α1-COP modulates plasmodesmata function through sphingolipid enzyme regulation.

Callose, a ß-1,3-glucan plant cell wall polymer, regulates symplasmic channel size at plasmodesmata (PD) and plays a crucial role in a variety of plant processes. However, elucidating the molecular mechanism of PD callose homeostasis is limited. We screened and identified an Arabidopsis mutant plant with excessive callose deposition at PD and found that the mutated gene was α1-COP, a member of the coat protein I (COPI) coatomer complex. We report that loss of function of α1-COP elevates the callose accumulation at PD by affecting subcellular protein localization of callose degradation enzyme PdBG2. This process is linked to the functions of ERH1, an inositol phosphoryl ceramide synthase, and glucosylceramide synthase through physical interactions with the α1-COP protein. Additionally, the loss of function of α1-COP alters the subcellular localization of ERH1 and GCS proteins, resulting in a reduction of GlcCers and GlcHCers molecules, which are key sphingolipid (SL) species for lipid raft formation. Our findings suggest that α1-COP protein, together with SL modifiers controlling lipid raft compositions, regulates the subcellular localization of GPI-anchored PDBG2 proteins, and hence the callose turnover at PD and symplasmic movement of biomolecules. Our findings provide the first key clue to link the COPI-mediated intracellular trafficking pathway to the callose-mediated intercellular signaling pathway through PD.

The trichothecene mycotoxin deoxynivalenol facilitates cell-to-cell invasion during wheat-tissue colonization by Fusarium graminearum.

Fusarium head blight disease on small-grain cereals is primarily caused by the ascomycete fungal pathogen Fusarium graminearum. Infection of floral spike tissues is characterized by the biosynthesis and secretion of potent trichothecene mycotoxins, of which deoxynivalenol (DON) is widely reported due to its negative impacts on grain quality and consumer safety. The TRI5 gene encodes an essential enzyme in the DON biosynthesis pathway and the single gene deletion mutant, ΔTri5, is widely reported to restrict disease progression to the inoculated spikelet. In this study, we present novel bioimaging evidence revealing that DON facilitates the traversal of the cell wall through plasmodesmata, a process essential for successful colonization of host tissue. Chemical complementation of ΔTri5 did not restore macro- or microscopic phenotypes, indicating that DON secretion is tightly regulated both spatially and temporally. A comparative qualitative and quantitative morphological cellular analysis revealed infections had no impact on plant cell wall thickness. Immunolabelling of callose at plasmodesmata during infection indicates that DON can increase deposits when applied exogenously but is reduced when F. graminearum hyphae are present. This study highlights the complexity of the interconnected roles of mycotoxin production, cell wall architecture and plasmodesmata in this highly specialized interaction.

C-Type LECTIN receptor-like kinase 1 and ACTIN DEPOLYMERIZING FACTOR 3 are key components of plasmodesmata callose modulation.

Plasmodesmata (PDs) are intercellular organelles carrying multiple membranous nanochannels that allow the trafficking of cellular signalling molecules. The channel regulation of PDs occurs dynamically and is required in various developmental and physiological processes. It is well known that callose is a critical component in regulating PD permeability or symplasmic connectivity, but the understanding of the signalling pathways and mechanisms of its regulation is limited. Here, we used the reverse genetic approach to investigate the role of C-type lectin receptor-like kinase 1 (CLRLK1) in the aspect of PD callose-modulated symplasmic continuity. Here, we found that loss-of-function mutations in CLRLK1 resulted in excessive PD callose deposits and reduced symplasmic continuity, resulting in an accelerated gravitropic response. The protein interactome study also found that CLRLK1 interacted with actin depolymerizing factor 3 (ADF3) in vitro and in plants. Moreover, mutations in ADF3 result in elevated PD callose deposits and faster gravitropic response. Our results indicate that CLRLK1 and ADF3 negatively regulate PD callose accumulation, contributing to fine-tuning symplasmic opening apertures. Overall, our studies identified two key components involved in the deposits of PD callose and provided new insights into how symplasmic connectivity is maintained by the control of PD callose homoeostasis.

