Synthesis of a Side Chain Alkyne Analogue of Sitosterol as a Chemical Probe for Imaging in Plant Cells.

Clickable chemical tools are essential for studying the localization and role of biomolecules in living cells. For this purpose, alkyne-based close analogs of the respective biomolecules are of outstanding interest. Here, in the field of phytosterols, we present the first alkyne derivative of sitosterol, which fulfills the crucial requirements for such a chemical tool as follows: very similar in size and lipophilicity to the plant phytosterols, and correct absolute configuration at C-24. The alkyne sitosterol FB-DJ-1 was synthesized, starting from stigmasterol, which comprised nine steps, utilizing a novel alkyne activation method, a Johnson-Claisen rearrangement for the stereoselective construction of a branched sterol side chain, and a Bestmann-Ohira reaction for the generation of the alkyne moiety.

Protein Detection and Localization in Plant Cells Using Spot-Tagging.

Fluorescent labelling of proteins enables the determination of their spatiotemporal localization but, sometimes, it can perturb their activity, native localization, and functionality. Spot-tag is a12-amino acid peptide recognized by a single-domain nanobody and could potentially resolve the issues associated with large fluorescence tags due to its small size. Here, using as an example the microtubule motor CENTROMERIC PROTEIN E-RELATED KINESIN 7.3 (KIN7.3), we introduce the spot-tag for protein labelling in fixed and living plant cells. Spot-tagging and detection by an anti-spot nanobody of ectopically expressed KIN7.3 did not interfere with its native localization. Most importantly, our spot-tagging pipeline facilitated the localization of KIN7.3 much more rapidly and likely accurately than labelling with large fluorescent proteins or even immunolocalization approaches. We should, though, note some limitations we have not resolved yet. Spot-tagging is functional only in fixed cells; it is available only as two fluorophores and may create a noisy background during imaging. However, we foresee that, besides the limitations of this method, spot-tagging will apply to many proteins, offsetting activity perturbations and low photon quantum yields of other protein-tagging approaches.

Melatonin Mitigates Lead-Induced Oxidative Stress and Modifies Phospholipid Profile in Tobacco BY-2 Suspension Cells.

Many studies have shown that melatonin (an indoleamine) is an important molecule in plant physiology. It is known that this indoleamine is crucial during plant stress responses, especially by counteracting secondary oxidative stress (efficient direct and indirect antioxidant) and switching on different defense plant strategies. In this report, we present exogenous melatonin's potential to protect lipid profile modification and membrane integrity in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) cell culture exposed to lead. There are some reports of the positive effect of melatonin on animal cell membranes; ours is the first to report changes in the lipid profile in plant cells. The experiments were performed in the following variants: LS: cells cultured on unmodified LS medium-control; (ii) MEL: BY-2 cells cultured on LS medium with melatonin added from the beginning of culture; (iii) Pb: BY-2 cells cultured on LS medium with Pb2+ added on the 4th day of culture; (iv) MEL+Pb: BY-2 cells cultured on LS medium with melatonin added from the start of culture and stressed with Pb2+ added on the 4th day of culture. Lipidomic analysis of BY-2 cells revealed the presence of 40 different phospholipids. Exposing cells to lead led to the overproduction of ROS, altered fatty acid composition and increased PLD activity and subsequently elevated the level of phosphatidic acid at the cost of dropping the phosphatidylcholine. In the presence of lead, double-bond index elevation, mainly by higher quantities of linoleic (C18:2) and linolenic (C18:3) acids in the log phase of growth, was observed. In contrast, cells exposed to heavy metal but primed with melatonin showed more similarities with the control. Surprisingly, the overproduction of ROS caused of lipid peroxidation only in the stationary phase of growth, although considerable changes in lipid profiles were observed in the log phase of growth-just 4 h after lead administration. Our results indicate that the pretreatment of BY-2 with exogenous melatonin protected tobacco cells against membrane dysfunctions caused by oxidative stress (lipid oxidation), but also findings on a molecular level suggest the possible role of this indoleamine in the safeguarding of the membrane lipid composition that limited lead-provoked cell death. The presented research indicates a new mechanism of the defense strategy of plant cells generated by melatonin.

SnapShot: Condensates in plant biology.

Plant cells share a number of biological condensates with cells from other eukaryotes. There are, however, a growing number of plant-specific condensates that support different cellular functions. Condensates operating in different plant tissues contribute to aspects of development and stress responses. To view this SnapShot, open or download the PDF.

Optimizing interleukin-6 and 8 expression, clarification and purification in plant cell packs and plants for application in advanced therapy medicinal products and cellular agriculture.

