Polysaccharide assemblies in fungal and plant cell walls explored by solid-state NMR.

Structural analysis of macromolecular complexes within their natural cellular environment presents a significant challenge. Recent applications of solid-state NMR (ssNMR) techniques on living fungal cells and intact plant tissues have greatly enhanced our understanding of the structure of extracellular matrices. Here, we selectively highlight the most recent progress in this field. Specifically, we discuss how ssNMR can provide detailed insights into the chemical composition and conformational structure of pectin, and the consequential impact on polysaccharide interactions and cell wall organization. We elaborate on the use of ssNMR data to uncover the arrangement of the lignin-polysaccharide interface and the macrofibrillar structure in native plant stems or during degradation processes. We also comprehend the dynamic structure of fungal cell walls under various morphotypes and stress conditions. Finally, we assess how the combination of NMR with other techniques can enhance our capacity to address unresolved structural questions concerning these complex macromolecular assemblies.

Analytical implications of different methods for preparing plant cell wall material.

Almost all plant cells are surrounded by a wall constructed of co-extensive networks of polysaccharides and proteoglycans. The capability to analyse cell wall components is essential for both understanding their complex biology and to fully exploit their numerous practical applications. Several biochemical and immunological techniques are used to analyse cell walls and in almost all cases the first step is the preparation of an alcohol insoluble residue (AIR). There is significant variation in the protocols used for AIR preparation, which can have a notable impact on the downstream extractability and detection of cell wall components. To explore these effects, we have formally compared ten AIR preparation methods and analysed polysaccharides subsequently extracted using high-performance anion exchange chromatography (HPAEC-PAD) and Micro Array Polymer Profiling (MAPP). Our results reveal the impact that AIR preparation has on downstream detection of cell wall components and the need for optimisation and consistency when preparing AIR.

Determination of the Qualitative Composition of Biologically-Active Substances of Extracts of In Vitro Callus, Cell Suspension, and Root Cultures of the Medicinal Plant Rhodiola rosea.

The results of the qualitative composition analysis of the dried biomass extracts of in vitro callus, cell suspension, and root cultures show that the main biologically active substances (BAS) in the medicinal plant, Rhodiola rosea, are 6-C-(1-(4-hydroxyphenyl)ethyl)aromadendrin (25 mg, yield 0.21%), 2-(3,7-dihydroxy-2-(2-hydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-yl)-6,7-dihydroxychroman-4-one (23 mg, yield 0.2%), 2-(3,4-dimethoxyphenyl)-5,7-dimethoxychroman-4-one (175 mg, yield 1.5%), 5,7-dihydroxy-2-(4-hydroxy-3-(2-(4-hydroxyphenyl)-4-oxo-4H-chromen-6-yl)phenyl)-4H-chromen-4-one (45 mg, yield 0.5%), 5,6,7,8-tetrahydroxy-4-methoxyflavone (0.35 mg, 0.5%). BAS from the dried biomass extracts of in vitro callus, cell suspension, and root cultures of Rhodiola rosea will be used for the production of pharmaceuticals and dietary supplements with antitumor, antimicrobial, and antioxidant effects.

The genetic basis of cytoplasmic male sterility and fertility restoration in wheat.

Hybrid wheat varieties give higher yields than conventional lines but are difficult to produce due to a lack of effective control of male fertility in breeding lines. One promising system involves the Rf1 and Rf3 genes that restore fertility of wheat plants carrying Triticum timopheevii-type cytoplasmic male sterility (T-CMS). Here, by genetic mapping and comparative sequence analyses, we identify Rf1 and Rf3 candidates that can restore normal pollen production in transgenic wheat plants carrying T-CMS. We show that Rf1 and Rf3 bind to the mitochondrial orf279 transcript and induce cleavage, preventing expression of the CMS trait. The identification of restorer genes in wheat is an important step towards the development of hybrid wheat varieties based on a CMS-Rf system. The characterisation of their mode of action brings insights into the molecular basis of CMS and fertility restoration in plants.

Plant cell cultures as food-aspects of sustainability and safety.

