The selenium-independent phospholipid hydroperoxide glutathione peroxidase from Theobroma cacao (TcPHGPX) protects plant cells against damages and cell death.

Proteins from the glutathione peroxidase (GPX) family, such as GPX4 or PHGPX in animals, are extensively studied for their antioxidant functions and apoptosis inhibition. GPXs can be selenium-independent or selenium-dependent, with selenium acting as a potential cofactor for GPX activity. However, the relationship of plant GPXs to these functions remains unclear. Recent research indicated an upregulation of Theobroma cacao phospholipid hydroperoxide glutathione peroxidase gene (TcPHGPX) expression during early witches' broom disease stages, suggesting the use of antioxidant mechanisms as a plant defense strategy to reduce disease progression. Witches' broom disease, caused by the hemibiotrophic fungus Moniliophthora perniciosa, induces cell death through elicitors like MpNEP2 in advanced infection stages. In this context, in silico and in vitro analyses of TcPHGPX's physicochemical and functional characteristics may elucidate its antioxidant potential and effects against cell death, enhancing understanding of plant GPXs and informing strategies to control witches' broom disease. Results indicated TcPHGPX interaction with selenium compounds, mainly sodium selenite, but without improving the protein function. Protein-protein interaction network suggested cacao GPXs association with glutathione and thioredoxin metabolism, engaging in pathways like signaling, peroxide detection for ABA pathway components, and anthocyanin transport. Tests on tobacco cells revealed that TcPHGPX reduced cell death, associated with decreased membrane damage and H2O2 production induced by MpNEP2. This study is the first functional analysis of TcPHGPX, contributing to knowledge about plant GPXs and supporting studies for witches' broom disease control.

Undifferentiated Cells of Tessaria absinthioides with High Nutritional Value and Health-Promoting Phytochemicals. An Approach Based on Plant Cellular Agriculture.

In vitro cultures of undifferentiated plant cells of Tessaria absinthioides, a native herb popularly recognized and used for its health benefits, were studied as potential food supplements. These tissues were incubated under two light conditions, and the biomass obtained was freeze-dried and oven-dried. To evaluate their nutritional value, their physicochemical and functional properties were determined. Although in some cases there were significant differences in the results according to the drying methodology applied, all these tissues presented a high proportion of proteins (23.6-28.3%), a low percentage of fats (< 2%) constituted mainly by phytosterols, and a significant amount of crude fibers (6.9-9.0%) and ashes (> 10%). In addition, the freeze-dried calli resulted in a product with better functional properties. On the other hand, their phytochemical profiles and antioxidant capacity were studied and compared with tissues from wild specimens and with green tea and chamomile as reference extracts.

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.

Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia.

Antibody-drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg-1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

Toxin type and domain sequence affect accumulation of recombinant immunotoxins.Transient expression in plant cell packs and intact plants correlates well.IC₅₀ values of toxicity correlate with the cell surface receptor concentration.

Apple metabolism under oxidative stress affects plant cell wall structure and mechanical properties.

Several studies have shown beneficial effects of short exposure to oxidative stress on stored fruit, such as better preservation, increased firmness, preservation of polyphenolic compounds, and reduced risk of postharvest disorders such as bitter pit and superficial scald in apples. In this study the effect of short-term oxidative stress conditions on the physiology of apple fruit was investigated. Apple fruit of three cultivars were exposed to hypoxic storage conditions of various lengths to induce anaerobiosis. The response of apple fruit to short-term oxidative stress was evaluated by means of cell wall immunolabeling and atomic force microscopy. In addition, the antioxidant capacity and antioxidative activity of apple peels was assessed. Through various techniques, it was shown that short-term oxidative stress conditions promote specific enzymatic activity that induces changes in the cell wall of apple fruit cells. Exposure to short-term stress resulted in the remodeling of cell wall pectic polysaccharides, observed as an increase in the size and complexity of extracted oxalate pectin. Structural changes in the cell wall were followed by an increase in Young's modulus (compressive stiffness of a solid material, expressed as the relationship between stress and axial strain) of the cell wall material. The data presented in this paper show in a novel way how storage under short-term oxidative stress modifies the cell wall of apple fruit at the molecular level.

