Towards optimization of plant cell detection in suspensions using impedance-based analyses and the unified equivalent circuit model.

An improved approach for comparative study of plant cells for long term and continuous monitoring using electrical impedance spectroscopy is demonstrated for tomato and tobacco plant cells (MSK8 and BY2) in suspensions. This approach is based on the locations and magnitudes of defining features in the impedance spectra of the recently reported unified equivalent circuit model. The ultra-wide range (4 Hz to 20 GHz) impedance spectra of the cell lines were measured using custom probes, and were analyzed using the unified equivalent circuit model, highlighting significant negative phase peaks in the ~ 1 kHz to ~ 10 MHz range. These peaks differ between the tomato and tobacco cells, and since they can be easily defined, they can potentially be used as the signal for differentiating between different cell cultures or monitoring them over time. These findings were further analysed, showing that ratios relating the resistances of the media and the resistance of the cells define the sensitivity of the method, thus affecting its selectivity. It was further shown that cell agglomeration is also an important factor in the impedance modeling in addition to the overall cell concentration. These results can be used for optimizing and calibrating electrical impedance spectroscopy-based sensors for long term monitoring of cell lines in suspension for a given specific cell and media types.

Changes in ploidy affect vascular allometry and hydraulic function in Mangifera indica trees.

The enucleated vascular elements of the xylem and the phloem offer an excellent system to test the effect of ploidy on plant function because variation in vascular geometry has a direct influence on transport efficiency. However, evaluations of conduit sizes in polyploid plants have remained elusive, most remarkably in woody species. We used a combination of molecular, physiological and microscopy techniques to model the hydraulic resistance between source and sinks in tetraploid and diploid mango trees. Tetraploids exhibited larger chloroplasts, mesophyll cells and stomatal guard cells, resulting in higher leaf elastic modulus and lower dehydration rates, despite the high water potentials of both ploidies in the field. Both the xylem and the phloem displayed a scaling of conduits with ploidy, revealing attenuated hydraulic resistance in tetraploids. Conspicuous wall hygroscopic moieties in the cells involved in transpiration and transport indicate a role in volumetric adjustments as a result of turgor change in both ploidies. In autotetraploids, the enlargement of organelles, cells and tissues, which are critical for water and photoassimilate transport at long distances, point to major physiological novelties associated with whole-genome duplication.

Crystal structures of a nicotine MATE transporter provide insight into its mechanism of substrate transport.

A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotiana tabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward-facing forms. The overall structure has a bilobate V-shape with pseudo-symmetrical assembly of the N- and C-lobes. In one crystal structure, the C-lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2-specific conformational transitions imply that an unprecedented movement of the transmembrane α-helix 7 is related to the release of the substrate into the vacuolar lumen.

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.

Interactions between cell wall polysaccharides and polyphenols: Effect of molecular internal structure.

Cell wall polysaccharides (CPSs) and polyphenols are major constituents of the dietary fiber complex in plant-based foods. Their digestion (by gut microbiota) and bioefficacy depend not only on their structure and quantity, but also on their intermolecular interactions. The composition and structure of these compounds vary with their dietary source (i.e., fruit or vegetable of origin) and can be further modified by food processing. Various components and structures of CPSs and polyphenols have been observed to demonstrate common and characteristic behaviors during interactions. However, at a fundamental level, the mechanisms that ultimately drive these interactions are still not fully understood. This review summarizes the current state of knowledge on the internal factors that influence CPS-polyphenol interactions, describes the different ways in which these interactions can be mediated by molecular composition or structure, and introduces the main methods for the analysis of these interactions, as well as the mechanisms involved. Furthermore, a comprehensive overview is provided of recent key findings in the area of CPS-polyphenol interactions. It is becoming clear that these interactions are shaped by a multitude of factors, the most important of which are the physicochemical properties of the partners: their morphology (surface area and porosity/pore shape), chemical composition (sugar ratio, solubility, and non-sugar components), and molecular architecture (molecular weight, degree of esterification, functional groups, and conformation). An improved understanding of the molecular mechanisms that drive interactions between CPSs and polyphenols may allow us to better establish a bridge between food processing and the bioavailability of colonic fermentation products from CPSs and antioxidant polyphenols, which could ultimately lead to the development of new guidelines for the design of healthier and more nutritious foods.