KEY MESSAGE: Sustainability and safety aspects of plant cell cultures as food are presented. Applicability of dairy side streams as carbon source and use of natural growth enhancers in cultivation are shown. Biotechnologically produced cellular products are currently emerging to replace and add into the portfolio of agriculturally derived commodities. Plant cell cultures used for food could supplement current food production. However, still many aspects need to be resolved before this new food concept can enter the market. Issues related to sustainability and safety for human consumption are relevant for both consumers and regulators. In this study, two plant cell cultures, deriving from arctic bramble (Rubus arcticus) and birch (Betula pendula), were cultivated using lactose-rich dairy side streams as alternative carbon sources to replace sucrose. Biomasses were comparable to those of original plant cell culture media when up to 83% and 75% of the original sucrose was replaced by these side streams for arctic bramble and birch cell cultures, respectively. Furthermore, nutritional composition or sensory properties were not compromised. Synthetic plant growth regulators were replaced by natural components, such as coconut water and IAA for several subculture cycles. Finally, it was shown that only trace amounts of free growth regulators are present in the cells at the harvesting point and assessment by freshwater crustaceans assay indicated that toxicity of the cells was not exceeding that of traditionally consumed bilberry fruit.

Intact endosperm cells in buckwheat flour limit starch gelatinization and digestibility in vitro.

The objective of this study was to determine the biophysical properties of buckwheat (BW) endosperm and their influences on detachment of intact cells, starch gelatinization and digestibility. The intact cells were isolated from BW kernels by dry milling and sieving. The microscopy and texture analysis showed intact endosperm cells could be detached easily due to the fragile structure and low hardness of BW endosperm. More than 70% intact cells were found in commercial light flour. The starch granules entrapped in intact cells exhibited a delay gelatinization and restricted swelling behavior (2-3 â„ƒ higher onset gelatinization temperature than isolated starch). Starch in BW flour had a markedly lower extent of digestion compared to the broken cells and isolated starch. This study provided a new mechanistic understanding of low glycemic index of BW food, and could guide the processing of BW flour to retain slow digestion properties.

Fluorescence visualization of cellulose and pectin in the primary plant cell wall.

Plant cell walls constitute the extracellular matrix surrounding plant cells and are composed mainly of polysaccharides. The chemical makeup of the primary plant cell wall, and specifically, the abundance, localization and arrangement of the constituting polysaccharides are intimately linked with growth, morphogenesis and differentiation in plant cells. Visualization of the cell wall components is, therefore, a crucial tool in plant cell developmental studies. In this technical update, we present protocols for fluorescence visualization of cellulose and pectin in selected plant tissues and illustrate examples of some of the available labels that hold promise for live imaging of plant cell wall expansion and morphogenesis.

Membrane Extracts from Plant Tissues.

The comparison of isolated plant cell membranous enclosures can be hampered if their extraction method differs, e.g., in regard to the utilized buffers, the tissue, or the developmental stage of the plant. Thus, for comparable results, different cellular compartments should be isolated synchronously in one procedure. Here, we devise a workflow to isolate different organelles from one tissue, which is applicable to different eudicots such as Medicago x varia and Solanum lycopersicum. We describe this method for the isolation of different organelles from one plant tissue for the example of Arabidopsis thaliana. All compartments are retrieved by utilizing differential centrifugation with organelle-specific parameters.

Innovative Approaches for Recovery of Phytoconstituents from Medicinal/Aromatic Plants and Biotechnological Production.

Continuously growing demand for plant derived therapeutic molecules obtained in a sustainable and eco-friendly manner favors biotechnological production and development of innovative extraction techniques to obtain phytoconstituents. What is more, improving and optimization of alternative techniques for the isolation of high value natural compounds are issues having both social and economic importance. In this critical review, the aspects regarding plant biotechnology and green downstream processing, leading to the production and extraction of increased levels of fine chemicals from both plant cell, tissue, and organ culture or fresh plant materials and the remaining by-products, are discussed.

Quantum dots are conventionally applicable for wide-profiling of wall polymer distribution and destruction in diverse cells of rice.

Plant cell walls represent enormous biomass resources for biofuels, and it thus becomes important to establish a sensitive and wide-applicable approach to visualize wall polymer distribution and destruction during plant growth and biomass process. Despite quantum dots (QDs) have been applied to label biological specimens, little is reported about its application in plant cell walls. Here, semiconductor QDs (CdSe/ZnS) were employed to label the secondary antibody directed to the epitopes of pectin or xylan, and sorted out the optimal conditions for visualizing two polysaccharides distribution in cell walls of rice stem. Meanwhile, the established QDs approach could simultaneously highlight wall polysaccharides and lignin co-localization in different cell types. Notably, this work demonstrated that the QDs labeling was sensitive to profile distinctive wall polymer destruction between alkali and acid pretreatments with stem tissues of rice. Hence, this study has provided a powerful tool to characterize wall polymer functions in plant growth and development in vivo, as well as their distinct roles during biomass process in vitro.

