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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.

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.

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.

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 μm

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.

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.

Identifying the direct targets of GRF1 and GRF3 provided new insights into their roles in mediating growth–defense antagonism and integrating environmental stimuli into developmental processes., Growth-regulating factors (GRFs) belong to a small family of transcription factors that are highly conserved in plants. GRFs regulate many developmental processes and plant responses to biotic and abiotic stimuli. Despite the importance of GRFs, a detailed mechanistic understanding of their regulatory functions is still lacking. In this study, we used ChIP sequencing (ChIP-seq) to identify genome-wide binding sites of Arabidopsis GRF1 and GRF3, and correspondingly their direct downstream target genes. RNA-sequencing (RNA-seq) analysis revealed that GRF1 and GRF3 regulate the expression of a significant number of the identified direct targets. The target genes unveiled broad regulatory functions of GRF1 and GRF3 in plant growth and development, phytohormone biosynthesis and signaling, and the cell cycle. Our analyses also revealed that clock core genes and genes with stress- and defense-related functions are most predominant among the GRF1- and GRF3-bound targets, providing insights into a possible role for these transcription factors in mediating growth–defense antagonism and integrating environmental stimuli into developmental programs. Additionally, GRF1 and GRF3 target molecular nodes of growth–defense antagonism and modulate the levels of defense- and development-related hormones in opposite directions. Taken together, our results point to GRF1 and GRF3 as potential key determinants of plant fitness under stress conditions.

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.

Plasmodesmata (PD) allow membrane and cytoplasmic continuity between plant cells, and they are essential for intercellular communication and signaling in addition to metabolite partitioning. Plant pathogens have evolved a variety of mechanisms to subvert PD to facilitate their infection of plant hosts. PD are implicated not only in local spread around infection sites but also in the systemic spread of pathogens and pathogen-derived molecules. In turn, plants have developed strategies to limit pathogen spread via PD, and there is increasing evidence that PD may also be active players in plant defense responses. The last few years have seen important advances in understanding the roles of PD in plant-pathogen infection. Nonetheless, several critical areas remain to be addressed. Here we highlight some of these, focusing on the need to consider the effects of pathogen-PD interaction on the trafficking of endogenous molecules, and the involvement of chloroplasts in regulating PD during pathogen defense. By their very nature, PD are recalcitrant to most currently used investigative techniques, therefore answering these questions will require creative imaging and novel quantification approaches.

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.

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 thePNPasehomologues, 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'.

Soybean nodulation is a highly controlled process that involves complex gene regulation at both transcriptional and post-transcriptional levels. In the present study, we profiled gene expression changes, alternative splicing events, and DNA methylation patterns during nodule formation, development, and senescence. The transcriptome data uncovered key transcription patterns of nodule development that included 9669 core genes and 7302 stage-specific genes. Alternative splicing analysis uncovered a total of 2323 genes that undergo alternative splicing events in at least one nodule developmental stage, with activation of exon skipping and repression of intron retention being the most common splicing events in nodules compared to roots. Approximately 40% of the differentially spliced genes were also differentially expressed at the same nodule developmental stage, implying a substantial association between gene expression and alternative splicing. Genome-wide-DNA methylation analysis revealed dynamic changes in nodule methylomes that were specific to each nodule stage, occurred in a sequence-specific manner, and impacted the expression of 1864 genes. An attractive hypothesis raised by our data is that increased DNA methylation may contribute to the efficiency of alternative splicing. Together, our results provide intriguing insights into the associations between gene expression, alternative splicing, and DNA methylation that may shape transcriptome complexity and proteome specificity in developing soybean nodules.