An optimised chromatin immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant.

All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the chromatin landscape of this unique collection will likely shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.

Covalent Self-Labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings.

Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.

Modeling of paclitaxel biosynthesis elicitation in Corylus avellana cell culture using adaptive neuro-fuzzy inference system-genetic algorithm (ANFIS-GA) and multiple regression methods.

Paclitaxel as a microtubule-stabilizing agent is widely used for the treatment of a vast range of cancers. Corylus avellana cell suspension culture (CSC) is a promising strategy for paclitaxel production. Elicitation of paclitaxel biosynthesis pathway is a key approach for improving its production in cell culture. However, optimization of this process is time-consuming and costly. Modeling of paclitaxel elicitation process can be helpful to predict the optimal condition for its high production in cell culture. The objective of this study was modeling and forecasting paclitaxel biosynthesis in C. avellana cell culture responding cell extract (CE), culture filtrate (CF) and cell wall (CW) derived from endophytic fungus, either individually or combined treatment with methyl-ß-cyclodextrin (MBCD), based on four input variables including concentration levels of fungal elicitors and MBCD, elicitor adding day and CSC harvesting time, using adaptive neuro-fuzzy inference system (ANFIS) and multiple regression methods. The results displayed a higher accuracy of ANFIS models (0.94-0.97) as compared to regression models (0.16-0.54). The great accordance between the predicted and observed values of paclitaxel biosynthesis for both training and testing subsets support excellent performance of developed ANFIS models. Optimization process of developed ANFIS models with genetic algorithm (GA) showed that optimal MBCD (47.65 mM) and CW (2.77% (v/v)) concentration levels, elicitor adding day (16) and CSC harvesting time (139 h and 41 min after elicitation) can lead to highest paclitaxel biosynthesis (427.92 µg l-1). The validation experiment showed that ANFIS-GA method can be a promising tool for selecting the optimal conditions for maximum paclitaxel biosynthesis, as a case study.

Nanosecond pulsed electrical fields enhance product recovery in plant cell fermentation.

The potential of pharmacologically active secondary plant metabolites is limited by the low yield from often rare plants, and the lack of economically feasible chemical synthesis of these complex compounds. Plant cell fermentation offers an alternative strategy to overcome these constraints. However, the efficiency of this approach is limited by intracellular sequestration of the products, such that continuous bioprocessing is not possible. As a precondition for such a, more attractive, continuous process, it is of great importance to stimulate the export of the product into the medium without impairing viability and, thus, the productivity of the cells. Using nicotine alkaloids of tobacco as a case study, an alternative strategy is explored, where nanosecond pulsed electric fields (nsPEFs) are applied for the efficient downstream recovery of the products. To maintain cell viability and allow for the further use of biomass, cells were exposed to strong (1-20 kV·cm-1), but very short (10-100 ns) electric pulses, which leads to a temporary permeabilisation of cell membranes. Using two transgenic cell lines, where two key genes involved in the metabolism of the anti-Alzheimer compound nornicotine were overexpressed, we could show that this nsPEF treatment improved the partitioning of some nicotine alkaloids to the culture medium without impairing viability, nor the synthesis of alkaloids. However, this release was only partial and did not work for nornicotine. Thus, nsPEFs produced a fractionation of alkaloids. We explain this electrofractionation by a working model considering the differential intracellular compartmentalization of nicotineic alkaloids.

Analysis of Plant Cell Walls Using High-Throughput Profiling Techniques with Multivariate Methods.

Plant cell walls are composed of a number of coextensive polysaccharide-rich networks (i.e., pectin, hemicellulose, protein). Polysaccharide-rich cell walls are important in a number of biological processes including fruit ripening, plant-pathogen interactions (e.g., pathogenic fungi), fermentations (e.g., winemaking), and tissue differentiation (e.g., secondary cell walls). Applying appropriate methods is necessary to assess biological roles as for example in putative plant gene functional characterization (e.g., experimental evaluation of transgenic plants). Obtaining datasets is relatively easy, using for example gas chromatography-mass spectrometry (GC-MS) methods for monosaccharide composition, Fourier transform infrared spectroscopy (FT-IR) and comprehensive microarray polymer profiling (CoMPP); however, analyzing the data requires implementing statistical tools for large-scale datasets. We have validated and implemented a range of multivariate data analysis methods on datasets from tobacco, grapevine, and wine polysaccharide studies. Here we present the workflow from processing samples to acquiring data to performing data analysis (particularly principal component analysis (PCA) and orthogonal projection to latent structure (OPLS) methods).

