection against pathogens associated with Rhizobia. Both AMF and Rhizobia utilize the same signaling pathways, which facilitate their association with host plants and enable nitrogen fixation within the soil ecosystem. A positive relationship has been established between AMF colonization and the diversity of soil microbial communities. Nitrogen-fixing rhizobia, mycorrhizal fungi, and root nodule symbioses typically exhibit synergistic interactions concerning infection rates and their effects on mineral nutrition and plant growth, thereby significantly enhancing the status of soil fertility, particularly with respect to soil quality characteristics.

Genome wide analysis of HMA gene family in Hydrangea macrophylla and characterization of HmHMA2 in response to aluminum stress

Aluminum toxicity poses a significant threat to plant growth, especially in acidic soils. Heavy metal ATPases (HMAs) are crucial for transporting heavy metal ions across plant cell membranes, yet their role in Al3+ transport remains unexplored. This study identified eight HmHMA genes in the genome of Hydrangea macrophylla, categorizing them into two major clades based on phylogenetic relationships. These genes were found unevenly distributed across six chromosomes. Detailed analysis of their physicochemical properties, collinearity, and gene structure was conducted. RNA-seq and qRT-PCR analyses revealed that specific HmHMA genes, notably HmHMA2, were predominantly expressed in roots and flowers under Al3+ stress, indicating their potential role in Al3+ tolerance. HmHMA2 showed significant expression in roots, especially under Al3+ stress conditions, and when expressed in yeast cells, it conferred resistance to aluminum and zinc but increased sensitivity to cadmium. Overexpression of HmHMA2 in hydrangea leaf discs significantly improved Al3+ tolerance, reduced oxidative stress markers like hydrogen peroxide and malondialdehyde, and enhanced antioxidant enzyme activity such as SOD, POD and CAT compared to controls. These findings shed lights on the potential role of HmHMAs in Al transport and tolerance in H. macrophylla.

Knockdown of β-conglycinin α' and α subunits alters seed protein composition and improves salt tolerance in soybean.

Soybean is an important plant source of protein worldwide. Increasing demands for soybean can be met by improving the quality of its seed protein. In this study, GmCG-1, which encodes the β-conglycinin α' subunit, was identified via combined genome-wide association study and transcriptome analysis. We subsequently knocked down GmCG-1 and its paralogues GmCG-2 and GmCG-3 with CRISPR-Cas9 technology and generated two stable multigene knockdown mutants. As a result, the β-conglycinin content decreased, whereas the 11S/7S ratio, total protein content and sulfur-containing amino acid content significantly increased. Surprisingly, the globulin mutant exhibited salt tolerance in both the germination and seedling stages. Little is known about the relationship between seed protein composition and the salt stress response in soybean. Metabonomics and RNA-seq analysis indicated that compared with the WT, the mutant was formed through a pathway that was more similar to that of active salicylic acid biosynthesis; however, the synthesis of cytokinin exhibited greater defects, which could lead to increased expression of plant dehydrin-related salt tolerance proteins and cell membrane ion transporters. Population evolution analysis suggested that GmCG-1, GmCG-2, and GmCG-3 were selected during soybean domestication. The soybean accessions harboring GmCG-1Hap1 presented relatively high 11S/7S ratios and relatively high salt tolerance. In conclusion, knockdown of the β-conglycinin α and α' subunits can improve the nutritional quality of soybean seeds and increase the salt tolerance of soybean plants, providing a strategy for designing soybean varieties with high nutritional value and high salt tolerance.