Signals and Their Perception for Remodelling, Adjustment and Repair of the Plant Cell Wall.

The integrity of the cell wall is important for plant cells. Mechanical or chemical distortions, tension, pH changes in the apoplast, disturbance of the ion homeostasis, leakage of cell compounds into the apoplastic space or breakdown of cell wall polysaccharides activate cellular responses which often occur via plasma membrane-localized receptors. Breakdown products of the cell wall polysaccharides function as damage-associated molecular patterns and derive from cellulose (cello-oligomers), hemicelluloses (mainly xyloglucans and mixed-linkage glucans as well as glucuronoarabinoglucans in Poaceae) and pectins (oligogalacturonides). In addition, several types of channels participate in mechanosensing and convert physical into chemical signals. To establish a proper response, the cell has to integrate information about apoplastic alterations and disturbance of its wall with cell-internal programs which require modifications in the wall architecture due to growth, differentiation or cell division. We summarize recent progress in pattern recognition receptors for plant-derived oligosaccharides, with a focus on malectin domain-containing receptor kinases and their crosstalk with other perception systems and intracellular signaling events.

A Brief Review of Plant Cell Transfection, Gene Transcript Expression, and Genotypic Integration for Enhancing Compound Production.

Plants possess a plethora of important secondary metabolites, which are unique sources of natural pigments, pharmaceutical compounds, food additives, natural pesticides, and other industrial components. The commercial significance of such metabolites/compounds has directed the research toward their production and exploration of methods for enhancement of production. Biotechnological tools are critical in selecting, integrating, multiplying, improving, and analyzing medicinal plants for secondary metabolite production. Out of many techniques that are being explored to enhance secondary metabolite production, "plant cell transfection" is the latest tool to achieve maximum output from the plant source. It is based upon the introduction of foreign DNA into the plant cell relying on physical treatment such as electroporation, cell squeezing, sonoporation, optical transfection nanoparticles, magnetofection, and chemical treatment or biological treatment that depends upon carrier. One of the promising tools that have been exploited is CRISPR-Cas9. Overall, the abovementioned tools focus on the stable transfection of desired gene transcripts. Since the integration and continuous expression of transfected gene of particular trait represents stable transfection of host cell genome, resulting from transfer of required trait to daughter cells ultimately leading to enhanced production of secondary metabolites of interest. This chapter will review a set of biotechnological tools that are candidates for achieving the enhanced bioactive compound production indicated here to be used for drug discovery.

Live Fluorescence Visualization of Cellulose and Pectin in Plant Cell Walls.

The plant cell wall comprises various types of macromolecules whose abundance and spatial distribution change dynamically and are crucial for plant architecture. High-resolution live cell imaging of plant cell wall components is, therefore, a powerful tool for plant cell biology and plant developmental biology. To acquire suitable data, the experimental setup for staining and imaging of non-fixed samples must be straightforward and avoid creating stress-induced artifacts. We present a detailed sample preparation and live image acquisition protocol for fluorescence visualization of cell wall components using commercially available probes and stains.

Metabolic and Transcriptional Stress Memory in Sorbus pohuashanensis Suspension Cells Induced by Yeast Extract.

Plant stress memory can provide the benefits of enhanced protection against additional stress exposure. Here, we aimed to explore the responses of recurrent and non-recurrent yeast extract (YE) stresses in Sorbus pohuashanensis suspension cells (SPSCs) at metabolomics and transcriptional levels. Biochemical analyses showed that the cell wall integrity and antioxidation capacity of SPSCs in the pretreated group were evidently improved. Metabolic analysis showed that there were 39 significantly altered metabolites in the pretreated group compared to the non-pretreated group. Based on the transcriptome analysis, 219 differentially expressed genes were obtained, which were highly enriched in plant-pathogen interaction, circadian rhythm-plant, oxidative phosphorylation, and phenylpropanoid biosynthesis. Furthermore, the correlation analysis of the transcriptome and metabolome data revealed that phenylpropanoid biosynthesis involved in the production of biphenyl phytoalexins may play a critical role in the memory response of SPSC to YE, and the key memory genes were also identified, including PAL1, BIS1, and BIS3. Collectively, the above results demonstrated that the memory responses of SPSC to YE were significant in almost all levels, which would be helpful for better understanding the adaptation mechanisms of medicinal plants in response to biotic stress, and laid a biotechnological foundation to accumulate favorable antimicrobial drug candidates from plant suspension cells.