Cell cycle-dependent dynamics of a plant intermediate filament motif protein with intracellular localization related to microtubules.

Although intermediate filaments (IFs) are biochemically and immunologically suggested to exist in plant cells, there are few molecular genetic studies related to the proteins that form these structures. In this study, Arabidopsis AT3G05270 was selected as a candidate gene for a protein constituting IF in plant cells. The protein encoded by AT3G05270 has a large α-helix as well as the IF protein motif indispensable for maintaining the structures of IF. Moreover, fluorescence signals of this protein fused with GFP exhibited cytoskeleton-like filamentous structures in plant cells. Thus, we named the protein encoded by AT3G05270 as Intermediate Filament Motif Protein 1 (IFMoP1). The structures composed of IFMoP1 and their localizations were examined in IFMoP1-GFP-expressing tobacco BY-2 cells whose cell cycle was synchronized using aphidicolin, a DNA synthesis inhibitor, and propyzamide, a microtubule-disrupting agent. The IFMoP1-GFP signals were present at the spindles and phragmoplasts in the mitotic phase. In addition, the frequency of cells with cytoskeleton-like filamentous structures composed of IFMoP1-GFP increased with the increase in cells that completed cell division, and then decreased after several hours. In terms of the relationship in intracellular localization between IFMoP1 and microtubules, the filamentous structures composed of IFMoP1 were present independently of microtubules during interphase. In living cells, these filamentous structures moved along with the nucleus. IFMoP1 co-localized with spindle and phragmoplast microtubules during mitosis, as well as with a part of the cortical microtubules in interphase.

Purified plant cell walls with adsorbed polyphenols alter porcine faecal bacterial communities during in vitro fermentation.

A substantial fraction of ingested polyphenols accumulate in the large intestine (LI), attached to undigested plant cell walls (PCW) (dietary fibre). Yet, whether these PCW-bound polyphenols alter the structure and function of the resident microbiota remains unclear. This study characterised bacterial populations during the in vitro fermentation of three standard polyphenols: ferulic acid (FER), (±)-catechin (CAT), and cyanidin-3-glucoside (CYAN), adsorbed individually or in combination to apple cell walls (ACW). During fermentation with porcine faeces, samples were collected at regular time-points (up to 72 hours) for bacterial 16S rRNA gene amplicon sequencing and fermentation end-product analyses (short-chain fatty acids and ammonium). The metabolic end-products differed to only a small extent between substrates, though significantly for propionate (P < 0.0001). Significant differences in microbial populations were noted between substrates tested (P < 0.0001). The presence of cyanidin-3-glucoside resulted in the most significant differences between bacterial communities during fermentation of the ACW substrate. Key microbes identified to be associated with the ACW with adsorbed polyphenols as well as individual polyphenols were: Phascolarctobacterium with ACW + FER and FER, the Lachnospiraceae family with ACW + CYAN, Parabacteroides with ACW + CYAN and CYAN, Collinsella and Coprococcus with ACW + CAT, and the Clostridiales order with ACW + CAT and CAT. This study has demonstrated the use of a simplified model to indicate any microbial effects of polyphenols associated with dietary fibre in whole fruits. This work has shown that individual polyphenols, or those adsorbed to PCW, have potentially very different effects on the gut bacteria. Future work could examine further polyphenols associated with a range of fresh fruits.

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.

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.

Studies to Elucidate the Effects of Furostanol Glycosides from Dioscorea deltoidea Cell Culture in a Rat Model of Endothelial Dysfunction.