Healthcare and nutrition are facing a paradigm shift in light of advanced therapy medicinal products (ATMPs) and cellular agriculture options respectively. Both options heavily rely on some sort of animal cell culture, e.g. autologous stem cells. These cultures require various growth factors, such as interleukin-6 and 8 (IL-6/8), in a pure, safe and sustainable form that can be provided in a scalable manner. Plants seem well suited for this task because purification of small proteins can be readily achieved by membrane separation, human/animal pathogens do not replicate in plants and production can be scaled up using in-door farming or agricultural practices. Here, we illustrate this capacity by first optimizing the codon usage of IL-6/8 for translation in Nicotiana spp., as well as testing the effect of untranslated regions and product targeting to different sub-cellular compartments on expression in a high-throughput plant cell pack (PCP) assay. In the chloroplast, IL-6 accumulated up to 6.9±3.8 (SD, n=2) and 14.4±7.4 mg kg-1 (SD, n=5) were observed in case of IL-8. When transferring IL-8 expression into whole plants, accumulation was 12.3±1.5 mg kg-1 (SD, n=3). After extraction and clarification, IL-8 was purified using a two-stage process consisting of an ultrafiltration/diafiltration step with 100 kDa and 10 kDa cut off membranes followed by an IMAC polishing step. The purity, yield and recovery were 97.8%, 6.6 mg kg-1 and 38%, respectively. We evaluated the ability of the proposed purification process to remove endotoxins to ensure the compatibility of plant-made growth factors with cell culture.

Structure-activity relationships of aniline-based squaraines for distinguishable staining and bright two-photon fluorescence bioimaging in plant cells.

An organelle-selective vision provides insights into the physiological response of plants and crops to environmental stresses in sustainable agriculture ecosystems. Biological applications often require two-photon excited fluorophores with low phototoxicity, high brightness, deep penetration, and tuneable cell entry. We obtained three aniline-based squaraines (SQs) tuned from hydrophobic to hydrophilic characteristics by modifying terminal pendant groups and substituents, and investigated their steady-state absorption and far-red-emitting fluorescence properties. The SQs exhibited two-photon absorption (2PA) ranging from 750 to 870 nm within the first biological spectral window; their structure-property relationships, corresponding to the 2PA cross sections (δ2PA), and structure differences were demonstrated. The maximum δ2PA value was ∼1220 GM at 800 nm for hydrophilic SQ3. Distinct biological staining efficiency and selective SQ bioimaging were evaluated utilizing the onion epidermal cell model. Contrary to the hydrophobic SQ1 results in the onion epidermal cell wall, amphiphilic SQ2 tagged the vacuole and nucleus and SQ3 tagged the vacuole. Distinguishable staining profiles in the roots and leaves were achieved. We believe that this study is the first to demonstrate distinct visualisation efficiency induced by the structure differences of two-photon excited SQs. Our results can help establish the versatile roles of novel near-infrared-emitting SQs in biological applications.

Spatiotemporal regulation of plant cell division.

Plant morphogenesis largely depends on the orientation and rate of cell division and elongation, and their coordination at all levels of organization. Despite recent progresses in the comprehension of pathways controlling division plane determination in plant cells, many pieces are missing to the puzzle. For example, we have a partial comprehension of formation, function and evolutionary significance of the preprophase band, a plant-specific cytoskeletal array involved in premitotic setup of the division plane, as well as the role of the nucleus and its connection to the preprophase band of microtubules. Likewise, several modeling studies point to a strong relationship between cell shape and division geometry, but the emergence of such geometric rules from the molecular and cellular pathways at play are still obscure. Yet, recent imaging technologies and genetic tools hold a lot of promise to tackle these challenges and to revisit old questions with unprecedented resolution in space and time.

Myosin XI, a model of its conserved role in plant cell tip growth.

In eukaryotic cells, organelle and vesicle transport, positioning, and interactions play crucial roles in cytoplasmic organization and function. These processes are governed by intracellular trafficking mechanisms. At the core of that trafficking, the cytoskeleton and directional transport by motor proteins stand out as its key regulators. Plant cell tip growth is a well-studied example of cytoplasm organization by polarization. This polarization, essential for the cell's function, is driven by the cytoskeleton and its associated motors. This review will focus on myosin XI, a molecular motor critical for vesicle trafficking and polarized plant cell growth. We will center our discussion on recent data from the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The biochemical properties and structure of myosin XI in various plant species are discussed, highlighting functional conservation across species. We further explore this conservation of myosin XI function in the process of vesicle transport in tip-growing cells. Existing evidence indicates that myosin XI actively organizes actin filaments in tip-growing cells by a mechanism based on vesicle clustering at their tips. A hypothetical model is presented to explain the essential function of myosin XI in polarized plant cell growth based on vesicle clustering at the tip. The review also provides insight into the in vivo localization and dynamics of myosin XI, emphasizing its role in cytosolic calcium regulation, which influences the polymerization of F-actin. Lastly, we touch upon the need for additional research to elucidate the regulation of myosin function.

