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The oxidation-reduction (redox) status of the cell influences or regulates transcription factors and enzymes involved in epigenetic changes, such as DNA methylation, histone protein modifications, and chromatin structure and remodeling. These changes are crucial regulators of chromatin architecture, leading to differential gene expression in eukaryotes. But the cell's redox homeostasis is difficult to sustain since the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is not equal in plants at different developmental stages and under abiotic stress conditions. Exceeding optimum ROS and RNS levels leads to oxidative stress and thus alters the redox status of the cell. Consequently, this alteration modulates intracellular epigenetic modifications that either mitigate or mediate the plant growth and stress response.Recent studies suggest that the altered redox status of the cell reform the cellular functions and epigenetic changes. Recent high-throughput techniques have also greatly advanced redox-mediated gene expression discovery, but the integrated view of the redox status, and its associations with epigenetic changes and subsequent gene expression in plants are still scarce. In this review, we accordingly focus on how the redox status of the cell affects epigenetic modifications in plants under abiotic stress conditions and during developmental processes. This is a first comprehensive review on the redox status of the cell covering the redox components and signaling, redox status alters the post-translational modification of proteins, intracellular epigenetic modifications, redox interplay during DNA methylation, redox regulation of histone acetylation and methylation, redox regulation of miRNA biogenesis, redox regulation of chromatin structure and remodeling and conclusion, future perspectives and biotechnological opportunities for the future development of the plants.The interaction of redox mediators such as ROS, RNS and antioxidants regulates redox homeostasis and redox-mediated epigenetic changes. We discuss how redox mediators modulate epigenetic changes and show the opportunities for smart use of the redox status of the cell in plant development and abiotic stress adaptation. However, how a redox mediator triggers epigenetic modification without activating other redox mediators remains yet unknown.

International audience; The plant cytoskeleton regulates fundamental biological processes, including cell division. How to experimentally perturb the cytoskeleton is a key question if one wants to understand the role of both actin filaments (AFs) and microtubules (MTs) in a given biological process. While a myriad of mutants are available, knock-out in cytoskeleton regulators, when nonlethal, often produce little or no phenotypic perturbation because such regulators are often part of a large family, leading to functional redundancy. In this review, alternative techniques to modify the plant cytoskeleton during plant cell division are outlined. The different pharmacological and genetic approaches already developed in cell culture, transient assays, or in whole organisms are presented. Perspectives on the use of optogenetics to perturb the plant cytoskeleton are also discussed.

New evidences are emerging to support the importance of viral replication complexes (VRCs) in not only viral replication, but also viral cell-to-cell movement. Currently, how VRCs grow in size and colocalize with viral movement proteins (MPs) remains unclear. Herein, we performed live-cell imaging of red clover necrotic mosaic virus (RCNMV) dsRNA by using reporter B2-GFP plants. Tiny granules of dsRNA were formed along the endoplasmic reticulum (ER) at an early stage of infection. Importantly, the colocalization of the dsRNA granules with the virus-encoded p27 replication protein showed that these structures are components of VRCs. These granules moved throughout the cytoplasm, driven by the acto-myosin system, and coalesced with each other to form larger aggregates; the MPs were not associated with these processes. Notably, the MPs colocalized preferentially with large dsRNA aggregates, rather than with tiny dsRNA granules, suggesting that the increase in the size of VRCs promotes their colocalization with MPs.

Phloem-feeding insects cause massive losses in agriculture and horticulture. Host plant resistance to phloem-feeding insects is often mediated by changes in phloem composition, which deter insect settling and feeding and decrease viability. Here, we report that rice plant resistance to the phloem-feeding brown planthopper (BPH) is associated with fortification of the sclerenchyma tissue, which is located just beneath the epidermis and a cell layer or two away from the vascular bundle in the rice leaf sheath. We found that BPHs prefer to feed on the smooth and soft region on the surface of rice leaf sheaths called the long-cell block. We identified Bph30 as a rice BPH resistance gene that prevents BPH stylets from reaching the phloem due to the fortified sclerenchyma. Bph30 is strongly expressed in sclerenchyma cells and enhances cellulose and hemicellulose synthesis, making the cell walls stiffer and sclerenchyma thicker. The structurally fortified sclerenchyma is a formidable barrier preventing BPH stylets from penetrating the leaf sheath tissues and arriving at the phloem to feed. Bph30 belongs to a novel gene family, encoding a protein with two leucine-rich domains. Another member of the family, Bph40, also conferred resistance to BPH. Collectively, the fortified sclerenchyma-mediated resistance mechanism revealed in this study expands our understanding of plant-insect interactions and opens a new path for controlling planthoppers in rice.

