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Intercellular communication in plants, as in other multicellular organisms, allows cells in tissues to coordinate their responses for development and in response to environmental stimuli. Much of this communication is facilitated by plasmodesmata (PD), consisting of membranes and cytoplasm, that connect adjacent cells to each other. PD have long been viewed as passive conduits for the movement of a variety of metabolites and molecular cargoes, but this perception has been changing over the last two decades or so. Research from the last few years has revealed the importance of PD as signaling hubs and as crucial players in hormone signaling. The adoption of advanced biochemical approaches, molecular tools and high-resolution imaging modalities have led to several recent breakthroughs in our understanding of the roles of PD, revealing the structural and regulatory complexity of these 'protoplasmic connecting threads'. We highlight several of these findings that we think well illustrate the current understanding of PD as functioning at the nexus of plant physiology, development, and acclimation to the environment.
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In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in N. benthamiana to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the Nicotiana and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of NbIPI1 led to defects in chloroplast ribosomal RNA processing and changes to stability of rpl16 transcripts, revealing conserved function with its maize ortholog. NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in N. benthamiana chloroplasts.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , RNA de Cloroplastos/metabolismo , Proteínas de Arabidopsis/genética , Zea mays/genética , Zea mays/metabolismo , RNA , Cloroplastos/genética , Cloroplastos/metabolismoRESUMO
Callose, a ß-(1,3)-d-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD or, conversely, by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing likely intercellular trafficking activity during plant immunity. Despite the popularity of this metric, there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying plasmodesmal callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescence microscopy to measure callose deposition in fixed tissue. Manual or semiautomated workflows for image analysis were also compared and found to produce similar results, although the semiautomated workflow produced a wider distribution of data points. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Glucanos , Nicotiana , Doenças das Plantas , Folhas de Planta , Plasmodesmos , Glucanos/metabolismo , Nicotiana/metabolismo , Plasmodesmos/metabolismo , Folhas de Planta/metabolismo , Doenças das Plantas/microbiologia , Compostos de Anilina/metabolismo , Imunidade Vegetal , Coloração e Rotulagem/métodosRESUMO
Reactive oxygen species (ROS) are important signalling molecules that influence many aspects of plant biology. One way in which ROS influence plant growth and development is by modifying intercellular trafficking through plasmodesmata (PD). Viruses have evolved to use PD for their local cell-to-cell spread between plant cells, so it is therefore not surprising that they have found ways to modulate ROS and redox signalling to optimise PD function for their benefit. This review examines how intracellular signalling via ROS and redox pathways regulate intercellular trafficking via PD during development and stress. The relationship between viruses and ROS-redox systems, and the strategies viruses employ to control PD function by interfering with ROS-redox in plants is also discussed.
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Comunicação Celular , Oxirredução , Plasmodesmos , Espécies Reativas de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Plasmodesmos/metabolismo , Plantas/virologia , Plantas/metabolismo , Vírus de Plantas/fisiologia , Transdução de Sinais , Células Vegetais/virologiaRESUMO
Callose, a beta-(1,3)-D-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD, or conversely by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during innate immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing the intercellular trafficking activity during plant immunity. Despite the popularity of this metric there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying PD callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescent microscopy to measure callose deposition in fixed tissue. Manual or semi-automated workflows for image analysis were also compared and found to produce similar results although the semi-automated workflow produced a wider distribution of data points.
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Interactions between plants and microbes are ubiquitous. The outcomes of these interactions involve interkingdom communication, with myriad, diverse signals moving between microbes and their potential plant hosts. Years of biochemical, genetic, and molecular biology research have provided an overview of the landscape of the repertoires of effectors and elicitors encoded by microbes that allow them to stimulate and manipulate responses from their potential plant hosts. Similarly, considerable insight into the plant machinery and capacity for responding to microbes has been gained. The advent of new bioinformatics and modeling approaches has greatly contributed to our understanding of how these interactions occur, and it is expected that these tools, coupled with burgeoning genome sequencing data, will eventually allow the prediction of the outcome of these interactions and whether they will result in a relationship that benefits one or both partners. As a complement to these studies, cell biological studies are elucidating how cells in the plant hosts behave in response to microbial signals. Such studies have brought new attention to the indispensable role of the plant endomembrane system in determining the outcome of plant-microbe interactions. This Focus Issue addresses not only how the plant endomembrane acts locally to mediate responses to microbes but, also, the importance of the plant endomembrane beyond the plant cell borders for cross-kingdom effects. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.