Currently, there is no doubt surrounding a theory that the cardiotropic effects of sex hormones can be due to their direct effect on the cardiovascular system. In recent years, interest in the study of steroid glycosides has increased. We studied the effects of furostanol glycosides (protodioscin and deltozid) from the cell culture of the Dioscorea deltoidea (laboratory code DM-05) on the physiological and biochemical parameters of vascular endothelial function in hypoestrogen-induced endothelial dysfunction after bilateral ovariectomy. It was shown that the use of DM-05 at a dose of 1 mg/kg makes it possible to prevent the development of arterial hypertension (the level of systolic blood pressure (SBP) decreases by 9.7% (p < 0.05) and diastolic blood pressure (DBP) by 8.2%), to achieve a decrease in the coefficient of endothelial dysfunction by 1.75 times against the background of a hypoestrogenic state. With DM-05, an increase in the concentration of stable nitric oxide metabolites (NOx) by 45.6% (p < 0.05) and an increase in mRNA endothelial nitric oxide synthase (eNOS) expression by 34.8% (p < 0.05) was established, which indicates a positive effect of furostanol glycosides on the metabolism of nitric oxide after ovariectomy. Positive dynamics in the histological structure of the heart and the abdominal aorta indicate the pronounced endothelio- and atheroprotective effects of DM-05.

The rolB plant oncogene affects multiple signaling protein modules related to hormone signaling and plant defense.

The rolB plant oncogene of Agrobacterium rhizogenes perturbs many biochemical processes in transformed plant cells, thereby causing their neoplastic reprogramming. The oncogene renders the cells more tolerant to environmental stresses and herbicides and inhibits ROS elevation and programmed cell death. In the present work, we performed a proteomic analysis of Arabidopsis thaliana rolB-expressing callus line AtB-2, which represents a line with moderate expression of the oncogene. Our results show that under these conditions rolB greatly perturbs the expression of some chaperone-type proteins such as heat-shock proteins and cyclophilins. Heat-shock proteins of the DnaK subfamily were overexpressed in rolB-transformed calli, whereas the abundance of cyclophilins, members of the closely related single-domain cyclophilin family was decreased. Real-time PCR analysis of corresponding genes confirmed the reliability of proteomics data because gene expression correlated well with the expression of proteins. Bioinformatics analysis indicates that rolB can potentially affect several levels of signaling protein modules, including effector-triggered immunity (via the RPM1-RPS2 signaling module), the miRNA processing machinery, auxin and cytokinin signaling, the calcium signaling system and secondary metabolism.

Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae.

In the present investigation, transgenic tobacco callus cultures and plants overexpressing the silicatein gene LoSilA1 from marine sponge Latrunculia oparinae were obtained and their bioreduction behaviour for the synthesis of silver nanoparticles (AgNPs) was studied. Synthesized nanoparticles were characterized using UV-visible spectroscopy, Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic flame electron microscopy (AFM) and nanoparticle tracking analysis (NTA). Our measurements showed that the reduction of silver nitrate produced spherical AgNPs with diameters in the range of 12-80 nm. The results of XRD analysis proved the crystal nature of the obtained AgNPs. FTIR analysis indicated that particles are reduced and stabilized in solution by the capping agent, which is likely to be proteins present in the callus extract. Interestingly, the reduction potential of LoSiLA1-transgenic callus line was increased three-fold compared with the empty vector-transformed calli. The synthesized AgNPs were found to exhibit strong antibacterial activity against Escherichia coli and Agrobacterium rhizogenes. The present study reports the first evidence for using genetic engineering for activation of the reduction potential of plant cells for synthesis of biocidal AgNPs.

Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition.

This article presents a new insight about TBY-2 cells; from extracellular polysaccharides secretion to cell wall composition during cell suspension culture. In the medium of cells taken 2 days after dilution (end of lag phase), a two unit pH decrease from 5.38 to 3.45 was observed and linked to a high uronic acid (UA) amount secretion (47.8%) while, in 4 and 7 day-old spent media, pH increased and UA amounts decreased 35.6 and 42.3% UA, respectively. To attain deeper knowledge of the putative link between extracellular polysaccharide excretion and cell wall composition, we determined cell wall UA and neutral sugar composition of cells from D2 to D12 cultures. While cell walls from D2 and D3 cells contained a large amount of uronic acid (twice as much as the other analysed cell walls), similar amounts of neutral sugar were detected in cells from lag to end of exponential phase cells suggesting an enriched pectin network in young cultures. Indeed, monosaccharide composition analysis leads to an estimated percentage of pectins of 56% for D3 cell wall against 45% D7 cell walls indicating that the cells at the mid-exponential growth phase re-organized their cell wall linked to a decrease in secreted UA that finally led to a stabilization of the spent medium pH to 5.4. In conclusion, TBY-2 cell suspension from lag to stationary phase showed cell wall remodeling that could be of interest in drug interaction and internalization study.