Shoot apical meristem (SAM) and root apical meristem (RAM) act as pools of stem cells to give rise to aboveground and underground tissues and organs in higher plants. Despite the structure differences between these two meristems, they evolve similar signaling pathways to maintain stem cell homeostasis. The CLAVATA3 (CLV3)-WUSCHEL (WUS) negative feedback loop acts as a core pathway in controlling SAM homeostasis, while CLV3/EMBRYO SURROUNDING REGION (ESR) 40 (CLE40) and WUSCHEL-RELATED HOMEOBOX5 (WOX5), homologues of CLV3 and WUS, direct columella stem cell fate. It has been shown that CLV3 INSENSITIVE KINASES (CIKs) are essential for maintaining SAM homeostasis. Whether CIKs regulate the distal root meristem remains to be elucidated. Here, we report that CIKs are indispensable for transducing the CLE40 signal to maintain homeostasis of the distal root meristem. The cik mutants displayed disrupted quiescent center and delayed columella stem cell (CSC) differentiation. Biochemical assays demonstrated that CIKs interact with ARABIDOPSIS CRINKLY4 (ACR4) in a ligand-independent manner and can be phosphorylated by ACR4 in vitro. In addition, the phosphorylation level of CIKs can be rapidly induced by CLE40, which partially depends on ACR4. Though CIKs act as conserved and redundant regulators in the SAM and RAM, our results demonstrated that they exhibit differentiated functions in these meristems.

A central role of cell division in the life of multicellular plants is underlined by the fact that plants cannot move, in contrast to multicellular animals. Hence, cell division in plants fulfills not only a developmental task sensu stricto (for example, formation of organs, tissues and cell types) but also is key for adaptation to environmental conditions, presumably more so than in animals, by enhancing, reducing, as well as redirecting cell divisions, and thus adjusting growth.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Plant exosome-like nanovesicles, being innately replete with bioactive lipids, proteins, RNA, and other pharmacologically active molecules, offer unique morphological and compositional characteristics as natural nanocarriers. Furthermore, their compelling physicochemical traits underpin their modulative role in physiological processes, all of which have fostered the concept that these nanovesicles may be highly proficient in the development of next-generation biotherapeutic and drug delivery nanoplatforms to meet the ever-stringent demands of current clinical challenges. This review systemically deals with various facets of plant exosome-like nanovesicles ranging from their origin and isolation to identification of morphological composition, biological functions, and cargo-loading mechanisms. Efforts are made to encompass their biotherapeutic roles by elucidating their immunological modulating, anti-tumor, regenerative, and anti-inflammatory roles. We also shed light on re-engineering these nanovesicles into robust, innocuous, and non-immunogenic nanovectors for drug delivery through multiple stringent biological hindrances to various targeted organs such as intestine and brain. Finally, recent advances centered around plant exosome-like nanovesicles along with new insights into transdermal, transmembrane and targeting mechanisms of these vesicles are also elucidated. We expect that the continuing development of plant exosome-like nanovesicle-based therapeutic and delivery nanoplatforms will promote their clinical applications.

Exploitation of enzymes in biocatalytic processes provides scope both in the synthesis and degradation of molecules. Enzymes have power not only in their catalytic efficiency, but their chemoselectivity, regioselectivity, and stereoselectivity means the reactions they catalyze are precise and reproducible. Focusing on carbohydrate processing enzymes, this review covers advances in biocatalysis involving carbohydrates over the last 2-3 years. Given the notorious difficulties in the chemical synthesis of carbohydrates, the use of enzymes for synthesis has potential for significant impact in the future. The use of catabolic enzymes in the degradation of biomass, which can be exploited in the production of biofuels to provide a sustainable and greener source of energy, and the synthesis of molecules that have a range of applications including in the pharmaceutical and food industries will be explored.

Fat-Specific Protein 27 (FSP27) belongs to a small group of vertebrate proteins containing a Cell-death Inducing DNA fragmentation factor-α-like Effector (CIDE)-C domain and is involved in lipid droplet (LD) accumulation and energy homeostasis. FSP27 is predominantly expressed in white and brown adipose tissues, as well as liver, and plays a key role in mediating LD-LD fusion. No orthologs have been identified in invertebrates or plants. In this study, we tested the function of mouse FSP27 in stably-transformed Arabidopsis thaliana leaves and seeds, as well as through transient expression in Nicotiana tabacum suspension-cultured cells and N. benthamiana leaves. Confocal microscopic analysis of plant cells revealed that, similar to ectopic expression in mammalian cells, FSP27 produced in plants 1) correctly localized to LDs, 2) accumulated at LD-LD contact sites, and 3) induced an increase in the number and size of LDs and also promoted LD clustering and fusion. Furthermore, FSP27 increased oil content in transgenic A. thaliana seeds. Given that plant oils have uses in human and animal nutrition as well as industrial uses such as biofuels and bioplastics, our results suggest that ectopic expression of FSP27 in plants represents a potential strategy for increasing oil content and energy density in bioenergy or oilseed crops.

The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of

In multicellular eukaryotic cells, metabolism and growth are sustained by the cooperative functioning of organelles in combination with cell-to-cell communication at the organism level. In land plants, multiple strategies have evolved to adapt to life outside water. As basal land plant, the moss Physcomitrella patens is used for comparative genomics, allowing to study lineage-specific features, as well as to track the evolution of fundamental parameters of plant cell organisation and physiology. P. patens is a versatile model for cell biology research, especially to investigate adaptive growth, stress biology as well as organelle dynamics and interactions. Recent advances include the use of genetically encoded biosensors for in vivo imaging of physiological parameters.

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.

Summary Plant cell growth is constrained by the rate of water uptake and wall extensibility. New research reveals that the topology and geometry of cells in a tissue predicts the pressures that ultimately determine growth.