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Interações entre Hospedeiro e Microrganismos , Plantas , Plantas/microbiologiaRESUMO
Viral synergism occurs when mixed infection of a susceptible plant by 2 or more viruses leads to increased susceptibility to at least 1 of the viruses. However, the ability of 1 virus to suppress R gene-controlled resistance against another virus has never been reported. In soybean (Glycine max), extreme resistance (ER) against soybean mosaic virus (SMV), governed by the Rsv3 R-protein, manifests a swift asymptomatic resistance against the avirulent strain SMV-G5H. Still, the mechanism by which Rsv3 confers ER is not fully understood. Here, we show that viral synergism broke this resistance by impairing downstream defense mechanisms triggered by Rsv3 activation. We found that activation of the antiviral RNA-silencing pathway and the proimmune mitogen-activated protein kinase 3 (MAPK3), along with the suppression of the proviral MAPK6, are hallmarks of Rsv3-mediated ER against SMV-G5H. Surprisingly, infection with bean pod mottle virus (BPMV) disrupted this ER, allowing SMV-G5H to accumulate in Rsv3-containing plants. BPMV subverted downstream defenses by impairing the RNA-silencing pathway and activating MAPK6. Further, BPMV reduced the accumulation of virus-related siRNAs and increased the virus-activated siRNA that targeted several defense-related nucleotide-binding leucine-rich repeat receptor (NLR) genes through the action of the suppression of RNA-silencing activities encoded in its large and small coat protein subunits. These results illustrate that viral synergism can result from abolishing highly specific R gene resistance by impairing active mechanisms downstream of the R gene.
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Glycine max , Potyvirus , Resistência à Doença/genética , Genes vpr , Potyvirus/fisiologia , RNA Interferente Pequeno , RNA de Cadeia Dupla , Mecanismos de Defesa , Doenças das PlantasRESUMO
In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 homologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. We examined the function of ISE2 and IPI1 in chloroplast RNA processing in N. benthamiana. A combination of deep sequencing and Sanger sequencing revealed C-to-U editing at 41 sites in 18 transcripts, with 34 sites conserved in the closely related N. tabacum. Virus induced gene silencing of NbISE2 or NbIPI1 led to defective C-to-U revealed that they have overlapping roles at editing a site in the rpoB transcript but have distinct roles in editing other transcripts. This finding contrasts with maize ppr103 mutants that showed no defects in editing. The results indicate that NbISE2 and NbIPI1 are important for C-to-U editing in N. benthamiana chloroplasts, and they may function in a complex to edit specific sites while having antagonistic effects on editing others. That NbIPI1, carrying a DYW domain, is involved in organelle C-to-U RNA editing supports previous work showing that this domain catalyzes RNA editing.
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Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.
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Arabidopsis , Carbono , Carbono/metabolismo , Glycine max/metabolismo , Sementes/metabolismo , Ácidos Graxos/metabolismo , Arabidopsis/genéticaRESUMO
Viruses are major pathogens of agricultural crops. Viral infections often start after the virus enters the outer layer of a tissue, and many successful viruses, after local replication in the infected tissue, are able to spread systemically. Quantitative details of virus dynamics in plants, however, are poorly understood, in part, because of the lack of experimental methods which allow the accurate measurement of the degree of infection in individual plant tissues. Recently, a group of researchers followed the kinetics of infection of individual cells in leaves of Nicotiana tabacum plants using Tobacco etch virus (TEV) expressing either Venus or blue fluorescent protein (BFP). Assuming that viral spread occurs from lower to upper leaves, the authors fitted a simple mathematical model to the frequency of cellular infection by the two viral variants found using flow cytometry. While the original model could accurately describe the kinetics of viral spread locally and systemically, we found that many alternative versions of the model, for example, if viral spread starts at upper leaves and progresses to lower leaves or when virus dissemination is stopped due to an immune response, fit the data with reasonable quality, and yet with different parameter estimates. These results strongly suggest that experimental measurements of the virus infection in individual leaves may not be sufficient to identify the pathways of viral dissemination between different leaves and reasons for viral control. We propose experiments that may allow discrimination between the alternatives. By analyzing the kinetics of coinfection of individual cells by Venus and BFP strains of TEV we found a strong deviation from the random infection model, suggesting cooperation between the two strains when infecting plant cells. Importantly, we showed that many mathematical models on the kinetics of coinfection of cells with two strains could not adequately describe the data, and the best fit model needed to assume (i) different susceptibility of uninfected cells to infection by two viruses locally in the leaf vs. systemically from other leaves, and (ii) decrease in the infection rate depending on the fraction of uninfected cells which could be due to a systemic immune response. Our results thus demonstrate the difficulty in reaching definite conclusions from extensive and yet limited experimental data and provide evidence of potential cooperation between different viral variants infecting individual cells in plants.
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Coinfecção , Vírus de Plantas , Modelos Teóricos , Doenças das Plantas , Folhas de Planta , Vírus de Plantas/genética , Potyvirus , NicotianaRESUMO
Plasmodesmata (PD) facilitate the exchange of nutrients and signaling molecules between neighboring plant cells, and they are therefore essential for proper growth and development. PD have been studied extensively in efforts to elucidate the ultrastructure of individual PD nanopores and the distribution of PD in a variety of cell walls. These studies often involved the use of serial ultrathin sections and manual quantification of PD by transmission electron microscopy (TEM). In recent years, a variety of techniques that offer more amenable approaches for quantifying PD distribution have been reported. Here, we describe the quantification of PD densities using the serial scanning electron microscopy technique called focused ion beam-scanning electron microscopy (FIB-SEM). For this, resin-embedded samples prepared by standard TEM methods undergo successive rounds of imaging by SEM interspersed with milling of the sample surface by a focused beam of gallium ions to reveal a new surface. In this way, the details of the sample are sequentially revealed and imaged. Over the course of a few hours, repetitive milling and imaging facilitates the automated collection of nanometer-resolution data of several µm of sample depth. FIB-SEM can be targeted to interrogate specific cell walls and cell wall junctions, and the subsequent three-dimensional renderings of the data can be used to visualize the ultrastructural details of the sample. PD densities can then be rapidly quantified by calculating the number of PD per µm2 of cell wall observed in the renderings.