It is now widely accepted that plant RNAs can have effects at sites far away from their sites of synthesis. Cellular mRNA transcripts, endogenous small RNAs and defense-related small RNAs all move from cell to cell via plasmodesmata (PD), and may even move long distances in the phloem. Despite their small size, PD have complicated substructures, and the area of the pore available for RNA trafficking can be remarkably small. The intent of this review is to bring into focus the role of PD in cell-to-cell and long distance communication in plants. We consider how cellular RNAs could move through the cell to the PD and thence through PD. The protein composition of PD and the possible roles of PD proteins in RNA trafficking are also discussed. Recent evidence for RNA metabolism in organelles acting as a factor in controlling PD flux is also presented, highlighting new aspects of plant intra- and intercellular communication. It is clear that while the phenomenon of RNA mobility is common and essential, many questions remain, and these have been highlighted throughout this review.

Plastids are critical organelles in plant cells that perform diverse functions and are central to many metabolic pathways. Beyond their major roles in primary metabolism, of which their role in photosynthesis is perhaps best known, plastids contribute to the biosynthesis of phytohormones and other secondary metabolites, store critical biomolecules, and sense a range of environmental stresses. Accordingly, plastid-derived signals coordinate a host of physiological and developmental processes, often by emitting signalling molecules that regulate the expression of nuclear genes. Several excellent recent reviews have provided broad perspectives on plastid signalling pathways. In this review, we will highlight recent advances in our understanding of chloroplast signalling pathways. Our discussion focuses on new discoveries illuminating how chloroplasts determine life and death decisions in cells and on studies elucidating tetrapyrrole biosynthesis signal transduction networks. We will also examine the role of a plastid RNA helicase, ISE2, in chloroplast signalling, and scrutinize intriguing results investigating the potential role of stromules in conducting signals from the chloroplast to other cellular locations.

The chloroplast-resident RNA helicase ISE2 (INCREASED SIZE EXCLUSION LIMIT2) can modulate the formation and distribution of plasmodesmata and intercellular trafficking. We have determined that ISE2 expression is induced by viral infection. Therefore, the responses of Nicotiana benthamiana plants with varying levels of ISE2 expression to infection by Tobacco mosaic virus and Turnip mosaic virus were examined. Surprisingly, increased or decreased ISE2 expression led to faster viral systemic spread and, in some cases, enhanced systemic necrosis. The contributions of RNA silencing and hormone-mediated immune responses to the increased viral susceptibility of these plants were assessed. In addition, Arabidopsis thaliana plants with increased ISE2 expression were found to be more susceptible to infection by the beet cyst nematode Heterodera schachtii. Our analyses provide intriguing insights into unexpected functional roles of a chloroplast protein in mediating plant-pathogen interactions. The possible roles of plasmodesmata in determining the outcomes of these interactions are also discussed.

INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is a chloroplast-localized RNA helicase that is indispensable for proper plant development. Chloroplasts in leaves with reduced ISE2 expression have previously been shown to exhibit reduced thylakoid contents and increased stromal volume, indicative of defective development. It has recently been reported that ISE2 is required for the splicing of group II introns from chloroplast transcripts. The current study extends these findings, and presents evidence for ISE2's role in multiple aspects of chloroplast RNA processing beyond group II intron splicing. Loss of ISE2 from Arabidopsis thaliana leaves resulted in defects in C-to-U RNA editing, altered accumulation of chloroplast transcripts and chloroplast-encoded proteins, and defective processing of chloroplast ribosomal RNAs. Potential ISE2 substrates were identified by RNA immunoprecipitation followed by next-generation sequencing (RIP-seq), and the diversity of RNA species identified supports ISE2's involvement in multiple aspects of chloroplast RNA metabolism. Comprehensive phylogenetic analyses revealed that ISE2 is a non-canonical Ski2-like RNA helicase that represents a separate sub-clade unique to green photosynthetic organisms, consistent with its function as an essential protein. Thus ISE2's evolutionary conservation may be explained by its numerous roles in regulating chloroplast gene expression.