Nanomaterial-based delivery systems can deliver functional genes or siRNA into intact plant cells and create transgene-free genetically engineered plants. This system allows highly efficient and organelle-specific delivery that can overcome host-range limitations. This approach will have a diverse range of applications in plant biotechnology and plant biology.

Plant growth and development are driven by extended phases of irreversible cell expansion generating cells that increase in volume from 10- to 100-fold. Some specialized cell types define cortical sites that reinitiate polarized growth and generate branched cell morphology. This structural specialization of individual cells has a major importance for plant adaptation to diverse environments and practical importance in agricultural contexts. The patterns of cell shape are defined by highly integrated cytoskeletal and cell wall systems. Microtubules and actin filaments locally define the material properties of a tough outer cell wall to generate complex shapes. Forward genetics, powerful live cell imaging experiments, and computational modeling have provided insights into understanding of mechanisms of cell shape control. In particular, finite element modeling of the cell wall provides a new way to discover which cell wall heterogeneities generate complex cell shapes, and how cell shape and cell wall stress can feedback on the cytoskeleton to maintain growth patterns. This review focuses on cytoskeleton-dependent cell wall patterning during cell branching, and how combinations of multi-scale imaging experiments and computational modeling are being used to unravel systems-level control of morphogenesis.

The use of methyl jasmonate, alone or in combination with cyclic oligosaccharides such as cyclodextrins, has proved to be a successful strategy for increasing the production of trans-resveratrol in Vitis vinifera cell cultures. However, understanding the intracellular signalling pathways involved in its production would improve the management of grapevine cells as biofactories of this high-value natural product. The results obtained herein confirm the involvement of hydrogen peroxide and nitric oxide in cyclodextrins and methyl jasmonate-induced trans-resveratrol production in grapevine cell cultures. In fact, methyl jasmonate led to maximal intracellular levels of hydrogen peroxide and nitric oxide after 24 h of treatment, but extracellular hydrogen peroxide was only detected in the culture medium when grapevine cells were treated with cyclodextrins. The results derived from the cytochemical detection of H2O2 in elicited grapevine cell cultures also suggested that the combined treatment with cyclodextrins and methyl jasmonate not only increased the production of H2O2 but also released cell wall fragments with electron-dense deposits. Moreover, nitric oxide was localized in all the cellular compartments, particularly in the nucleus and cytoplasmic organelles, whereas hydrogen peroxide was mainly found in cytoplasmic areas close to the cell wall, and in the nucleoplasm.

In the last decade, recent advances in single-cell RNA sequencing coupled with computational algorithms have opened new avenues to study the cell type composition of tissues and organs as well as to infer cell developmental trajectories. These technologies have been used to resolve and map atlases of tissues and organs in many animal species as well as to further order cell developmental trajectories. Despite these advances in animals, many of the current plant cell type expression profiles confound multiple developmental stages preventing an accurate monitoring of cell lineage. In this review, we propose how the application of single-cell sequencing will improve our molecular understanding of cell type differentiation. Using root vascular cells as a model, we highlight the potential of single cell transcriptomics as well as its limitations to monitor the progression of vascular maturation. By comparing cell morphology, functionality and gene expression, we aim to provide a new perspective of plant cell type differentiation.

Lignin research has long been motivated by the outstanding importance of wood for human societies. The annual, non-woody Arabidopsis thaliana, has nevertheless contributed greatly to our understanding of lignification, due to its unrivalled genetic resources. Arabidopsis is also great for cell and developmental biology, allowing precise imaging and tracking of cell types. Root endodermis differentiation involves the precise lignification of the Casparian Strip, as an apoplastic barrier; while barrier damage triggers a less localized, compensatory lignification. Transcriptional reprogramming and peptide-induced signalling emerge as promising tools for the study of endodermal lignification. We argue that endodermis lignification is an attractive model complementary to equally powerful, cellular xylem differentiation systems, as it might better represent the restricted - often localized - lignification seen in non-vascular cells.

Aluminum (Al) toxicity in the acid soils (pH ≤ 5) is the major limiting abiotic factor affecting the productivity of crops. Boron (B) has been reported to alleviate Al toxicity. In spite of recent advances, it is not clear how B relieves Al toxicity. Results demonstrated that Al toxicity hampered the root elongation. Moreover, lumogallion fluorescent molecular probe unequivocally localized mostly bound Al to the periphery of the cell wall (CW) and to the nuclei. Additionally, Al toxicity induced variations in the CW components through the accumulation of pectin and hemicellulose. Nevertheless, B supply reduced callose deposition, increased root growth and reduced changes in the CW components under Al toxicity. Moreover, B supply reduced the un-methylated pectin while increased the degree of methyl esterification of pectin. These results imply that B due to its role in the CW formation could reduce aluminum-induced negative effects on plant growth by attenuating apoplastic Al3+ and changes in the CW components which ultimately results in the improved root growth.