AtHVA22a, a plant-specific homologue of Reep/DP1/Yop1 family proteins is involved in turnip mosaic virus propagation.

The movement of potyviruses, the largest genus of single-stranded, positive-sense RNA viruses responsible for serious diseases in crops, is very complex. As potyviruses developed strategies to hijack the host secretory pathway and plasmodesmata (PD) for their transport, the goal of this study was to identify membrane and/or PD-proteins that interact with the 6K2 protein, a potyviral protein involved in replication and cell-to-cell movement of turnip mosaic virus (TuMV). Using split-ubiquitin membrane yeast two-hybrid assays, we screened an Arabidopsis cDNA library for interactors of TuMV6K2. We isolated AtHVA22a (Hordeum vulgare abscisic acid responsive gene 22), which belongs to a multigenic family of transmembrane proteins, homologous to Receptor expression-enhancing protein (Reep)/Deleted in polyposis (DP1)/Yop1 family proteins in animal and yeast. HVA22/DP1/Yop1 family genes are widely distributed in eukaryotes, but the role of HVA22 proteins in plants is still not well known, although proteomics analysis of PD fractions purified from Arabidopsis suspension cells showed that AtHVA22a is highly enriched in a PD proteome. We confirmed the interaction between TuMV6K2 and AtHVA22a in yeast, as well as in planta by using bimolecular fluorescence complementation and showed that TuMV6K2/AtHVA22a interaction occurs at the level of the viral replication compartment during TuMV infection. Finally, we showed that the propagation of TuMV is increased when AtHVA22a is overexpressed in planta but slowed down upon mutagenesis of AtHVA22a by CRISPR-Cas9. Altogether, our results indicate that AtHVA22a plays an agonistic effect on TuMV propagation and that the C-terminal tail of the protein is important in this process.

'Emerging into the World' - the regulation and control of dormancy and sprouting in geophytes.

Geophytic plants synchronize growth and quiescence with the external environment to survive and thrive under changing seasons. Besides seasonal growth adaptation, dormancy and sprouting are critical factors determining crop yield and market supply as various geophytes also serve as major food, floriculture, and ornamental crops. Dormancy in such crops decides crop availability in the market, as most of such crops are consumed during the dormant stage. On the other hand, uniform/maximal sprouting is crucial for maximum yield. Thus, dormancy and sprouting regulation have great economic importance. Dormancy-sprouting cycles in geophytes are regulated by genetic, exogenous (environmental), and endogenous (genetic, metabolic and hormonal, etc.) factors. Comparatively, the temperature is more dominant in regulating dormancy and sprouting in geophytes, unlike aboveground tissues, where both photoperiod and temperature control are involved. Despite huge economic importance, studies concerning the regulation of dormancy and sprouting are scarce in the majority of geophytes. To date, only a few molecular factors involved in the process have been suggested. Recently, omics studies on molecular and metabolic factors involved in dormancy and growth regulations of underground vegetative tissues have provided more insight into the mechanism. Here, we discuss current knowledge of the environmental and molecular regulation and control of dormancy and sprouting in geophytes and discuss challenges/questions that need to be addressed in the future for crop improvement.

Arabidopsis PDLP7 modulated plasmodesmata function is related to BG10-dependent glucosidase activity required for callose degradation.