Are vacuolar dynamics crucial factors for plant cell division and differentiation?

Vacuoles are the largest membrane-bound organelles in plant cells, critical for development and environmental responses. Vacuolar dynamics indicate reversible changes of vacuoles in morphology, size, or numbers. In this review, we summarize current understandings of vacuolar dynamics in different types of plant cells, biological processes associated with vacuolar dynamics, and regulators controlling vacuolar dynamics. Specifically, we point out the possibility that vacuolar dynamics play key roles in cell division and differentiation, which are controlled by the nucleus. Finally, we propose three routes through which vacuolar dynamics actively participate in nucleus-controlled cellular activities.

Engineering Plant Cell Fates and Functions for Agriculture and Industry.

Many plant species are grown to enable access to specific organs or tissues, such as seeds, fruits, or stems. In some cases, a value is associated with a molecule that accumulates in a single type of cell. Domestication and subsequent breeding have often increased the yields of these target products by increasing the size, number, and quality of harvested organs and tissues but also via changes to overall plant growth architecture to suit large-scale cultivation. Many of the mutations that underlie these changes have been identified in key regulators of cellular identity and function. As key determinants of yield, these regulators are key targets for synthetic biology approaches to engineer new forms and functions. However, our understanding of many plant developmental programs and cell-type specific functions is still incomplete. In this Perspective, we discuss how advances in cellular genomics together with synthetic biology tools such as biosensors and DNA-recording devices are advancing our understanding of cell-specific programs and cell fates. We then discuss advances and emerging opportunities for cell-type-specific engineering to optimize plant morphology, responses to the environment, and the production of valuable compounds.

Metal-Organic Framework-Mediated Delivery of Nucleic Acid across Intact Plant Cells.

Plant synthetic biology is applied in sustainable agriculture, clean energy, and biopharmaceuticals, addressing crop improvement, pest resistance, and plant-based vaccine production by introducing exogenous genes into plants. This technique faces challenges delivering genes due to plant cell walls and intact cell membranes. Novel approaches are required to address this challenge, such as utilizing nanomaterials known for their efficiency and biocompatibility in gene delivery. This work investigates metal-organic frameworks (MOFs) for gene delivery in intact plant cells by infiltration. Hence, small-sized ZIF-8 nanoparticles (below 20 nm) were synthesized and demonstrated effective DNA/RNA delivery into Nicotiana benthamiana leaves and Arabidopsis thaliana roots, presenting a promising and simplified method for gene delivery in intact plant cells. We further demonstrate that small-sized ZIF-8 nanoparticles protect RNA from RNase degradation and successfully silence an endogenous gene by delivering siRNA in N. benthamiana leaves.

Roles of Reactive Carbonyl Species (RCS) in Plant Response to Abiotic Stress.

Abiotic and biotic stress conditions lead to production of reactive carbonyl species (RCS) which are lipid peroxide derivatives and have detrimental effects on plant cells especially at high concentrations. There are several molecules that can be classified in RCS; among them, 4-hydroxy-(E)-2-nonenal (HNE) and acrolein are widely recognized and studied because of their toxicity. The toxicity mechanisms of RCS are well known in animals but their roles in plant systems especially signaling aspects in metabolism need to be addressed. This chapter focuses on the production mechanisms of RCS in plants as well as how plants scavenge and modify them to prevent irreversible damage in the cell. We aimed to get a comprehensive look at the literature to summarize the signaling roles of RCS in plant metabolism and their interaction with other signaling mechanisms such as highly recognized reactive oxygen species (ROS) signaling. Changing climate promotes more severe abiotic stress effects on plants which also decrease yield on the field. The effects of abiotic stress conditions on RCS metabolism are also gathered in this chapter including their signaling roles during abiotic stresses. Different methods of measuring RCS in plants are also presented in this chapter to draw more attention to the study of RCS metabolism in plants.

[Spatial transcriptomics techniques and its applications in plant research].

The heterogeneity of gene expression in plant cells plays a crucial role in determining the functional differences among tissues. Recent advancements in spatial transcriptome (ST) technology have significantly contributed to the study of specific biological questions in plants. This technology has been successfully applied to examine cell development, identification, and stress resistance. This review aims to explore the application of ST technology in plants by reviewing three aspects: the development of ST technology, its current application in plants, and future research directions. The review provides a systematic description of the development process of ST technology, with a focus on analyzing its progress in studying plant cell growth and differentiation, plant cell identification, and stress resistance. In addition, the challenges faced by ST technology in plant applications are summarized, along with proposed future directions for plant research, including the advantages of combining other omics technologies with ST technology to tackle scientific challenges in the field of plants.