The recent Ebola virus disease (EVD) outbreaks make the development of efficacious and low cost vaccines against Ebola virus (EBOV) an urgent goal. Multiepitopic vaccines allow a rational design rendering vaccines able to induce proper immune responses in terms of polarization and potency. In addition, the pathogen variants can be easily covered by including epitopes conserved among relevant isolates. Other important aspects to consider in vaccination are the costs associated to production, distribution, and administration of the vaccine. Plants provide an advantageous platform for this purpose, since they yield biomass at very low costs and some species can be used to formulate purification-free oral vaccines. In the present study, a multiepitopic protein called Zerola, which carries epitopes from the EBOV glycoprotein (GP), was designed based on immunoinformatic approaches and current experimental evidence on B cell protective GP epitopes. Moreover, expression studies performed in nuclear-transformed tobacco lines confirmed the capacity of the plant cell to synthetize the Zerola antigenic protein. The generation of this plant-based candidate vaccine is a step forward in the development of highly efficient and low cost EBOV vaccines.

Light is the most important physical factor in growth and development of plants. Light intensity is directly proportional to the growth and accumulation of natural antioxidants during in vitro cultures of various medicinal plants. The present research study was designed to determine the effect of different light intensities i.e. normal light (2000–2500 lx), diffused light (500–1000 lx) and complete dark (0 lx) on callus growth dynamics and production of natural antioxidants in olive cult. Arbosana. Highest callus induction frequency (50%) was observed in the stem explants pre-treated with silver nanoparticles suspension (AgNPs: 50 ppm) and cultured on MS media supplemented with combination of 6-Benzylaminopurine (BAP: 2 mg/l), Gibberellic acid (GA3: 1.5 mg/l) plus Naphthalene acetic acid (NAA: 0.5 mg/l). Maximum callus biomass (FW = 1414 mg/l) was recorded when the cultured explants were incubated initially for seven days in complete darkness, followed by transference to diffused light for one week and then finally placed under normal light in total fifty six days culture period. Moreover, phytochemical analysis of the callus cultures showed significantly higher activities of antioxidant enzymes i.e. SOD, POD, CAT and APx (2.45, 2.96, 2.57 and 1.67 U/mg. protein) in the callus cultures grown under dark condition as compared with other light treatments. For non-enzymatic antioxidant potential, maximum activity of TPC, TFC, PAL and DPPH (2.42 mg GAE/g, 1.50 mg QAE/g, 3.95 U/mg and 75%) were recorded in the calli raised in vitro under diffused light. This is the first report on the production of natural antioxidants in response to different light intensities in callus cultures of Olea europaea. Future studies should focus on large scale production of callus cultures in order to yield maximum biomass from this high valued plant.

Plant cells use autophagy to degrade their own cytoplasm in vacuoles, thereby not only recycling their breakdown products, but also ensuring the homeostasis of essential cytoplasmic constituents and organelles. Plants and other eukaryotes have a conserved set of core Autophagy-related (ATG) genes involved in the biogenesis of the autophagosome, the main autophagic compartment destined for the lytic vacuole. In the past decade, the core ATG genes were isolated from several plant species. The core ATG proteins include the components of the VACUOLAR PROTEIN SORTING 34 (VPS34) complex that is responsible for the local production of phosphatidylinositol 3-phosphate (PI3P) at the site of autophagosome formation. Dissecting the roles of PI3P and its effectors in autophagy is challenging, because of the multi-faceted links between autophagosomal and endosomal systems. This review highlights recent studies on putative plant PI3P effectors involved in autophagosome dynamics. Molecular mechanisms underlying the requirement of PI3P for autophagosome biogenesis and trafficking are also discussed.

International audience; The formation of spatial and temporal patterns is an essential component of organismal development. Patterns can be observed on every level from subcellular to organismal and may emerge from local rules that correspond to the interactions between molecules, cells, or tissues. The emergence of robust patterns may seem in contradiction with the prominent heterogeneity at subcellular and cellular scales, however it has become increasingly clear that heterogeneity can be instrumental for pattern formation. Here we review recent examples in plant development, involving genetic regulation, cell arrangement, growth and signal gradient. We discuss how patterns emerge from local rules, whether heterogeneity is stochastic or can be patterned, and whether stochastic noise is amplified or requires filtering for robust patterns to be achieved. We also stress the importance of modelling in investigating such questions.

The growth of individual cells underlies the development of biological forms. In plants, cells are interconnected by rigid walls, fixing their position with respect to one another and generating mechanical feedbacks between cells. Current research is shedding new light on how plant growth is controlled by physical inputs at the level of individual cells and growing tissues. In this review, we discuss recent progress in our understanding of the cellular basis of growth from a biomechanical perspective. We describe the role of the cell wall and turgor pressure in growth and highlight the often-overlooked role of cell geometry in this process. It is becoming apparent that a combination of experimental and theoretical approaches is required to answer new emerging questions in the biomechanics of plant morphogenesis. We summarise how this multidisciplinary approach brings us closer to a unified understanding of the generation of biological forms in plants.