Thioredoxin (Trx) f and NADPH-dependent Trx reductase C (NTRC) have both been proposed as major redox regulators of starch metabolism in chloroplasts. However, little is known regarding the specific role of each protein in this complex mechanism. To shed light on this point, tobacco plants that were genetically engineered to overexpress the NTRC protein from the chloroplast genome were obtained and compared to previously generated Trx f-overexpressing transplastomic plants. Likewise, we investigated the impact of NTRC and Trx f deficiency on starch metabolism by generating Nicotiana benthamiana plants that were silenced for each gene. Our results demonstrated that NTRC overexpression induced enhanced starch accumulation in tobacco leaves, as occurred with Trx f. However, only Trx f silencing leads to a significant decrease in the leaf starch content. Quantitative analysis of enzyme activities related to starch synthesis and degradation were determined in all of the genotypes. Zymographic analyses were additionally performed to compare the amylolytic enzyme profiles of both transplastomic tobacco plants. Our findings indicated that NTRC overexpression promotes the accumulation of transitory leaf starch as a consequence of a diminished starch turnover during the dark period, which seems to be related to a significant reductive activation of ADP-glucose pyrophosphorylase and/or a deactivation of a putative debranching enzyme. On the other hand, increased starch content in Trx f-overexpressing plants was connected to an increase in the capacity of soluble starch synthases during the light period. Taken together, these results suggest that NTRC and the ferredoxin/Trx system play distinct roles in starch turnover. This research was funded by the by the Spanish Ministry of Science and Innovation, grants number AGL2010-15107 and AGL2016-79868. M.A. was supported by FPU pre-doctoral fellowship from Spanish Ministry of Education, Culture and Sports. T.B.-S. was supported by NSF 1456761.

Transport of salicylic acid is important for systemic immunity and is regulated by osmotic pressure and cuticle permeability., The plant cuticle is often considered a passive barrier from the environment. We show that the cuticle regulates active transport of the defense hormone salicylic acid (SA). SA, an important regulator of systemic acquired resistance (SAR), is preferentially transported from pathogen-infected to uninfected parts via the apoplast. Apoplastic accumulation of SA, which precedes its accumulation in the cytosol, is driven by the pH gradient and deprotonation of SA. In cuticle-defective mutants, increased transpiration and reduced water potential preferentially routes SA to cuticle wax rather than to the apoplast. This results in defective long-distance transport of SA, which in turn impairs distal accumulation of the SAR-inducer pipecolic acid. High humidity reduces transpiration to restore systemic SA transport and, thereby, SAR in cuticle-defective mutants. Together, our results demonstrate that long-distance mobility of SA is essential for SAR and that partitioning of SA between the symplast and cuticle is regulated by transpiration.

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.

Chloroplasts retain part of their ancestral genomes and the machinery for expression of those genomes. The nucleus-encoded chloroplast RNA helicase INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is required for chloroplast ribosomal RNA processing and chloro-ribosome assembly. To further elucidate ISE2's role in chloroplast translation, two independent approaches were used to identify its potential protein partners. Both a yeast two-hybrid screen and a pull-down assay identified plastid ribosomal protein L15, uL15c (formerly RPL15), as interacting with ISE2. The interaction was confirmed in vivo by co-immunoprecipitation. Interestingly, we found that rpl15 null mutants do not complete embryogenesis, indicating that RPL15 is an essential gene for autotrophic growth of Arabidopsis thaliana. Arabidopsis and Nicotiana benthamiana plants with reduced expression of RPL15 developed chlorotic leaves, had reduced photosynthetic capacity and exhibited defective chloroplast development. Processing of chloroplast ribosomal RNAs and assembly of ribosomal subunits were disrupted by reduced expression of RPL15. Chloroplast translation was also decreased, reducing accumulation of chloroplast-encoded proteins, in such plants compared to wild-type plants. Notably, knockdown of RPL15 expression increased intercellular trafficking, a phenotype also observed in plants with reduced ISE2 expression. This finding provides further evidence for chloroplast function in modulating intercellular trafficking via plasmodesmata.