Resource allocation modeling for autonomous prediction of plant cell phenotypes.

Predicting the plant cell response in complex environmental conditions is a challenge in plant biology. Here we developed a resource allocation model of cellular and molecular scale for the leaf photosynthetic cell of Arabidopsis thaliana, based on the Resource Balance Analysis (RBA) constraint-based modeling framework. The RBA model contains the metabolic network and the major macromolecular processes involved in the plant cell growth and survival and localized in cellular compartments. We simulated the model for varying environmental conditions of temperature, irradiance, partial pressure of CO2 and O2, and compared RBA predictions to known resource distributions and quantitative phenotypic traits such as the relative growth rate, the C:N ratio, and finally to the empirical characteristics of CO2 fixation given by the well-established Farquhar model. In comparison to other standard constraint-based modeling methods like Flux Balance Analysis, the RBA model makes accurate quantitative predictions without the need for empirical constraints. Altogether, we show that RBA significantly improves the autonomous prediction of plant cell phenotypes in complex environmental conditions, and provides mechanistic links between the genotype and the phenotype of the plant cell.

A robust transformer-based pipeline of 3D cell alignment, denoise and instance segmentation on electron microscopy sequence images.

Germline cells are critical for transmitting genetic information to subsequent generations in biological organisms. While their differentiation from somatic cells during embryonic development is well-documented in most animals, the regulatory mechanisms initiating plant germline cells are not well understood. To thoroughly investigate the complex morphological transformations of their ultrastructure over developmental time, nanoscale 3D reconstruction of entire plant tissues is necessary, achievable exclusively through electron microscopy imaging. This paper presents a full-process framework designed for reconstructing large-volume plant tissue from serial electron microscopy images. The framework ensures end-to-end direct output of reconstruction results, including topological networks and morphological analysis. The proposed 3D cell alignment, denoise, and instance segmentation pipeline (3DCADS) leverages deep learning to provide a cell instance segmentation workflow for electron microscopy image series, ensuring accurate and robust 3D cell reconstructions with high computational efficiency. The pipeline involves five stages: the registration of electron microscopy serial images; image enhancement and denoising; semantic segmentation using a Transformer-based neural network; instance segmentation through a supervoxel-based clustering algorithm; and an automated analysis and statistical assessment of the reconstruction results, with the mapping of topological connections. The 3DCADS model's precision was validated on a plant tissue ground-truth dataset, outperforming traditional baseline models and deep learning baselines in overall accuracy. The framework was applied to the reconstruction of early meiosis stages in the anthers of Arabidopsis thaliana, resulting in a topological connectivity network and analysis of morphological parameters and characteristics of cell distribution. The experiment underscores the 3DCADS model's potential for biological tissue identification and its significance in quantitative analysis of plant cell development, crucial for examining samples across different genetic phenotypes and mutations in plant development. Additionally, the paper discusses the regulatory mechanisms of Arabidopsis thaliana's germline cells and the development of stamen cells before meiosis, offering new insights into the transition from somatic to germline cell fate in plants.

Production of specialized metabolites in plant cell and organo-cultures: the role of gamma radiation in eliciting secondary metabolism.

PURPOSE: To provide an updated summary of recent advances in the application of gamma irradiation to elicit secondary metabolism and for induction of mutations in plant cell and organ cultures for the production of industrially important specialized metabolites (SMs). CONCLUSIONS: Research on the application of gamma radiation with plants has contributed a lot to microbial decontamination of seeds, and the promotion of physiological processes such as seed germination, seedling vigor, plant growth, and development. Various studies have demonstrated the influence of gamma rays on the morphology, physiology, and biochemistry of plants. Recent research efforts have also shown that low-dose gamma (5-100 Gy) irradiation can be utilized as an expedient solution to alleviate the deleterious effect of abiotic stresses and to obtain better yields of plants. Inducing mutagenesis using gamma irradiation has also evolved as a better option for inducing genetic variability in crops, vegetables, medicinal and ornamentals for their genetic improvement. Plant SMs are gaining increasing importance as pharmaceutical, therapeutic, cosmetic, and agricultural products. Plant cell, tissue, and organ cultures represent an attractive alternative to conventional methods of procuring useful SMs. Among the varied approaches the elicitor-induced in vitro culture techniques are considered an efficient tool for studying and improving the production of SMs. This review focuses on the utilization of low-dose gamma irradiation in the production of high-value SMs such as phenolics, terpenoids, and alkaloids. Furthermore, we present varied successful examples of gamma-ray-induced mutations in the production of SMs.