A Mechanistic Approach on Structural, Analytical and Pharmacological Potential of Beta-sitosterol: A Promising Nutraceutical

Abstract: Phytosterols are bioactive substances that are found spontaneously in the cell membranes of plants and have an atomic composition similar to cholesterol produced by vertebrate cells. They are widely distributed in dietary lipids from plants such as nuts, seeds, and beans with olive oil. β-sitosterol has a variation of pharmacological belongings, with analgesic, immunomodulatory, antiseptic, antineoplastic, anti-inflammatory, cholesterol decreasing, hepatoprotective, and protecting action concerning respiratory and non-alcoholic fatty liver disease illnesses, antioxidant, and anti-diabetic activity. Clinical studies on humans have shown that it works against prostate cancer and has anti-inflammatory and anti-cancer properties. Pharmacological testing of β-sitosterol demonstrated a range of actions including antibacterial, anti-inflammatory, antinociceptive, anticancer, antifertility, angiogenic, antioxidant, immunomodulatory, diabetes-fighting, and anticancer without significant toxicity. Several formulations have been created by numerous authors, but there are few scholarly reviews of the analytical, pharmacology, and phytochemistry methodologies for this molecule. In this review the literature on β-sitosterol, its biosynthesis, pharmacology, nutraceutical applications, toxicity, formulations, and analytical techniques are all highlighted.

Aspects of β-sitosterol's Pharmacology, Nutrition and Analysis.

Phytosterols are bioactive substances found naturally in the cell membranes of plants and have an arrangement of molecules similar to that of fat, which is produced by mammalian cells. They are widely distributed as dietary sources of lipids in plants, such as nuts, seeds, olive oil, and legumes. This review provides a summary of the efficacy of BS in treating lifestyle problems, as well as an appraisal of previous research. Data was collected from PubMed, ScienceDirect, Scopus, and Google scholar (1968 -2024) using standard keywords "β-sitosterol," "Classification," "Biosynthesis," "Pharmacokinetics," "Herbal nutraceutical," "Analytical," "Structure," "Pharmacological effect." A total of 222 studies were included in this review. Numerous in vitro and in vivo investigations have shown that BSs exhibit several biological properties such as calming and anxiolytic effects; narcotic and immune-stimulating effects; antibacterial, antineoplastic, inflammation-causing, lipid-lowering, and hepatoprotective effects; and antioxidant, anti-diabetic, and wound-healing effects in contrast to respiratory and non-alcoholic fatty liver disease illnesses. β-sitosterol is a promising natural substance for the management of cholesterol and inflammation. However, further studies are needed to understand its pharmacological consequences and determine its best use in clinical applications. β-Sitosterol, also known as "plant sterol ester," is often present in plants and has several applications, notably in medicine and the food industry. Experimental research on β-sitosterol provides unequivocal evidence that phytosterol can be supplemented with other methods to combat serious illnesses. Such a high potential identifies this substance as a noteworthy medication for the future based on its composition. Although β-sitosterol has anticancer and anti-inflammatory properties and is useful in human clinical trials for enlarged prostates, its mechanism of action remains unclear.

Contrasting Retromer with a Newly Described Retriever in Arabidopsis thaliana.

The tight regulation of protein composition within the plasma membranes of plant cells is crucial for the proper development of plants and for their ability to respond to a changing environment. Upon being endocytosed, integral membrane proteins can be secreted, sorted into multivesicular bodies/late endosomes, and degraded in the lytic vacuole, or recycled back to the plasma membrane to continue functioning. The evolutionarily conserved retromer complex has attracted the interest of plant cell biologists for over a decade as it has emerged as a key regulator of the trafficking of endocytosed integral plasma membrane proteins. Recently, a related recycling complex that shares a subunit with retromer was described in metazoan species. Named "retriever", homologs to the proteins that comprise this new recycling complex and its accessory proteins are found within plant lineages. Initial experiments indicate that there is conservation of function between metazoan and plant retriever proteins, suggesting that it is prudent to re-evaluate the available plant retromer data with the added potential of a plant retriever complex.