DNA methylation is a dynamic and reversible type of epigenetic mark that contributes to cellular physiology by affecting transcription activity, transposon mobility and genome stability. When plants are infected with pathogens, plant DNA methylation patterns can change, indicating an epigenetic interplay between plant host and pathogen. In most cases methylation can change susceptibility. While DNA hypomethylation appears to be a common phenomenon during the susceptible interaction, the levels and patterns of hypomethylation in transposable elements and genic regions may mediate distinct responses against various plant pathogens. The effect of DNA methylation on the plant immune response and other cellular activities and molecular functions is established by localized differential DNA methylation via cis-regulatory mechanisms as well as through trans-acting mechanisms. Understanding the epigenetic differences that control the phenotypic variations between susceptible and resistant interactions should facilitate the identification of new sources of resistance mediated by epigenetic mechanisms, which can be exploited to endow pathogen resistance to crops.

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa and 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. We conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.

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. Additional content is available on the Focus on Cell Biology of Virus-Plant and Virus-Vector Interactions. New Technologies for Studying Negative-Strand RNA Viruses in Plant and Arthropod Hosts A Non-Conserved p33 Protein of Citrus Tristeza Virus Interacts with Multiple Viral Partners

Plant cells are surrounded by cellulosic cell walls, creating a potential challenge to resource sharing and information exchange between individual cells. To overcome this, plants have evolved channels called plasmodesmata that provide cytoplasmic continuity between each cell and its immediate neighbors. We first review plasmodesmata basics—their architecture, their origin, the types of cargo they transport, and their molecular components. The bulk of this review discusses the regulation of plasmodesmata formation and function. Historically, plasmodesmata research has focused intensely on uncovering regulatory or structural proteins that reside within or immediately adjacent to plasmodesmata. Recent findings, however, underscore that plasmodesmata are exquisitely sensitive to signals far removed from the plasmodesmal channel itself. Signals originating from molecules and pathways that regulate cellular homeostasis—such as reactive oxygen species, organelle-organelle signaling, and organelle-nucleus signaling—lead to astonishing alterations in gene expression that affect plasmodesmata formation and function.

In plants, plasmodesmata (PD) serve as channels for micromolecular and macromolecular cell-to-cell transport. Based on structure, PD in immature tissues are classified into two types, simple and branched (X- and Y-shaped) or twinned. The maximum size of molecules capable of PD transport defines PD aperture, known as the PD size exclusion limit. Here we report an Arabidopsis mutation, decreased size exclusion limit1 ( dse1 ), that exhibits reduced cell-to-cell transport of the small (524 Da) fluorescent tracer 8-hydroxypyrene-1,3,6-trisulfonic acid at the midtorpedo stage of embryogenesis. Correspondingly, the fraction of X- and Y-shaped and twinned PD was reduced in dse1 embryos compared with WT embryos at this stage, suggesting that the frequency of PD is related to transport capability. dse1 is caused by a point mutation in At4g29860 (previously termed TANMEI ) at the last donor splice site of its transcript, resulting in alternative splicing in both the first intron and the last intron. AtDSE1 is a conserved eukaryotic 386-aa WD-repeat protein critical for Arabidopsis morphogenesis and reproduction. Similar to its homologs in mouse, null mutants are embryo-lethal. The weak loss-of-function mutant dse1 exhibits pleiotropic phenotypes, including retarded vegetative growth, delayed flowering time, dysfunctional male and female organs, and delayed senescence. Finally, silencing of DSE1 in Nicotiana benthamiana leaves leads to reduced movement of GFP fused to tobacco mosaic virus movement protein. Thus, DSE1 is important for regulating PD transport between plant cells.

Plasmodesmata (PD) structure and function vary temporally and spatially during all stages of plant development. PD that originate during, or post, cell division are designated as primary or secondary according to classical terminology. PD structure may be simple, twinned, or branched. Studies of PD during leaf, root, and embryo development have lead to the generalization that cells in less mature tissues contain predominantly simple PD. New quantitative analyses reveal that twinned and branched PD also occur in immature tissues. New data also highlight the versatility of viral movement proteins as tags for labeling PD in immature tissues as well as PD in mature tissues. A summary of the formation and function of primary, secondary, and branched PD during leaf, trichome, embryo, apical meristem, vascular cambium, and root development underscores the remarkable and indispensible plant-specific intercellular communication system that is mediated by PD.