HISTONE DEACETYLASE 9 transduces heat signal in plant cells.

Heat stress limits plant growth, development, and crop yield, but how plant cells precisely sense and transduce heat stress signals remains elusive. Here, we identified a conserved heat stress response mechanism to elucidate how heat stress signal is transmitted from the cytoplasm into the nucleus for epigenetic modifiers. We demonstrate that HISTONE DEACETYLASE 9 (HDA9) transduces heat signals from the cytoplasm to the nucleus to play a positive regulatory role in heat responses in Arabidopsis. Heat specifically induces HDA9 accumulation in the nucleus. Under heat stress, the phosphatase PP2AB'ß directly interacts with and dephosphorylates HDA9 to protect HDA9 from 26S proteasome-mediated degradation, leading to the translocation of nonphosphorylated HDA9 to the nucleus. This heat-induced enrichment of HDA9 in the nucleus depends on the nucleoporin HOS1. In the nucleus, HDA9 binds and deacetylates the target genes related to signaling transduction and plant development to repress gene expression in a transcription factor YIN YANG 1-dependent and -independent manner, resulting in rebalance of plant development and heat response. Therefore, we uncover an HDA9-mediated positive regulatory module in the heat shock signal transduction pathway. More important, this cytoplasm-to-nucleus translocation of HDA9 in response to heat stress is conserved in wheat and rice, which confers the mechanism significant implication potential for crop breeding to cope with global climate warming.

Building an Artificial Plant Cell Wall on a Lipid Bilayer by Assembling Polysaccharides and Engineered Proteins.

The cell wall constitutes a fundamental structural component of plant cells, providing them with mechanical resistance and flexibility. Mimicking this wall is a critical step in the conception of an experimental model of the plant cell. The assembly of cellulose/hemicellulose in the form of cellulose nanocrystals and xyloglucans as a representative model of the plant cell wall has already been mastered; however, these models lacked the pectin component. In this work, we used an engineered chimeric protein designed for bridging pectin to the cellulose/hemicellulose network, therefore achieving the assembly of complete cell wall mimics. We first engineered a carbohydrate-binding module from Ruminococcus flavefaciens able to bind oligogalacturonan, resulting in high-affinity polygalacturonan receptors with Kd in the micromolar range. A Janus protein, with cell wall gluing property, was then designed by assembling this carbohydrate-binding module with a Ralstonia solanacearum lectin specific for fucosylated xyloglucans. The resulting supramolecular architecture is able to bind fucose-containing xyloglucans and homogalacturonan, ensuring high affinity for both. A two-dimensional assembly of an artificial plant cell wall was then built first on synthetic polymer and then on the supported lipid bilayer. Such an artificial cell wall can serve as a basis for the development of plant cell mechanical models and thus deepen the understanding of the principles underlying various aspects of plant cells and tissues.

Pectin self-assembly and its disruption by water: insights into plant cell wall mechanics.