Electroosmotic extraction coupled to mass spectrometry analysis of metabolites in live cells.

Quantitative mass spectrometry analysis of metabolites at a single-cell level is critical to understanding the cell functionality and heterogeneity. To preserve cell viability after extraction, the extracted volume needs to be precisely controlled at a subpicoliter-to-picoliter level. Recently, we developed a volume-controlled, and highly sensitive approach for live cell analysis at a single-cell level by integrating electroosmotic extraction and nano-electrospray ionization mass spectrometry (nanoESI MS) analysis. Herein, we use outer epidermal cells of Allium cepa as a model system to present the details of our workflow, including detailed descriptions of the experimental setup for live cell analysis, preparation of the extraction nanopipette, establishment of calibration curves, and extraction and quantification of glucose in an individual onion cell. The capability of this procedure for quantitative live cell analysis has been demonstrated by accurate quantification of glucose in Allium cepa. In principle, our approach is applicable to identification and quantification of metabolites in live mammalian cells.

Microfluidic capillary zone electrophoresis mass spectrometry analysis of alkaloids in Lobelia cardinalis transgenic and mutant plant cell cultures.

Application of a microfluidic CE* device for CZE-MS allows for fast, rapid, and in-depth analysis of large sample sets. This microfluidic CZE-MS device, the 908 Devices ZipChip, involves minimal sample preparation and is ideal for small cation analytes, such as alkaloids. Here, we evaluated the microfluidic device for the analysis of alkaloids from Lobelia cardinalis hairy root cultures. Extracts from wild-type, transgenic, and selected mutant plant cultures were analyzed and data batch processed using the mass spectral processing software MZmine2 and the statistical software Prism 8. In total 139 features were detected as baseline resolved peaks via the MZmine2 software optimized for the electrophoretic separations. Statistically significant differences in the relative abundance of the primary alkaloid lobinaline (C27 H34 N2 ), along with several putative "lobinaline-like" molecules were observed utilizing this approach. Additionally, a method for performing both targeted and untargeted MS/MS experiments using the microfluidic device was developed and evaluated. Coupling data-processing software with CZE-MS data acquisition has enabled comprehensive metabolomic profiles from plant cell cultures to be constructed within a single working day.

Cell wall polysaccharides degradation and ultrastructure modification of apricot during storage at a near freezing temperature.

The effects of near freezing temperature (NFT) storage at -1.9 °C on cell wall degradation of 'Shushanggan' apricot was studied comparing to 0 °C and 5 °C storage. Our results indicated that NFT storage strongly inhibited the solubilization of Na2CO3-soluble pectin and cellulose, by the suppression of cell wall modifying enzymes (polygalacturonase, ß-Galactosidase, pectin methyl esterase and cellulase) and related genes expressions. The loss of side chains was the main modification in CDTA (Cyclohexane-diamine-tetraacetic Acid)-soluble pectin during storage and made the main contribution to the softening of apricot, while the loss of side chain was suppressed by NFT storage. Microscopic observation showed that NFT storage delayed the degradation of pectin fraction and protected cell wall structure from loosing. This study proves that NFT storage is an effective technology to suppress the cell wall polysaccharides degradation and ultrastructure modification of apricot.

Transient Gene Expression as a Tool to Monitor and Manipulate the Levels of Acidic Phospholipids in Plant Cells.

Anionic phospholipids represent only minor fraction of cell membranes lipids but they are critically important for many membrane-related processes, including membrane identity, charge, shape, the generation of second messengers, and the recruitment of peripheral proteins. The main anionic phospholipids of the plasma membrane are phosphoinositides phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), phosphatidylserine (PS), and phosphatidic acid (PA). Recent insights in the understanding of the nature of protein-phospholipid interactions enabled the design of genetically encoded fluorescent molecular probes that can interact with various phospholipids in a specific manner allowing their imaging in live cells. Here, we describe the use of transiently transformed plant cells to study phospholipid-dependent membrane recruitment.

Cellular Force Microscopy to Measure Mechanical Forces in Plant Cells.

Cellular force microscopy (CFM) is a noninvasive microindentation method used to measure plant cell stiffness in vivo. CFM is a scanning probe microscopy technique similar in operation to atomic force microscopy (AFM); however, the scale of movement and range of forces are much larger, making it suitable for stiffness measurements on turgid plant cells in whole organs. CFM experiments can be performed on living samples over extended time periods, facilitating the exploration of the dynamics of processes involving mechanics. Different sensor technologies can be used, along with a variety of probe shapes and sizes that can be tailored to specific applications. Measurements can be made for specific indentation depths, forces and timing, allowing for very precise mechanical stimulation of cells with known forces. High forces with sharp tips can also be used for mechanical ablation of cells with force feedback.