The microdomains of plasmodesmata, specialized cell-wall channels responsible for communications between neighboring cells, are composed of various plasmodesmata-located proteins (PDLPs) and lipids. Here, we found that, among all PDLP or homologous proteins in Arabidopsis thaliana genome, PDLP5 and PDLP7 possessed a C-terminal sphingolipid-binding motif, with the latter being the only member that was significantly upregulated upon turnip mosaic virus and cucumber mosaic virus infections. pdlp7 mutant plants exhibited significantly reduced callose deposition, larger plasmodesmata diameters, and faster viral transmission. These plants exhibited increased glucosidase activity but no change in callose synthase activity. PDLP7 interacted specifically with glucan endo-1,3-ß-glucosidase 10 (BG10). Consistently, higher levels of callose deposition and slower virus transmission in bg10 mutants were observed. The interaction between PDLP7 and BG10 was found to depend on the presence of the Gnk2-homologous 1 (GnK2-1) domain at the N terminus of PDLP7 with Asp-35, Cys-42, Gln-44, and Leu-116 being essential. In vitro supplementation of callose was able to change the conformation of the GnK2-1 domain. Our data suggest that the GnK2-1 domain of PDLP7, in conjunction with callose and BG10, plays a key role in plasmodesmata opening and closure, which is necessary for intercellular movement of various molecules.

Molecular Advances of Bud Dormancy in Trees.

Seasonal bud dormancy in perennial woody plants is a crucial and intricate process that is vital for the survival and development of plants. Over the past few decades, significant advancements have been made in understanding many features of bud dormancy, particularly in model species, where certain molecular mechanisms underlying this process have been elucidated. In this review, we provide an overview of recent molecular progress in understanding bud dormancy in trees, with a specific emphasis on the integration of common signaling and molecular mechanisms identified across different tree species. Additionally, we address some challenges that have emerged in the in-depth understanding of bud dormancy and offer insights for future studies.

Tracking the messengers: Emerging advances in mRNA-based plant communication.

Messenger RNAs (mRNAs) are the templates for protein translation but can also act as non-cell-autonomous signaling molecules. Plants input endogenous and exogenous cues to mobile mRNAs and output them to local or systemic target cells and organs to support specific plant responses. Mobile mRNAs form ribonucleoprotein (RNP) complexes with proteins during transport. Components of these RNP complexes could interact with plasmodesmata (PDs), a major mediator of mRNA transport, to ensure mRNA mobility and transport selectivity. Based on advances in the last two to three years, this review summarizes mRNA transport mechanisms in local and systemic signaling from the perspective of RNP complex formation and PD transport. We also discuss the physiological roles of endogenous mRNA transport and the recently revealed roles of non-cell-autonomous mRNAs in inter-organism communication.

Distributing Plant Developmental Regulatory Proteins via Plasmodesmata.

During plant development, mobile proteins, including transcription factors, abundantly serve as messengers between cells to activate transcriptional signaling cascades in distal tissues. These proteins travel from cell to cell via nanoscopic tunnels in the cell wall known as plasmodesmata. Cellular control over this intercellular movement can occur at two likely interdependent levels. It involves regulation at the level of plasmodesmata density and structure as well as at the level of the cargo proteins that traverse these tunnels. In this review, we cover the dynamics of plasmodesmata formation and structure in a developmental context together with recent insights into the mechanisms that may control these aspects. Furthermore, we explore the processes involved in cargo-specific mechanisms that control the transport of proteins via plasmodesmata. Instead of a one-fits-all mechanism, a pluriform repertoire of mechanisms is encountered that controls the intercellular transport of proteins via plasmodesmata to control plant development.

Effect of gibberellin on crown root development in the mutant of the rice plasmodesmal Germin-like protein OsGER4.

Rice is an essential but highly stress-susceptible crop, whose root system plays an important role in plant development and stress adaptation. The rice root system architecture is controlled by gene regulatory networks involving different phytohormones including auxin, jasmonate, and gibberellin. Gibberellin is generally known as a molecular clock that interacts with different pathways to regulate root meristem development. The exogenous treatment of rice plantlets with Gibberellin reduced the number of crown roots, whilst the exogenous jasmonic acid treatment enhanced them by involving a Germin-like protein OsGER4. Due to those opposite effects, this study aims to investigate the effect of Gibberellin on crown root development in the rice mutant of the plasmodesmal Germin-like protein OsGER4. Under exogenous gibberellin treatment, the number of crown roots significantly increased in osger4 mutant lines and decreased in the OsGER4 overexpressed lines. GUS staining showed that OsGER4 was strongly expressed in rice root systems, particularly crown and lateral roots under GA3 application. Specifically, OsGER4 was strongly expressed from the exodermis, epidermis, sclerenchyma to the endodermis layers of the crown root, along the vascular bundle and throughout LR primordia. The plasmodesmal protein OsGER4 is suggested to be involved in crown root development by maintaining hormone homeostasis, including Gibberillin.