Plant cell walls undergo multiple cycles of dehydration and rehydration during their life. Calcium crosslinked low methoxy pectin is a major constituent of plant cell walls. Understanding the dehydration-rehydration behavior of pectin gels may shed light on the water transport and mechanics of plant cells. In this work, we report the contributions of the microstructure to the mechanics of pectin-Ca gels subjected to different extents of dehydration and subsequent rehydration. This is investigated using a pectin gel composition that forms 'egg-box bundles', a characteristic feature of the microstructure of low methoxy pectin-Ca gels. Large amplitude oscillatory shear (LAOS) rheology along with small angle neutron scattering and near infrared (NIR) spectroscopy on pectin gels is used to elucidate the mechanical and microstructural changes during dehydration-rehydration cycles. As the extent of dehydration increases, the reswelling ability, strain-stiffening behavior and yield strain decrease. These effects are more prominent at faster rates of dehydration and are not completely reversible upon rehydration to the initial undried state. Microstructural changes due to the aggregation of egg-box bundles and single chains and the associated changes in the water configurations lead to these irreversible changes.

Elastic shell theory for plant cell wall stiffness reveals contributions of cell wall elasticity and turgor pressure in AFM measurement.

The stiffness of a plant cell in response to an applied force is determined not only by the elasticity of the cell wall but also by turgor pressure and cell geometry, which affect the tension of the cell wall. Although stiffness has been investigated using atomic force microscopy (AFM) and Young's modulus of the cell wall has occasionally been estimated using the contact-stress theory (Hertz theory), the existence of tension has made the study of stiffness more complex. Elastic shell theory has been proposed as an alternative method; however, the estimation of elasticity remains ambiguous. Here, we used finite element method simulations to verify the formula of the elastic shell theory for onion (Allium cepa) cells. We applied the formula and simulations to successfully quantify the turgor pressure and elasticity of a cell in the plane direction using the cell curvature and apparent stiffness measured by AFM. We conclude that tension resulting from turgor pressure regulates cell stiffness, which can be modified by a slight adjustment of turgor pressure in the order of 0.1 MPa. This theoretical analysis reveals a path for understanding forces inherent in plant cells.

A comprehensive study on the interaction of Eu(III) and U(VI) with plant cells (Daucus carota) in suspension.

Daucus carota suspension cells showed a high affinity towards Eu(III) and U(VI) based on a single-step bioassociation process with an equilibrium after 48-72 h. Cells responded with an increased metabolic activity towards heavy metal stress. Luminescence spectroscopy pointed to multiple species for both f-block elements in the culture media, providing initial hints of their interaction with cells and released metabolites. Using nuclear magnetic resonance spectroscopy, we could prove that malate, as an released metabolite in the culture medium, was found to complex with U. Luminescence spectroscopy also showed that Eu(III)-EDTA species are interacting with the cells. Furthermore, Eu(III) and U(VI) coordination is dominated by phosphate groups provided by the cells. We found that Ca ion channels of D. carota cells were involved in the uptake of U(VI), which led to a bioprecipitation of U(VI) in the vacuole of the cells, most probably as uranyl(VI) phosphates along with an intracellular sorption of U(VI) on biomembranes by lipid structures. Eu(III) could be found locally concentrated in the cell wall and in the cytoplasm with a co-localization with phosphorous and oxygen.

Isolation of Specialized Plant Cells by Fluorescence-Activated Cell Sorting.

Plant organs are built of different cell types, characterized by specific transcription programs and metabolic profiles. The possibility of isolation of such cell types to perform differential transcriptomic, proteomic and metabolomic analyses is highly important to understand many aspects of plant physiology, namely, the structure and regulation of economically valuable specialized metabolic pathways. Here, we describe the isolation of idioblast leaf protoplasts of the medicinal plant Catharanthus roseus by fluorescence-activated cell sorting, taking advantage of the differential autofluorescence properties of those specialized cells.

Arabidopsis exocyst subunit SEC6 is involved in cell plate formation during Microgametogenesis.

Cytokinesis during pollen mitosis I is critical for cell division and differentiation in the male gametophyte development, but the vesicle trafficking mechanisms in this process are largely unknown. Exocyst is an octameric tethering complex which plays multiple important roles in plant cell vesicle trafficking. Here we report the characterization of exocyst subunit SEC6 in the cytokinesis during pollen mitosis I. We found that significantly amount of pollen from two sec6/+ mutant alleles arrested at the transition from unicelluar stage microspore to bicellular stage. Further analysis showed that sec6 mutation impaired cell plate formation and led to vesicles accumulation in cytoplasm. The localization of KNOLLE on the cell plate was compromised. Consistently, SEC6 gene was expressed start from early pollen development stage and SEC6-GFP localized to the cell plate. These results indicated that SEC6 participated in the cell plate formation during pollen mitosis I, where it might help to tether the vesicles before fusion.