International audience; The plasma membrane (PM) is the biological membrane that separates the interior of all cells from the outside. The PM is constituted of a huge diversity of proteins and lipids. In this review, we will update the diversity of molecular species of lipids found in plant PM. We will further discuss how lipids govern global properties of the plant PM, explaining that plant lipids are unevenly distributed and are able to organize PM in domains. From that observation, it emerges a complex picture showing a spatial and multiscale segregation of PM components. Finally, we will discuss how lipids are key players in the function of PM in plants, with a particular focus on plant-microbe interaction, transport and hormone signaling, abiotic stress responses, plasmodesmata function. The last chapter is dedicated to the methods that the plant membrane biology community needs to develop to get a comprehensive understanding of membrane organization in plants.

Specification of cell polarity is vital to normal cell growth, morphogenesis, and function. As other eukaryotes, plants generate cellular polarity that is coordinated with tissue polarity and organ axes. In development, new cell types are generated by stem-cell division and differentiation, a process often involving proteins that are polarized to cortical domains at the plasma membrane. In the past decade, pioneering work using the model plant Arabidopsis identified multiple proteins that are polarized in dividing cells to instruct divisional behaviors and/or specify cell fates. In this review, we use these polarized cell-division regulators as example to summarize key mechanisms underlying protein polarization in plant cells. Recent progress underscores that self-organizing amplification processes are commonly involved in establishing cell polarity, and cellular polarity is influenced by both tissue-level and local mechano-chemical cues. In addition, protein polarization during asymmetric cell division shows a distinct feature of temporal control in the stomatal lineage. We further discuss possible coordination between protein polarization and the progression of cell cycle in this developmental context.

The endomembrane system is critical for plant growth and development and understanding its function and regulation is of great interest for plant biology research. Small-molecule targeting distinctive endomembrane components have proven powerful tools to dissect membrane trafficking in plant cells. However, unambiguous elucidation of the complex and dynamic trafficking processes requires chemical probes with enhanced precision. Determination of the mechanism of action of a compound, which is facilitated by various chemoproteomic approaches, opens new avenues for the improvement of its specificity. Moreover, rational molecule design and reverse chemical genetics with the aid of virtual screening and artificial intelligence will enable us to discover highly precise chemical probes more efficiently. The next decade will witness the emergence of more such accurate tools, which together with advanced live quantitative imaging techniques of subcellular phenotypes, will deepen our insights into the plant endomembrane system.

Assembly of polysaccharide-based walls by plant cells involves the rapid synthesis, trafficking, and deposition of complex biopolymers, but how these events are controlled and coordinated to achieve a strong, resilient extracellular matrix has remained obscure for decades. Recent quantitative analyses of fluorescence microscopy data have revealed details of the trafficking and synthetic activity of cellulose synthases, and new methods for labeling matrix polymers have unveiled aspects of their regulated deposition in the wall. Detailed studies of the identity, architecture, activity, and trafficking of the proteins and protein complexes that synthesize wall polymers, combined with advances in image acquisition and analysis, will aid future efforts to dissect wall assembly.

Autophagy mediates the delivery of cytoplasmic content to vacuoles or lysosomes for degradation or storage. The best characterized autophagy route called macroautophagy involves the sequestration of cargo in double-membrane autophagosomes and is conserved in eukaryotes, including plants. Recently, several new receptors, some of them plant-specific, that select cargo for macroautophagy have been identified. Some of these receptors appear to participate in regulation of competing catabolic pathways, for example proteasome-mediated versus autophagic degradation under specific stress conditions. Vacuolar microautophagy, a process by which the vacuole directly engulf cytoplasmic material, also occurs in plants but its underlying molecular mechanisms are yet to be elucidated.

The phragmoplast is a plant-specific secretory module that partitions daughter cells during cytokinesis by constructing a cell plate from membranes and oligosaccharides. The cell plate is typically a long structure, which requires the phragmoplast to expand to complete cytokinesis. The phragmoplast expands by coordinating microtubule dynamics with membrane trafficking. Each step in phragmoplast expansion involves the establishment of anti-parallel microtubule overlaps that are enriched with the protein MAP65, which recruits cytokinetic vesicles through interaction with the tethering factor, TRAPPII. Cell plate assembly triggers dissolution of the anti-parallel overlaps and stabilization of microtubule plus ends through association with the cell plate assembly machinery. This opinion article discusses processes that drive phragmoplast expansion as well as highlights key questions that remain for better understanding its role in plant cell division.

Research on mosses has provided answers to many fundamental questions in the life sciences, with the model moss Physcomitrella patens spearheading the field. Recent breakthroughs in cell biology were obtained in the quantification of chlorophyll fluorescence, signalling via calcium waves, the creation of designer organelles, gene identification in cellular reprogramming, reproduction via motile sperm and egg cells, asymmetric cell division, visualization of the actin cytoskeleton, identification of genes responsible for the shift from 2D to 3D growth, the structure and importance of the cell wall, and in the live imaging and modelling of protein networks in general. Highly standardized growth conditions, simplicity of most moss tissues, and an outstandingly efficient gene editing facilitate quantitative moss cell biology.

The process of shape change in cells and tissues inevitably involves the modification of structural elements, therefore it is necessary to integrate mechanics with biochemistry to develop a full understanding of morphogenesis. Here, we discuss recent findings on the role of biomechanics and biochemical processes in plant cell growth and development. In particular, we focus on how the plant cytoskeleton components, which are known to regulate morphogenesis, are influenced by biomechanical stress. We also discuss new insights into the role that pectin plays in biomechanics and morphogenesis. Using the jigsaw-shaped pavement cells of the leaf as a case study, we review new findings on the biomechanics behind the morphogenesis of these intricately-shaped cell types. Finally, we summarize important quantitative techniques that has allowed for the testing and the generation of hypotheses that link biomechanics to morphogenesis.