Wall porosity in isolated cells from food plants: Implications for nutritional functionality.

Macronutrients in whole plant foods are enclosed inside cells. The metabolic response from these entrapped nutrients may depend on cell-wall porosity, by controlling the passage of digestive enzymes. As non-interacting size mimics of digestive enzymes, we investigated the diffusion of fluorescently-labelled probes across the walls of isolated plant cells from potato tuber, red kidney bean and banana. Diffusion properties of permeable probes, dextran (20-kDa and 70-kDa) and albumin, were quantified, using fluorescence recovery after photobleaching. The consistent reduction of diffusion rate in the presence of cell walls (around 40%) compared to free-diffusion rate was attributed to the limiting porosity of the wall matrix. A combination of the physical barrier effects demonstrated here and non-catalytic binding of enzymes to cell walls limits the hydrolysis of intracellular macronutrients. This and further understanding of the structural basis for the physical barrier properties would help to design foods from plant materials with enhanced nutrition.

Swelling behavior and satiating effect of the gel microparticles obtained from callus cultures pectins.

Gel microparticles were prepared from pectins of campion (SVCgel) and duckweed (LMCgel) callus cultures, as well as from commercial apple pectin (APgel) by emulsion dehydration techniques with successive ionotropic gelation. The morphology and swelling behavior of the microparticles were determined after successive incubation in simulated gastric (SGF), intestinal (SIF), and colonic (SCF) fluids. Both SVCgel and LMCgel microparticles were found to swell in SGF and SIF gradually, and at oral administration decreased food intake by laboratory mice during the first 5 h of free-feeding. The SVCgel microparticles demonstrated the higher stability in SCF within 24 h than LMCgel ones. Only the SVCgel microparticles were shown to decrease food intake by 24% during the 21 h of free-feeding and decreased body weight of mice by 4% during 24 h after oral administration. The APgel microparticles lost their shape in SIF, then fully disintegrated after 0.5 h of incubation in SCF, and failed to affect food intake or mice body weight. The data obtained indicated that sustainability and swelling of the gel microparticles from the SVC pectin in the colonic fluid may provide the stronger satiating effect compared to that of the LMCgel microparticles.

Comparative metabolomics of scab-resistant and susceptible apple cell cultures in response to scab fungus elicitor treatment.

Apple scab disease caused by the fungus Venturia inaequalis is a devastating disease that seriously affects quality and yield of apples. In order to understand the mechanisms involved in scab resistance, we performed gas chromatography-mass spectrometry based metabolomics analysis of the cell culture of scab resistant cultivar 'Florina' and scab susceptible cultivar 'Vista Bella' both prior -to and -following treatment with V. inaequalis elicitor (VIE). A total 21 metabolites were identified to be altered significantly in 'Florina' cell cultures upon VIE-treatment. Among 21 metabolites, formation of three new specialized metabolites aucuparin, noraucuparin and eriobofuran were observed only in resistant cultivar 'Florina' after the elicitor treatment. The score plots of principal component analysis (PCA) exhibited clear discrimination between untreated and VIE-treated samples. The alteration in metabolite levels correlated well with the changes in the transcript levels of selected secondary metabolite biosynthesis genes. Aucuparin, noraucuparin and eriobofuran isolated from the 'Florina' cultures showed significant inhibitory effect on the conidial germination of V. inaequalis. The results expand our understanding of the metabolic basis of scab-resistance in apple and therefore are of interest in apple breeding programs to fortify scab resistance potential of commercially grown apple cultivars.

Plant cell culture technology in the cosmetics and food industries: current state and future trends.

The production of drugs, cosmetics, and food which are derived from plant cell and tissue cultures has a long tradition. The emerging trend of manufacturing cosmetics and food products in a natural and sustainable manner has brought a new wave in plant cell culture technology over the past 10 years. More than 50 products based on extracts from plant cell cultures have made their way into the cosmetics industry during this time, whereby the majority is produced with plant cell suspension cultures. In addition, the first plant cell culture-based food supplement ingredients, such as Echigena Plus and Teoside 10, are now produced at production scale. In this mini review, we discuss the reasons for and the characteristics as well as the challenges of plant cell culture-based productions for the cosmetics and food industries. It focuses on the current state of the art in this field. In addition, two examples of the latest developments in plant cell culture-based food production are presented, that is, superfood which boosts health and food that can be produced in the lab or at home.