Production of bioactive plant secondary metabolites through in vitro technologies-status and outlook.

Medicinal plants have been used by mankind since ancient times, and many bioactive plant secondary metabolites are applied nowadays both directly as drugs, and as raw materials for semi-synthetic modifications. However, the structural complexity often thwarts cost-efficient chemical synthesis, and the usually low content in the native plant necessitates the processing of large amounts of field-cultivated raw material. The biotechnological manufacturing of such compounds offers a number of advantages like predictable, stable, and year-round sustainable production, scalability, and easier extraction and purification. Plant cell and tissue culture represents one possible alternative to the extraction of phytochemicals from plant material. Although a broad commercialization of such processes has not yet occurred, ongoing research indicates that plant in vitro systems such as cell suspension cultures, organ cultures, and transgenic hairy roots hold a promising potential as sources for bioactive compounds. Progress in the areas of biosynthetic pathway elucidation and genetic manipulation has expanded the possibilities to utilize plant metabolic engineering and heterologous production in microorganisms. This review aims to summarize recent advances in the in vitro production of high-value plant secondary metabolites of medicinal importance.Key points• Bioactive plant secondary metabolites are important for current and future use in medicine• In vitro production is a sustainable alternative to extraction from plants or costly chemical synthesis• Current research addresses plant cell and tissue culture, metabolic engineering, and heterologous production.

Plant-microbial interaction: The mechanism and the application of microbial elicitor induced secondary metabolites biosynthesis in medicinal plants.

Plants and microbes interact with each other via different chemical signaling pathways. At the risophere level, the microbes can secrete molecules, called elicitors, which act on their receptors located in plant cells. The so-called elicitor molecules as well as their actions differ according to the mcirobes and induce different bilogical responses in plants such as the synthesis of secondary metabolites. Microbial compounds induced phenotype changes in plants are known as elicitors and signaling pathways which integrate elicitor's signals in plants are called elicitation. In this review, the impact of microbial elicitors on the synthesis and the secretion of secondary metabolites in plants was highlighted. Moreover, biological properties of these bioactive compounds were also highlighted and discussed. Indeed, several bacteria, fungi, and viruses release elicitors which bind to plant cell receptors and mediate signaling pathways involved in secondary metabolites synthesis. Different phytochemical classes such as terpenoids, phenolic acids and flavonoids were synthesized and/or increased in medicinal plants via the action of microbial elicitors. Moreover, these compounds compounds exhibit numerous biological activities and can therefore be explored in drugs discovery.

Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes.

Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins. APs from cardoon flowers display a great degree of heterogeneity, resulting in variable milk clotting activities and directly influencing the final product. Producing these APs using alternative platforms such as bacteria or yeast has proven challenging, which is hampering their implementation on an industrial scale. We have developed tobacco BY2 cell lines as an alternative plant-based platform for the production of cardosin B. These cultures successfully produced active cardosin B and a purification pipeline was developed to obtain isolated cardosin B. The enzyme displayed proteolytic activity towards milk caseins and milk clotting activity under standard cheese manufacturing conditions. We also identified an unprocessed form of cardosin B and further investigated its activation process. The use of protease-specific inhibitors suggested a possible role for a cysteine protease in cardosin B processing. Mass spectrometry analysis identified three cysteine proteases containing a granulin-domain as candidates for cardosin B processing. These findings suggest an interaction between these two groups of proteases and contribute to an understanding of the mechanisms behind the regulation and processing of plant APs. This work also paves the way for the use of tobacco BY2 cells as an alternative production system for active cardosins and represents an important advancement towards the industrial production of cardoon APs.