Subtilisin-like proteases from Fusarium graminearum induce plant cell death and contribute to virulence.

Fusarium head blight (FHB), caused by Fusarium graminearum, causes huge annual economic losses in cereal production. To successfully colonize host plants, pathogens secrete hundreds of effectors that interfere with plant immunity and facilitate infection. However, the roles of most secreted effectors of F. graminearum in pathogenesis remain unclear. We analyzed the secreted proteins of F. graminearum and identified 255 candidate effector proteins by liquid chromatography-mass spectrometry (LC-MS). Five subtilisin-like family proteases (FgSLPs) were identified that can induce cell death in Nicotiana benthamiana leaves. Further experiments showed that these FgSLPs induced cell death in cotton (Gossypium barbadense) and Arabidopsis (Arabidopsis thaliana). A signal peptide and light were not essential for the cell death-inducing activity of FgSLPs. The I9 inhibitor domain and the entire C-terminus of FgSLPs were indispensable for their self-processing and cell death-inducing activity. FgSLP-induced cell death occurred independent of the plant signal transduction components BRI-ASSOCIATED KINASE 1 (BAK1), SUPPRESSOR OF BIR1 1 (SOBIR1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), and PHYTOALEXIN DEFICIENT 4 (PAD4). Reduced virulence was observed when FgSLP1 and FgSLP2 were simultaneously knocked out. This study reveals a class of secreted toxic proteins essential for F. graminearum virulence.

Heat-induced SUMOylation differentially affects bacterial effectors in plant cells.

Bacterial pathogens deliver effectors into host cells to suppress immunity. How host cells target these effectors is critical in pathogen-host interactions. SUMOylation, an important type of posttranslational modification in eukaryotic cells, plays a critical role in immunity, but its effect on bacterial effectors remains unclear in plant cells. In this study, using bioinformatic and biochemical approaches, we found that at least 16 effectors from the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 are SUMOylated by the enzyme cascade from Arabidopsis thaliana. Mutation of SUMOylation sites on the effector HopB1 enhances its function in the induction of plant cell death via stability attenuation of a plant receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1. By contrast, SUMOylation is essential for the function of another effector, HopG1, in the inhibition of mitochondria activity and jasmonic acid signaling. SUMOylation of both HopB1 and HopG1 is increased by heat treatment, and this modification modulates the functions of these 2 effectors in different ways in the regulation of plant survival rates, gene expression, and bacterial infection under high temperatures. Therefore, the current work on the SUMOylation of effectors in plant cells improves our understanding of the function of dynamic protein modifications in plant-pathogen interactions in response to environmental conditions.

Plant cell size: Links to cell cycle, differentiation and ploidy.

Cell size affects many processes, including exchange of nutrients and external signals, cell division and tissue mechanics. Across eukaryotes, cells have evolved mechanisms that assess their own size to inform processes such as cell cycle progression or gene expression. Here, we review recent progress in understanding plant cell size regulation and its implications, relating these findings to work in other eukaryotes. Highlights include use of DNA contents as reference point to control the cell cycle in shoot meristems, a size-dependent cell fate decision during stomatal development and insights into the interconnection between ploidy, cell size and cell wall mechanics.

Plasmodesmata and intercellular molecular traffic control.

Plasmodesmata are plasma membrane-lined connections that join plant cells to their neighbours, establishing an intercellular cytoplasmic continuum through which molecules can travel between cells, tissues, and organs. As plasmodesmata connect almost all cells in plants, their molecular traffic carries information and resources across a range of scales, but dynamic control of plasmodesmal aperture can change the possible domains of molecular exchange under different conditions. Plasmodesmal aperture is controlled by specialised signalling cascades accommodated in spatially discrete membrane and cell wall domains. Thus, the composition of plasmodesmata defines their capacity for molecular trafficking. Further, their shape and density can likewise define trafficking capacity, with the cell walls between different cell types hosting different numbers and forms of plasmodesmata to drive molecular flux in physiologically important directions. The molecular traffic that travels through plasmodesmata ranges from small metabolites through to proteins, and possibly even larger mRNAs. Smaller molecules are transmitted between cells via passive mechanisms but how larger molecules are efficiently trafficked through plasmodesmata remains a key question in plasmodesmal biology. How plasmodesmata are formed, the shape they take, what they are made of, and what passes through them regulate molecular traffic through plants, underpinning a wide range of plant physiology.