The cellular responses to abiotic and biotic stress rely on the regulation of vesicle trafficking to ensure the correct localization of proteins specialized in sensing stress stimuli and effecting the response. Several studies have implicated the plant trans-Golgi network (TGN)-mediated trafficking in different types of biotic and abiotic stress responses; however, the underlying molecular mechanisms are poorly understood. Further, the identity, specialization and stress-relevant cargo transported by the TGN subcompartments involved in stress responses await more in depth characterization. This review presents TGN trafficking players implicated in stress and discusses potential avenues to understand the role of this dynamic network under such extreme circumstances.

Cyclotides are a class of ribosomally-synthesized plant peptides that function in plants as a defense against insects and fungal pathogens. Their unique structure comprises a cyclized peptide backbone threaded by three disulfide bonds, that imparts structural stability, a desirable quality for peptide-based therapeutics or insecticides. Producing these peptides synthetically is challenging due to the amount of chemical waste produced and inefficiency of folding certain cyclotides. Thus, it is desirable to develop a means to access cyclotide biosynthesis in their native hosts, cultured in defined conditions, at both laboratory and commercial scale. Here we developed suspension cell cultures from two species previously unexplored for cyclotide production in suspension cells, Clitoria ternatea L., Hybanthus enneaspermus F. Muell., as well as with Oldenlandia affinis (Roem.Schult.) DC., a species reported previously to accumulate cyclotides in cell suspensions. We assessed the growth rate, cyclotide production and gene expression for the various species. We found that while many cyclotides had reduced expression in Oldenlandia affinis suspension cells when compared to plant organs, those in Clitoria ternatea and Hybanthus enneaspermus maintained or increased expression levels. The cyclotides that continued to be expressed in suspension cultures shared similar sequence and biophysical properties as a group, regardless of phylogenetic origin of the host. Of particular interest was the discovery of inducibility by NaCl of cyclotide expression in O. affinis, cycloviolacin O2 expression in O. affinis, and the scale up of cycloviolacin O2 production in H. enneaspermus. Together the results presented here highlight the utility of plant cell suspensions as modalities to produce macrocyclic peptides.

While the pathways that produce plant pigments have been well studied for decades, the use by plants of nanoscale structures to produce colour effects has only recently begun to be studied. A variety of plants from across the plant kingdom have been shown to use different mechanism to generate structural colours in tissues as diverse as leaves, flowers and fruits. In this review we explore the cellular mechanisms by which these nanoscale structures are built and discuss the first insights that have been published into the genetic pathways underpinning these traits.

Mechanical forces were arguably among the first stimuli to be perceived by cells, and they continue to shape the evolution of all organisms. Great strides have been made in recent years in the field of plant cell and molecular mechanobiology, in part owing to focused efforts on key model systems. Here, we propose to enrich such work through evolutionary mechanobiology, or 'evo-mechano', and describe three major themes that could drive research in this area. We use plastid evo-mechano as a case study, describing how plastids from different lineages perceive their mechanical environments, how their mechanical properties vary across lineages, and their distinct roles in graviperception. Finally, we argue that future research into the biomechanical properties and mechanobiological signaling mechanisms that have been elaborated by green species over the past 1.5 billion years will help us understand both the universal and the unique adaptations of plants to their physical environment.

Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones. We therefore used a design of experiments approach to test the suitability of three plant-based systems for the expression of satisfactory quantities of recombinant PHACTR1, namely transient expression in tobacco (Nicotiana tabacum) BY-2 plant cell packs (PCPs), whole N. benthamiana leaves and BY-2 cell lysate (BYL). The highest yield was achieved using the BYL: up to 120 mg product kg-1 biomass equivalent within 48 h of translation. This was 1.3-fold higher than transient expression in N. benthamiana together with the silencing inhibitor p19, and 6-fold higher than the PCP system. The presence of Triton X-100 in the extraction buffer increased the recovery of PHACTR1 by 2-200-fold depending on the conditions. PHACTR1 was incompatible with biomass blanching and was stable for less than 16 h in raw plant extracts. Purification using a DDK-tag proved inefficient whereas 15% purity was achieved by immobilized metal affinity chromatography.

The plasma membrane is a sophisticated, organized, and highly heterogeneous structure that compartmentalizes cellular processes. To decipher the biological processes involving membrane proteins, it is necessary to analyze their spatiotemporal dynamics. However, it is difficult to directly assess the dynamics and interactions of biomolecules in living cells using traditional biochemical methods. Single-particle tracking (SPT) methods for imaging and tracking single particles conjugated with fluorescent probes offer an ideal approach to acquire valuable and complementary information about dynamic intracellular processes. SPT can be used to quantitatively monitor the diverse motions of individual particles in living cells. SPT also provides super-spatiotemporal resolution that allows early-stage or rapid response information to be obtained for a better understanding of molecular basis of associated signal transduction processes. More importantly, SPT can be used to detect the motion paths of individual biomolecules in vivo and in situ, thus unveiling the dynamic behavior of the biomolecules that support developmental processes in living cells. In this review, we give an overview of SPT methods, from image acquisition to the detection of single particles, as well as tracking and data analysis. We also discuss recent applications of SPT methods in the field of plant biology to reveal the complex biological functions of membrane proteins.