Iron Toxicity in Plants: A Review

Iron (Fe) is an essential micronutrient for plant growth and development. However, increased absorption of iron leads to iron toxicity. This leads to damage to plant cell membranes, decreased growth, productivity, and thus general health. The presence of free iron in cells can lead to an imbalance Redox in cells Pro-oxidant state, thus generating oxidative stress. In plants, iron is involved in chlorophyll synthesis, and is therefore essential for maintaining the structure and function of chloroplasts. Micronutrients in the soil can limit plant growth, even if all other nutrients are available in sufficient quantities. Micronutrient deficiencies are widespread due to the nature of the soil, high pH, low organic matter, salt stress, and persistent drought. High levels of bicarbonate in irrigation water, as well as unbalanced use of fertilizers. Iron (Fe) toxicity is a widespread nutritional disorder. It grows in acidic, sulphurous soils, as well as low sandy soils (i.e. iron is easily reducible) that contain a high percentage of organic matter. Iron toxicity can be reduced by using rice that is tolerant to high levels of iron.

A Rapid Method for Screening Pathogen-Associated Molecular Pattern-Triggered Immunity-Intensifying Microbes.

PAMP-triggered immunity (PTI) is the first layer of plant defense response that occurs on the plant plasma membrane. Recently, the application of a rhizobacterium, Bacillus amyloliquefaciens strain PMB05, has been demonstrated to enhance flg22Pst- or harpin-triggered PTI response such as callose deposition. This PTI intensification by PMB05 further contributes to plant disease resistance to different bacterial diseases. Under the demand for rapid and large-scale screening, it has become critical to establish a non-staining technology to identify microbial strains that can enhance PTI responses. Firstly, we confirmed that the expression of the GSL5 gene, which is required for callose synthesis, can be enhanced by PMB05 during PTI activation triggered by flg22 or PopW (a harpin from Ralstonia solanacearum). The promoter region of the GSL5 gene was further cloned and fused to the coding sequence of gfp. The constructed fragments were used to generate transgenic Arabidopsis plants through a plant transformation vector. The transgenic lines of AtGSL5-GFP were obtained. The analysis was performed by infiltrating flg22Pst or PopW in one homozygous line, and the results exhibited that the green fluorescent signals were observed until after 8 h. In addition, the PopW-induced fluorescent signal was significantly enhanced in the co-treatment with PMB05 at 4 h after inoculation. Furthermore, by using AtGSL5-GFP to analyze 13 Bacillus spp. strains, the regulation of PopW-induced fluorescent signal was observed. And, the regulation of these fluorescent signals was similar to that performed by callose staining. More importantly, the Bacillus strains that enhance PopW-induced fluorescent signals would be more effective in reducing the occurrence of bacterial wilt. Taken together, the technique by using AtGSL5-GFP would be a promising platform to screen plant immunity-intensifying microbes to control bacterial wilt.

The cytoskeleton controls the dynamics of plasma membrane proteins and facilitates their endocytosis in plants.

Plasma membranes (PMs) are highly dynamic structures where lipids and proteins can theoretically diffuse freely. However, reports indicate that PM proteins do not freely diffuse within their planes but are constrained by cytoskeleton networks, though the mechanisms for how the cytoskeleton restricts lateral diffusion of plant PM proteins are unclear. Through single-molecule tracking, we investigated the dynamics of 6 Arabidopsis (Arabidopsis thaliana) PM proteins with diverse structures and found distinctions in sizes and dynamics among these proteins. Moreover, we showed that the cytoskeleton, particularly microtubules, limits the diffusion of PM proteins, including transmembrane and membrane-anchoring proteins. Interestingly, the microfilament skeleton regulates intracellular transport of endocytic cargo. Therefore, these findings indicate that the cytoskeleton controls signal transduction by limiting diffusion of PM proteins in specific membrane compartments and participating in transport of internalized cargo vesicles, thus actively regulating plant signal transduction.