Summary Plasmodesmata (PD) transport developmentally important nucleic acids and proteins between plant cells [1–3]. Primary PD form during cell division and are simple, linear channels [4, 5]. Secondary PD form in existing cell walls after cell division and are simple, twinned, or branched [4]. PD function undergoes a marked reduction at the mid-torpedo stage of Arabidopsis embryogenesis [6]. Two mutants, increased size exclusion limit ( ise ) 1 and ise2, fail to undergo this transition, and their null mutations are embryonically lethal [7, 8]. We investigated the ultrastructure of PD in early-, mid-, and late-torpedo-stage embryos and in young leaves. Wild-type (WT) embryos contain twinned and branched (T/B) PD at all stages, but ise1 and ise2 embryos contain significantly higher proportions of T/B PD than WT embryos. WT T/B PD formation occurs in a stage- and tissue-specific pattern that is reversed in ise1 embryos. Silencing ISE1 in Nicotiana benthamiana leaves increases the frequency of secondary PD in existing cell walls. Silencing ISE2 increases the proportion of T/B secondary PD formed. Silenced tissues exhibit increased PD-mediated movement of green fluorescent protein tracers. Thus, silencing of ISE1 and ISE2 phenocopies ise1 and ise2 mutant embryos: when wild-type ISE1 and ISE2 functions are lost, de novo production of PD occurs, leading to increased intercellular transport.

Plant cells are encased in cellulose precluding direct contact. To enable intercellular communication, plants evolved cell wall-spanning channels called plasmodesmata. Plasmodesmata are essential to facilitate transport of small molecules such as photosynthate, as well as critical signaling macromolecules such as transcription factors and RNAs. Plasmodesmata are indispensible for all stages of plant development, from embryogenesis, through vegetative and reproductive development. Plasmodesmata are not passive channels, but instead they are highly dynamic and change their apertures in response to intracellular signals such as reactive oxygen species, hormones, and chloroplast and mitochondrial homeostasis. To date the best-known mechanism for controlling the degree of plasmodesmata transport is the reversible deposition of callose polysaccharides in the cell wall immediately surrounding plasmodesmata channels. Plant viruses have evolved to counteract innate plasmodesmata regulatory mechanisms and are well-known pirates of plasmodesmata during infectious spread.

Plant innate immunity is mediated by Resistance (R) proteins, which bear a striking resemblance to animal molecules of similar function. Tobacco N is a TIR-NB-LRR R gene that confers resistance to Tobacco mosaic virus, specifically the p50 helicase domain. An intriguing question is how plant R proteins recognize the presence of pathogen-derived Avirulence (Avr) elicitor proteins. We have used biochemical cell fraction and immunoprecipitation in addition to confocal fluorescence microscopy of living tissue to examine the association between N and p50. Surprisingly, both N and p50 are cytoplasmic and nuclear proteins, and N's nuclear localization is required for its function. We also demonstrate an in planta association between N and p50. Further, we show that N's TIR domain is critical for this association, and indeed, it alone can associate with p50. Our results differ from current models for plant innate immunity that propose detection is mediated solely through the LRR domains of these molecules. The data we present support an intricate process of pathogen elicitor recognition by R proteins involving multiple subcellular compartments and the formation of multiple protein complexes.

