The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts.

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.

Comparing Methods for Detection and Quantification of Plasmodesmal Callose in Nicotiana benthamiana Leaves During Defense Responses.

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.

Roles of ROS and redox in regulating cell-to-cell communication: Spotlight on viral modulation of redox for local spread.

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.

Reliable detection and quantification of plasmodesmal callose in Nicotiana benthamiana leaves during defense responses.

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.

Focus on the Plant Endomembrane System in Molecular Plant-Microbe Interactions.

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.

The dicot homolog of maize PPR103 carries a C-terminal DYW domain and is required for C-to-U editing of chloroplast RNA transcripts.

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.

Mathematical Modeling Suggests Cooperation of Plant-Infecting Viruses.

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.

Focused Ion Beam-Scanning Electron Microscopy for Investigating Plasmodesmal Densities.

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.

Investigating Plasmodesmata Function in Arabidopsis Thaliana Using a Low-Pressure Bombardment System and GFP Movement Assay.

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.

A glossary of plant cell structures: Current insights and future questions.

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.

Approaches for investigating plasmodesmata and effective communication.

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.

An Emerging Role for Chloroplasts in Disease and Defense.

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.

Poly(styrene-co-maleic acid)-mediated isolation of supramolecular membrane protein complexes from plant thylakoids.

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.

Organelles-nucleus-plasmodesmata signaling (ONPS): an update on its roles in plant physiology, metabolism and stress responses.

Plasmodesmata allow movement of metabolites and signaling molecules between plant cells and are, therefore, critical players in plant development and physiology, and in responding to environmental signals and stresses. There is emerging evidence that plasmodesmata are controlled by signaling originating from other organelles, primarily the chloroplasts and mitochondria. These signals act in the nucleus to alter expression of genetic pathways that control both trafficking via plasmodesmata and the plasmodesmatal pores themselves. This control circuit was dubbed organelle-nucleus-plasmodesmata signaling (ONPS). Here we discuss how ONPS arose during plant evolution and highlight the discovery of an ONPS-like module for regulating stomata. We also consider recent findings that illuminate details of the ONPS circuit and its roles in plant physiology, metabolism, and defense.

Chloroplast-to-nucleus retrograde signalling controls intercellular trafficking via plasmodesmata formation.

The signalling pathways that regulate intercellular trafficking via plasmodesmata (PD) remain largely unknown. Analyses of mutants with defects in intercellular trafficking led to the hypothesis that chloroplasts are important for controlling PD, probably by retrograde signalling to the nucleus to regulate expression of genes that influence PD formation and function, an idea encapsulated in the organelle-nucleus-PD signalling (ONPS) hypothesis. ONPS is supported by findings that point to chloroplast redox state as also modulating PD. Here, we have attempted to further elucidate details of ONPS. Through reverse genetics, expression of select nucleus-encoded genes with known or predicted roles in chloroplast gene expression was knocked down, and the effects on intercellular trafficking were then assessed. Silencing most genes resulted in chlorosis, and the expression of several photosynthesis and tetrapyrrole biosynthesis associated nuclear genes was repressed in all silenced plants. PD-mediated intercellular trafficking was changed in the silenced plants, consistent with predictions of the ONPS hypothesis. One striking observation, best exemplified by silencing the PNPase homologues, was that the degree of chlorosis of silenced leaves was not correlated with the capacity for intercellular trafficking. Finally, we measured the distribution of PD in silenced leaves and found that intercellular trafficking was positively correlated with the numbers of PD. Together, these results not only provide further support for ONPS but also point to a genetic mechanism for PD formation, clarifying a longstanding question about PD and intercellular trafficking. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Focus on Cell Biology of Virus-Plant and Virus-Vector Interactions.

A successful viral infection requires complex, compatible molecular interactions between the invading virus and the host. A better understanding of such interactions may assist in the development of novel approaches to control viral diseases for sustainable crop production. In the past decade, the cell biology of virus-host and virus-vector interactions has been one of the most exciting areas of research in the molecular plant-microbe field. This is partially attributed to the availability of powerful cell biology techniques, including imaging tools like confocal microscopy and electron microscopy and tomography. As a result, there has been an unprecedented increase in knowledge in the areas of the bi- and tripartite interactions of virus, host, and vector. We now have a much clearer picture of viral virulence mechanisms, virus-induced host defenses, viral counteracting strategies, and viral circulations in the insect vectors. This Focus Issue highlights molecular virus-plant and virus-vector interactions in the areas of cell biology and closely related disciplines and explores biotechnology-based antiviral strategies using knowledge generated from these research areas.

Viruses Reveal the Secrets of Plasmodesmal Cell Biology.

Plasmodesmata (PD) are essential for intercellular trafficking of molecules required for plant life, from small molecules like sugars and ions to macromolecules including proteins and RNA molecules that act as signals to regulate plant development and defense. As obligate intracellular pathogens, plant viruses have evolved to manipulate this communication system to facilitate the initial cell-to-cell and eventual systemic spread in their plant hosts. There has been considerable interest in how viruses manipulate the PD that connect the protoplasts of neighboring cells, and viruses have yielded invaluable tools for probing the structure and function of PD. With recent advances in biochemistry and imaging, we have gained new insights into the composition and structure of PD in the presence and absence of viruses. Here, we first discuss viral strategies for manipulating PD for their intercellular movement and examine how this has shed light on our understanding of native PD function. We then address the controversial role of the cytoskeleton in trafficking to and through PD. Finally, we address how viruses could alter PD structure and consider possible mechanisms of the phenomenon described as 'gating'. This discussion supports the significance of virus research in elucidating the properties of PD, these persistently enigmatic plant organelles.

Chloroplasts as mediators of plant biotic interactions over short and long distances.

In nature, plants interact with numerous other organisms. Some interactions benefit both the plant and the other organism(s), while others lead to disease or even death of the plant hosts. The traditional focus of research into plant biotic interactions has been on the negative effects on plants and the strategies plants use to mitigate or prevent these. Over the last several years there has been increasing appreciation for the diversity and importance of plant biotic interactions in plant success as well as the evolution and stabilization of ecosystems. With this new perspective, it is also becoming clear that the metabolic output of chloroplasts in plants is critical to establishing and maintaining these interactions. Here we highlight the roles of chloroplasts in diverse biotic interactions. Photosynthetic chloroplasts are the source of hormones, small molecules and a prodigious number of secondary metabolites, a significant portion of which influence plant biotic interactions. Importantly, the effects of chloroplasts on these interactions are not limited to sites of direct association or contact but also act at a distance in systemic leaves and roots, in the rhizosphere, in the air surrounding a plant and in neighboring plants, and they can persist over several years.