Cell integrity maintenance and genetic transfection of protoplasts in an acoustofluidic system

Hydrodynamic force loading platforms based on acoustofluidics have been developed to study the mechanical deformation of cancer cells and to control cell behavior. However, so far there have been no experimental measurements on living plant cells using such an acoustic approach. Unique structures, including cell walls, allow plant cells to exhibit more variation in mechanical resistance. In this work, we analyzed plant cell deformation and membrane permeability using a gigahertz (GHz) acoustofluidic system. By recording the proportion of intact cells in the cell population, we evaluated the mechanical resistance of the protoplasts to the hydrodynamic forces of the acoustic streaming. The results showed that a regenerated primary cell wall (PCW) could significantly improve the mechanical resistance of individual plant cells within 24 h compared to the freshly prepared protoplasts without walls. The results of enzymatic degradation showed that three main components of the primary cell wall contribute to different degrees to the improvement of the mechanical properties of the cells, in decreasing order: cellulose, hemicellulose, and pectin. Furthermore, we have shown that such an acoustofluidic system can alter the permeability of the protoplast membrane in a controllable manner for transient gene expression.

Mechanisms of Cancerprotective Effects of Phytosterols. Literature Review

Cancer is recognized as the second leading cause of mortality in the Russian Federation. Prolonging the life of oncology patients involves treatment with toxic drugs, causing multiple side effects. Today, scientists around the world are striving to find non-toxic drugs. The present study explores phytosterols. Phytosterols refer to a class of steroids widely distributed in plants as an essential component of plant cell membranes. They include stigmasterol, beta-sitosterol, and campesterol. Stigmasterol has been found to increase the expression of proapoptotic genes (Bax, p53) and decrease the expression of the antiapoptotic gene Bcl-2 in HepG2 liver cancer cells. Stigmasterol is able to induce cell arrest in G0-G1 phase (stationary phase), resulting in fewer cells in the G2/M phase (division phase). It induces apoptosis and protective autophagy in gastric cancer cells while inhibiting the Akt/mTOR signaling pathway. β-sitosterol exhibits growth inhibitory and cytotoxic effects against a number of established cancer cell lines in vitro and in vivo, and remains free from acute/subacute toxic effects. β-sitosterol is widely used to treat chronic prostate diseases. In 2020, spendings on dietary supplements rich in beta-sitosterol accounted for $24 827 065 in the USA. Campesterol induces cell apoptosis via the mitochondrial pathway. It appears cytotoxic to U937 hepatocellular cancer cells. Campesterol induces cell apoptosis and activates proapoptotic signaling in ovarian cancer cell lines of a person. The present literature review demonstrates that specific substances in this group, beta-sitosterol, stigmasterol, and campesterol, provide pronounced antitumor effects.

Research Progress on Plant Shaker K+ Channels.

Plant growth and development are driven by intricate processes, with the cell membrane serving as a crucial interface between cells and their external environment. Maintaining balance and signal transduction across the cell membrane is essential for cellular stability and a host of life processes. Ion channels play a critical role in regulating intracellular ion concentrations and potentials. Among these, K+ channels on plant cell membranes are of paramount importance. The research of Shaker K+ channels has become a paradigm in the study of plant ion channels. This study offers a comprehensive overview of advancements in Shaker K+ channels, including insights into protein structure, function, regulatory mechanisms, and research techniques. Investigating Shaker K+ channels has enhanced our understanding of the regulatory mechanisms governing ion absorption and transport in plant cells. This knowledge offers invaluable guidance for enhancing crop yields and improving resistance to environmental stressors. Moreover, an extensive review of research methodologies in Shaker K+ channel studies provides essential reference solutions for researchers, promoting further advancements in ion channel research.

A combined lipidomic and proteomic profiling of Arabidopsis thaliana plasma membrane.

The plant plasma membrane (PM) plays a key role in perception of environmental signals, and set-up of adaptive responses. An exhaustive and quantitative description of the whole set of lipids and proteins constituting the PM is necessary to understand how these components allow to fulfill such essential physiological functions. Here we provide by state-of-the-art approaches the first combined reference of the plant PM lipidome and proteome from Arabidopsis thaliana suspension cell culture. We identified and quantified a reproducible core set of 2165 proteins, which is by far the largest set of available data concerning this plant PM proteome. Using the same samples, combined lipidomic approaches, allowing the identification and quantification of an unprecedented repertoire of 414 molecular species of lipids showed that sterols, phospholipids, and sphingolipids are present in similar proportions in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were further determined, allowing to establish the complete lipidome of Arabidopsis PM, and highlighting specific characteristics of the different molecular species of lipids. Results obtained point to a finely tuned adjustment of the molecular characteristics of lipids and proteins. More than a hundred proteins related to lipid metabolism, transport, or signaling have been identified and put in perspective of the lipids with which they are associated. This set of data represents an innovative resource to guide further research relative to the organization and functions of the plant PM.