The most conspicuous function of plastids is the oxygenic photosynthesis of chloroplasts, yet plastids are super-factories that produce a plethora of compounds that are indispensable for proper plant physiology and development. Given their origins as free-living prokaryotes, it is not surprising that plastids possess their own genomes whose expression is essential to plastid function. This semi-autonomous character of plastids requires the existence of sophisticated regulatory mechanisms that provide reliable communication between them and other cellular compartments. Such intracellular signaling is necessary for coordinating whole-cell responses to constantly varying environmental cues and cellular metabolic needs. This is achieved by plastids acting as receivers and transmitters of specific signals that coordinate expression of the nuclear and plastid genomes according to particular needs. In this review we will consider the so-called retrograde signaling occurring between plastids and nuclei, and between plastids and other organelles. Another important role of the plastid we will discuss is the involvement of plastid signaling in biotic and abiotic stress that, in addition to influencing retrograde signaling, has direct effects on several cellular compartments including the cell wall. We will also review recent evidence pointing to an intriguing function of chloroplasts in regulating intercellular symplasmic transport. Finally, we consider an intriguing yet less widely known aspect of plant biology, chloroplast signaling from the perspective of the entire plant. Thus, accumulating evidence highlights that chloroplasts, with their complex signaling pathways, provide a mechanism for exquisite regulation of plant development, metabolism and responses to the environment. As chloroplast processes are targeted for engineering for improved productivity the effect of such modifications on chloroplast signaling will have to be carefully considered in order to avoid unintended consequences on plant growth and development.

Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like Nicotiana benthamiana and tomato (Lycopersicon esculentum). The method described here gives efficient silencing in Arabidopsis ecotype Columbia-0. We show that VIGS can be used to silence genes involved in general metabolism and defense and it is also effective at knocking down expression of highly expressed transgenes. A marker system to monitor the progress and efficiency of VIGS is also described.

Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes.

SGT1 and Rar1 are important signaling components of resistance (R) gene-mediated plant innate immune responses. Here we report that SGT1 and Rar1 associate with the molecular chaperone Hsp90. In addition, we show that Hsp90 associates with the resistance protein N that confers resistance to tobacco mosaic virus. This suggests that Hsp90-SGT1-Rar1 and R proteins might exist in one complex. Suppression of Hsp90 in Nicotiana benthamiana plants shows that it plays an important role in plant growth and development. In addition, Hsp90 suppression in NN plants compromises N-mediated resistance to tobacco mosaic virus. Our results reveal a new role for SGT1- and Rar1-associated chaperone machinery in R gene-mediated defense signaling.

Analysis of cellular ultrastructure has been dominated by transmission electron microscopy (TEM), so images collected by this technique have shaped our current understanding of cellular structure. More recently, three-dimensional (3D) analysis of organelle structures has typically been conducted using TEM tomography. However, TEM tomography application is limited by sample thickness. Focused ion beam-scanning electron microscopy (FIB-SEM) uses a dual beam system to perform serial sectioning and imaging of a sample. Thus FIB-SEM is an excellent alternative to TEM tomography and serial section TEM tomography. Animal tissue samples have been more intensively investigated by this technique than plant tissues. Here, we show that FIB-SEM can be used to study the 3D ultrastructure of plant tissues in samples previously prepared for TEM via commonly used fixation and embedding protocols. Reconstruction of FIB-SEM sections revealed ultra-structural details of the plant tissues examined. We observed that organelles packed tightly together in Nicotiana benthamiana Domin leaf cells may form membrane contacts. 3D models of soybean nodule cells suggest that the bacteroids in infected cells are contained within one large membrane-bound structure and not the many individual symbiosomes that TEM thin-sections suggest. We consider the implications of these organelle arrangements for intercellular signalling.