Actin is one of the three major cytoskeletal components in eukaryotic cells. Myosin XI is an actin-based motor protein in plant cells. Organelles are attached to myosin XI and translocated along the actin filaments. This dynamic actin-myosin XI system plays a major role in subcellular organelle transport and cytoplasmic streaming. Previous studies have revealed that myosin-driven transport and the actin cytoskeleton play essential roles in plant cell growth. Recent data have indicated that the actin-myosin XI cytoskeleton is essential for not only cell growth but also reproductive processes and responses to the environment. In this review, we have summarized previous reports regarding the role of the actin-myosin XI cytoskeleton in cytoplasmic streaming and plant development and recent advances in the understanding of the functions of actin-myosin XI cytoskeleton in Arabidopsis thaliana.

Cell division is a fundamental biological process that has been extensively investigated in different systems. Similar to most eukaryotic cells, plant cells assemble a mitotic spindle to separate replicated chromosomes. In contrast, to complete cell division, plant cells assemble a phragmoplast, which is composed of aligned microtubules and actin filaments. This structure helps transport vesicles containing new cell wall material, which then fuse to form the cell plate; the cell plate will expand to create the new dividing cell wall. Because vesicles are known to be transported by myosin motors during interphase, we hypothesized this could also be the case during cell division and we investigated the localization of the plant homologue of myosin V - myosin XI, in cell division. In this work, we used the protonemal cells of the moss Physcomitrella patens as a model, because of its simple cellular morphology and ease to generate transgenic cell lines expressing fluorescent tagged proteins. Using a fluorescent protein fusion of myosin XI, we found that, during mitosis, this molecule appears to associate with the kinetochores immediately after nuclear envelope breakdown. Following metaphase, myosin XI stays associated with the spindle's midzone during the rest of mitosis, and when the phragmoplast is formed, it concentrates at the cell plate. Using an actin polymerization inhibitor, latrunculin B, we found that the association of myosin XI with the mitotic spindle and the phragmoplast are only partially dependent on the presence of filamentous actin. We also showed that myosin XI on the spindle partially overlaps with a v-SNARE vesicle marker but is not co-localized with the endoplasmic reticulum and a RabA vesicle marker. These observations suggest an actin-dependent and an actin-independent behavior of myosin XI during cell division, and provide novel insights to our understanding of the function of myosin XI during plant cell division.

Maize mosaic virus (MMV), similar to other nucleorhabdoviruses, replicates in divergent hosts: plants and insects. To compare MMV protein localization and interactions, we visualized autofluorescent protein fusions in both cell types. Nucleoprotein (N) and glycoprotein (G) localized to the nucleus and cytoplasm, phosphoprotein (P) was only found in the nucleus, and 3 (movement) and matrix (M) were present in the cytoplasm. This localization pattern is consistent with the model of nucleorhabdoviral replication of N, P, L and viral RNA forming a complex in the nucleus and the subvirion associating with M and then G during budding into perinuclear space. The comparable localization patterns in both organisms indicates a similar replication cycle. Changes in localization when proteins were co-expressed suggested viral proteins interact thus altering organelle targeting. We documented a limited number of direct protein interactions indicating host factors play a role in the virus protein interactions during the infection cycle.

Genetic engineering of plants has enhanced crop productivity in the face of climate change and a growing global population by conferring desirable genetic traits to agricultural crops. Efficient genetic transformation in plants remains a challenge due to the cell wall, a barrier to exogenous biomolecule delivery. Conventional delivery methods are inefficient, damaging to tissue, or are only effective in a limited number of plant species. Nanoparticles are promising materials for biomolecule delivery, owing to their ability to traverse plant cell walls without external force and highly tunable physicochemical properties for diverse cargo conjugation and broad host range applicability. With the advent of engineered nuclease biotechnologies, we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering.

Manipulation in the light regimes combined with the effects of plant growth regulators (PGRs) and elicitors through plant cell culture technology is a promising strategy for enhancing the yield of medicinally important secondary metabolites. In this study, the effects of interplay between PGRs, elicitors and light regimes on cell cultures of F. indica have been investigated. The results showed that callus cultures resulted in maximum biomass formation (13.2 g/L) when incubated on solid MS (Murashige and Skoog) medium containing 1.0 mg/L BA under continuous light for 4 weeks. Among the other PGRs, compared with the auxins such as 2,4-D, and IBA, TDZ resulted in higher biomass accumulation (12.1 g/L). Elicitors (Me-J and PAA) resulted in a lower growth response, when compared with cytokinins and a higher response than auxins under all the light regimes on solid MS media. However, in liquid media, no significant increase in biomass was observed in response to the combined effects of PGRs and photoperiod regimes. Further, the highest phenolic content (TPC = 6.8 mg) and flavonoid content (TFC = 5.2 mg) were detected in the dark-grown cell cultures raised in vitro at 0.5 mg/L Me-J. The highest antioxidant activity (88%) was recorded in the dark-grown cell cultures harvested from LOG phase of the growth cycle supplemented with 0.5 mg/L Me-J. Furthermore, BA resulted in considerable flavonoids production (TFC = 4.7 mg) in the cell cultures grown under continuous light. However, overall dark treatment and elicitation with Me-J resulted in the optimal metabolic response in terms of secondary metabolites accumulation in cell suspension cultures of F. indica.