Genome-Wide Identification and Analysis of Plasma Membrane H+-ATPases Associated with Waterlogging in Prunus persica (L.) Batsch

Plant plasma membrane H+-ATPase is a transport protein that is generally located on the plasma membrane and generates energy by hydrolyzing adenosine triphosphate (ATP) to pump hydrogen ions (H+) in the cytoplasm out of the cell against a concentration gradient. The plasma membrane H+-ATPases in plants are encoded by a multigene family and potentially play a fundamental role in regulating plant responses to various abiotic stresses, thus contributing to plant adaptation under adverse conditions. To understand the characteristics of the plasma membrane H+-ATPase family in peach (Prunus persica), this study analyzed the plasma membrane H+-ATPase family genes in peach. The results showed that there were 27 members of the plasma membrane H+-ATPase family in peach with amino acid sequences ranging from 943 to 1327. Subcellular localization showed that 23 of the 27 members were located on the cell membrane, and the phylogenetic tree analysis indicated that peach plasma membrane H+-ATPase members were divided into five groups. There were four genes with tandem repeat relationships, and six plasma membrane H+-ATPase genes were differentially expressed after 5 days of flooding and under non-flooding conditions based on the RNA-seq and RT-qPCR analyses. This study also investigated the characteristics and possible functions of the plasma membrane H+-ATPase family members in peach. The results provide theoretical support for further studies on their biological functions in peach.

Molecular engineering of fluorescent dyes for long-term specific visualization of the plasma membrane based on alkyl-chain-regulated cell permeability

Long-term visualization of changes in plasma membrane dynamics during important physiological processes can provide intuitive and reliable information in a 4D mode. However, molecular tools that can visualize plasma membranes over extended periods are lacking due to the absence of effective design rules that can specifically track plasma membrane fluorescent dye molecules over time. Using plant plasma membranes as a model, we systematically investigated the effects of different alkyl chain lengths of FMR dye molecules on their performance in imaging plasma membranes. Our findings indicate that alkyl chain length can effectively regulate the permeability of dye molecules across plasma membranes. The study confirms that introducing medium-length alkyl chains improves the ability of dye molecules to target and anchor to plasma membranes, allowing for long-term imaging of plasma membranes. This provides useful design rules for creating dye molecules that enable long-term visualization of plasma membranes. Using the amphiphilic amino-styryl-pyridine fluorescent skeleton, we discovered that the inclusion of short alkyl chains facilitated rapid crossing of the plasma membrane by the dye molecules, resulting in staining of the cell nucleus and indicating improved cell permeability. Conversely, the inclusion of long alkyl chains hindered the crossing of the cell wall by the dye molecules, preventing staining of the cell membrane and demonstrating membrane impermeability to plant cells. The FMR dyes with medium-length alkyl chains rapidly crossed the cell wall, uniformly stained the cell membrane, and anchored to it for a long period without being transmembrane. This allowed for visualization and tracking of the morphological dynamics of the cell plasma membrane during water loss in a 4D mode. This suggests that the introduction of medium-length alkyl chains into amphiphilic fluorescent dyes can transform them from membrane-permeable fluorescent dyes to membrane-staining fluorescent dyes suitable for long-term imaging of the plasma membrane. In addition, we have successfully converted a membrane-impermeable fluorescent dye molecule into a membrane-staining fluorescent dye by introducing medium-length alkyl chains into the molecule. This molecular engineering of dye molecules with alkyl chains to regulate cell permeability provides a simple and effective design rule for long-term visualization of the plasma membrane, and a convenient and feasible means of chemical modification for efficient transmembrane transport of small molecule drugs.

Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids.

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.