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.

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.

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.

Viral synergism suppresses R gene-mediated resistance by impairing downstream defense mechanisms in soybean.

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.

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.

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.

Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans.

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.

Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector.

Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50 kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a prerecognition complex containing the p50 effector and NRIP1.

Interactions of gene expression, alternative splicing, and DNA methylation in determining nodule identity.

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.

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.

A role for Arabidopsis growth-regulating factors 1 and 3 in growth-stress antagonism.

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.

The essential chloroplast ribosomal protein uL15c interacts with the chloroplast RNA helicase ISE2 and affects intercellular trafficking through plasmodesmata.

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.

Correction: A Novel Role for the TIR Domain in Association with Pathogen-Derived Elicitors.

[This corrects the article DOI: 10.1371/journal.pbio.0050068.].

The chloroplast RNA helicase ISE2 is required for multiple chloroplast RNA processing steps in Arabidopsis thaliana.

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.

Phytopathogen-induced changes to plant methylomes.

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.

Altered Expression of a Chloroplast Protein Affects the Outcome of Virus and Nematode Infection.

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.