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

We use Arabidopsis thaliana embryogenesis as a model system for studying intercellular transport via plasmodesmata (PD). A forward genetic screen for altered PD transport identified increased size exclusion limit ( ise) 1 and ise2 mutants with increased intercellular transport of fluorescent 10-kDa tracers. Both ise1 and ise2 exhibit increased formation of twinned and branched PD. ISE1 encodes a mitochondrial DEAD-box RNA helicase, whereas ISE2 encodes a DEVH-type RNA helicase. Here, we show that ISE2 foci are localized to the chloroplast stroma. Surprisingly, plastid development is defective in both ise1 and ise2 mutant embryos. In an effort to understand how RNA helicases that localize to different organelles have similar impacts on plastid and PD development/function, we performed whole-genome expression analyses. The most significantly affected class of transcripts in both mutants encode products that target to and enable plastid function. These results reinforce the importance of plastid-mitochondria-nucleus cross-talk, add PD as a critical player in the plant cell communication network, and thereby illuminate a previously undescribed signaling pathway dubbed organelle–nucleus-plasmodesmata signaling. Several genes with roles in cell wall synthesis and modification are also differentially expressed in both mutants, providing new targets for investigating PD development and function.

The Arabidopsis embryo is a single symplast where all its cells allow intercellular communication via plasmodesmata (PD). However, PD apertures are dynamic and alter during embryonic development, both in terms of their general transport capacity within the entire embryo, and in mediating intercellular transport capacity between different regions of the embryo. Endogenous expression of 1X, 2X, and 3XGFP tracers illustrates that early embryos (up to the heart stage) traffic very large molecules such as 2XGFP (54 kDa). The embryo remains a single symplast for even 1XGFP (27 kDa) until the late-torpedo stage. Sub-division of the embryo into different symplastic sub-domains as a function of PD aperture is revealed at the mid-torpedo stage by more restricted movement of larger tracers; 2XGFP cannot move into the cotyledons, and 3XGFP cannot move down into the root tip. Tobacco mosaic virus (TMV) movement protein, P30, moves cell-to-cell via PD in embryos. P30-GFP targets to PD and forms puncta, just as it has been shown to do in older leaf tissues, and P30-GFP (57 kDa) exhibits more movement than similarly sized 2XGFP (54 kDa). As the embryo predominantly contains simple PD (∼90% of embryonic PD are simple) these data demonstrate that P30 also targets to simple (unbranched) PD. Two mutants allowed increased movement of exogenously added 10 kDa F-dextrans, called increased size exclusion limit 1 and ise2, and result in the increased formation of twinned and branched PD. Gene silencing of ISE1 and ISE2 leads to de novo production of secondary PD in young leaves, and concomitant increased cell-to-cell transport. ise1 and ise2 null mutants are lethal attesting to their essential function. ISE1 localizes to mitochondria and ISE2 localizes to chloroplasts. Thus, cellular homeostasis as mediated by organelle function, dramatically affects PD formation and function.

Plants have intercellular channels, plasmodesmata (PD), that span the cell wall to enable cell-to-cell transport of micro- and macromolecules. We identified an Arabidopsis thaliana embryo lethal mutant increased size exclusion limit 1 ( ise1 ) that results in increased PD-mediated transport of fluorescent tracers. The ise1 mutants have a higher frequency of branched and twinned PD than wild-type embryos. Silencing of ISE1 in mature Nicotiana benthamiana leaves also leads to increased PD transport, as monitored by intercellular movement of a GFP fusion to the tobacco mosaic virus movement protein. ISE1 encodes a putative plant-specific DEAD-box RNA helicase that localizes specifically to mitochondria. The N-terminal 100 aa of ISE1 specify mitochondrial targeting. Mitochondrial metabolism is compromised severely in ise1 mutant embryos, because their mitochondrial proton gradient is disrupted and reactive oxygen species production is increased. Although mitochondria are essential for numerous cell-autonomous functions, the present studies demonstrate that mitochondrial function also regulates the critical cell non-cell-autonomous function of PD.

Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are among the most popular for VIGS. We have developed a TRV RNA2 vector that allows the insertion of gene silencing fragments by ligation-independent cloning (LIC). This new vector has several advantages over previous vectors, particularly for applications involving the analysis of large numbers of sequences, since TRV-LIC vectors containing the desired insert are obtained with 100% efficiency. Importantly, this vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination, as is the case with GATEWAY-based vectors. We generated a collection of silencing vectors based on 400 tomato (Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity. This TRV-LIC vector should be a valuable resource to the plant community.