Light is an important physical factor necessary for the growth, morphogenesis and production of bioactive compounds in plants. In this study, effects of different photoperiod regimes and hormonal elicitors were investigated on the accumulation of biomass, antioxidant potential and biosynthesis of secondary volatiles in the cell cultures of Ajuga bracteosa. Maximum accumulation of biomass (13.2 g/L) was recorded in cell cultures established at 1.0 mg/L benzylaminopurine (BA) in exposure to continuous dark. Biochemical assays showed that in the presence of 0.5 methyl jasmonate (Me-J), cell cultures grown under continuous dark had the higher activities of superoxide dismutase (SOD: 4.5 U/mg), peroxidase (POD: 3.1 U/mg), total phenolic content (TPC: 8.1 mg GAE/g of DW) and total flavonoid content (TFC: 5.2 mg QE/g of DW) respectively. Nonetheless, the free radical scavenging activity (FRSA) was found correlated with the phenyl ammonia lyase (PAL) activity in the dark grown cell cultures. Analysis through gas chromatography-mass spectrometry (GC–MS) showed, biosynthesis of 29 compounds in the in vitro raised cell cultures. The major identified compounds consisted of monoterpene hydrocarbons such as β-pinene (2.1–9.5%), β-ocimene (1.4–8.3%), 1-terpinene-4-ol (5.8–9.6%), caryophyllene (1.3–6.2%), β-farnesene (0.82–7.8), oxygenated monoterpenes including myrtenal (2.2–8.4%), citronellyl acetate (2.1–7.3%) and sesquiterpenes such as caryophyllene oxide (1.5–5.5) and β-elemene (2.2–8.8%). This protocol has the potential for commercial production of important secondary volatiles.

Stilbenes are naturally scarce high-added-value plant compounds with chemopreventive, pharmacological and cosmetic properties. Bioproduction strategies include engineering the metabolisms of bacterial, fungal and plant cell systems. Strikingly, one of the most effective strategies consists in the elicitation of wild grapevine cell cultures, which leads to vast stilbene resveratrol accumulation in the extracellular medium. The combination of both cell culture elicitation and metabolic engineering strategies to produce resveratrol analogs proved more efficient for the hydroxylated derivative piceatannol than for the dimethylated derivative pterostilbene, for which human hydroxylase HsCYP1B1- and grapevine O-methyltransferase VvROMT-transformed cell cultures were respectively used. Rose orcinol O-methyltransferase (OOMT) displays enzymatic properties, which makes it an appealing candidate to substitute VvROMT in the combined strategy to enhance the pterostilbene production level by engineered grapevine cells upon elicitation. Here we cloned a Rosa hybrida OOMT gene, and created a genetic construction suitable for Agrobacterium-mediated plant transformation. OOMT’s ability to catalyze the conversion of resveratrol into pterostilbene was first assessed in vitro using protein extracts of agroinfiltrated N. benthamiana leaves and transformed grapevine callus. The grapevine cell cultures transformed with RhOOMT produced about 16 mg/L culture of pterostilbene and reached an extracellular distribution of up to 34% of total production at the best, which is by far the highest production reported to date in a plant system. A bonus large resveratrol production of ca. 1500–3000 mg/L was simultaneously obtained. Our results demonstrate a viable successful metabolic engineering strategy to produce pterostilbene, a resveratrol analog with enhanced pharmacological properties. This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2014-51861-R and BIO2017-82374-R), The Spanish Ministry of Economy and Competitiveness (PEJ-2014-A-90762/PEJ-2014-P-00289), Generalitat de Catalunya (2014SGR215) and European Funds for Regional Development (FEDER).

Bacteriophage T7 promoter and RNA polymerase (T7-Pol) are widely used for recombinant protein expression in bacteria. In plants, there exists conflicting results regarding the efficacy of protein expression from T7-Pol-derived mRNAs. To reconcile these contradictory observations, the expression of green fluorescent protein (GFP) from T7 constructs was evaluated in tobacco protoplasts. T7 constructs transcribed by a nuclearly targeted T7-Pol did not express GFP in plant protoplasts, however T7-Pol lacking a nuclear targeting signal was able to translate cytosolically transcribed mRNAs, but only if the messages contained a viral translation enhancer. GFP expression was further evaluated at the plant level by using agroinfiltration-mediated transient expression system. Unlike for cytosolic expression, nuclear T7 transcripts containing a viral translation enhancer element did not express GFP, and modifications designed to stabilize and facilitate export of T7 transcripts to the cytosol did not improve the expression. We conclude that expression of nuclear T7 constructs is not feasible in tobacco cells, but cytosolic transcription provides an alternative means to over-express RNAs directly in the cytosol.