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Parede Celular , Plasmodesmos , Íons , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de TransmissãoRESUMO
Plasmodesmata are nanopores in the plant cell wall that allow direct cell-to-cell communication. They are key for plant growth, development, and defense. However, studying these pores is challenging due to their small size, with diameters of 30-50 nm and lengths that match cell wall thickness. One particular challenge is measuring how much cell-to-cell trafficking is facilitated by the plasmodesmata in a tissue or between particular cells. Here, we present an approach for studying plasmodesmata-mediated trafficking in the model plant Arabidopsis thaliana by using an easy-to-build and affordable low-pressure particle bombardment apparatus. Using low-pressure particle bombardment at around 60 psi, we are able to transform individual cells in the leaf epidermis and study by confocal fluorescence microscopy the subsequent cell-to-cell trafficking of the diffusible molecule green fluorescent protein (GFP). The technique and equipment could be used by any plant biologist to measure intercellular trafficking through plasmodesmata under varying growth conditions including exposure to different stresses, light conditions, chemical treatments, or in various mutant backgrounds.
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Arabidopsis , Plasmodesmos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Folhas de Planta/metabolismo , Plantas/metabolismo , Plasmodesmos/metabolismoRESUMO
In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.
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Membrana Celular/metabolismo , Parede Celular/metabolismo , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Plantas/metabolismo , Organelas/metabolismo , Células Vegetais/metabolismoRESUMO
Plasmodesmata (PD) are integral plant cell wall components that provide routes for intercellular communication, signaling, and resource sharing. They are therefore essential for plant growth and survival. Much effort has been put forth to understand how PD are generated and their structure is refined for function and to determine how they regulate intercellular trafficking. This review provides an overview of some of the approaches that have been used to study PD structure and function, highlighting those that may be more widely adopted to address questions of PD cell biology and function. Extending our focus on the importance of communication, we address how effective communication strategies can increase diversity and accessibility in the research laboratory, focusing on challenges faced by our deaf/hard-of-hearing colleagues, and highlight successful approaches to including them in the research laboratory.
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Parede Celular , Plasmodesmos , Comunicação Celular , Transdução de SinaisRESUMO
Chloroplasts are key players in plant immune signaling, contributing to not only de novo synthesis of defensive phytohormones but also the generation of reactive oxygen and nitrogen species following activation of pattern recognition receptors or resistance (R) proteins. The local hypersensitive response (HR) elicited by R proteins is underpinned by chloroplast-generated reactive oxygen species. HR-induced lipid peroxidation generates important chloroplast-derived signaling lipids essential to the establishment of systemic immunity. As a consequence of this pivotal role in immunity, pathogens deploy effector complements that directly or indirectly target chloroplasts to attenuate chloroplast immunity (CI). Our review summarizes the current knowledge of CI signaling and highlights common pathogen chloroplast targets and virulence strategies. We address emerging insights into chloroplast retrograde signaling in immune responses and gaps in our knowledge, including the importance of understanding chloroplast heterogeneity and chloroplast involvement in intraorganellular interactions in host immunity.
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Cloroplastos , Doenças das Plantas , Reguladores de Crescimento de Plantas , Imunidade Vegetal , Plantas , Transdução de SinaisRESUMO
Derivatives of poly(styrene-co-maleic acid) (pSMA), have recently emerged as effective reagents for extracting membrane protein complexes from biological membranes. Despite recent progress in using SMAs to study artificial and bacterial membranes, very few reports have addressed their use in studying the highly abundant and well characterized thylakoid membranes. Recently, we tested the ability of twelve commercially available SMA copolymers with different physicochemical properties to extract membrane protein complexes (MPCs) from spinach thylakoid membrane. Based on the efficacy of both protein and chlorophyll extraction, we have found five highly efficient SMA copolymers: SMA® 1440, XIRAN® 25010, XIRAN® 30010, SMA® 17352, and SMA® PRO 10235, that show promise in extracting MPCs from chloroplast thylakoids. To further advance the application of these polymers for studying biomembrane organization, we have examined the composition of thylakoid supramolecular protein complexes extracted by the five SMA polymers mentioned above. Two commonly studied plants, spinach (Spinacia oleracea) and pea (Pisum sativum), were used for extraction as model biomembranes. We found that the pSMAs differentially extract protein complexes from spinach and pea thylakoids. Based on their differential activity, which correlates with the polymer chemical structure, pSMAs can be divided into two groups: unfunctionalized polymers and ester derivatives.