Plasmodesmata and intercellular molecular traffic control.

Plasmodesmata are plasma membrane-lined connections that join plant cells to their neighbours, establishing an intercellular cytoplasmic continuum through which molecules can travel between cells, tissues, and organs. As plasmodesmata connect almost all cells in plants, their molecular traffic carries information and resources across a range of scales, but dynamic control of plasmodesmal aperture can change the possible domains of molecular exchange under different conditions. Plasmodesmal aperture is controlled by specialised signalling cascades accommodated in spatially discrete membrane and cell wall domains. Thus, the composition of plasmodesmata defines their capacity for molecular trafficking. Further, their shape and density can likewise define trafficking capacity, with the cell walls between different cell types hosting different numbers and forms of plasmodesmata to drive molecular flux in physiologically important directions. The molecular traffic that travels through plasmodesmata ranges from small metabolites through to proteins, and possibly even larger mRNAs. Smaller molecules are transmitted between cells via passive mechanisms but how larger molecules are efficiently trafficked through plasmodesmata remains a key question in plasmodesmal biology. How plasmodesmata are formed, the shape they take, what they are made of, and what passes through them regulate molecular traffic through plants, underpinning a wide range of plant physiology.

Organelle Ca2+/CAM1-SELTP confers somatic cell embryogenic competence acquisition and transformation in plant regeneration.

Somatic cell totipotency in plant regeneration represents the forefront of the compelling scientific puzzles and one of the most challenging problems in biology. How somatic embryogenic competence is achieved in regeneration remains elusive. Here, we discover uncharacterized organelle-based embryogenic differentiation processes of intracellular acquisition and intercellular transformation, and demonstrate the underlying regulatory system of somatic embryogenesis-associated lipid transfer protein (SELTP) and its interactor calmodulin1 (CAM1) in cotton as the pioneer crop for biotechnology application. The synergistic CAM1 and SELTP exhibit consistent dynamical amyloplast-plasmodesmata (PD) localization patterns but show opposite functional effects. CAM1 inhibits the effect of SELTP to regulate embryogenic differentiation for plant regeneration. It is noteworthy that callus grafting assay reflects intercellular trafficking of CAM1 through PD for embryogenic transformation. This work originally provides insight into the mechanisms responsible for embryogenic competence acquisition and transformation mediated by the Ca2+/CAM1-SELTP regulatory pathway, suggesting a principle for plant regeneration and cell/genetic engineering.

Callose in leptoid cell walls of the moss Polytrichum and the evolution of callose synthase across bryophytes.

Introduction: Leptoids, the food-conducting cells of polytrichaceous mosses, share key structural features with sieve elements in tracheophytes, including an elongated shape with oblique end walls containing modified plasmodesmata or pores. In tracheophytes, callose is instrumental in developing the pores in sieve elements that enable efficient photoassimilate transport. Aside from a few studies using aniline blue fluorescence that yielded confusing results, little is known about callose in moss leptoids. Methods: Callose location and abundance during the development of leptoid cell walls was investigated in the moss Polytrichum commune using aniline blue fluorescence and quantitative immunogold labeling (label density) in the transmission electron microscope. To evaluate changes during abiotic stress, callose abundance in leptoids of hydrated plants was compared to plants dried for 14 days under field conditions. A bioinformatic study to assess the evolution of callose within and across bryophytes was conducted using callose synthase (CalS) genes from 46 bryophytes (24 mosses, 15 liverworts, and 7 hornworts) and one representative each of five tracheophyte groups. Results: Callose abundance increases around plasmodesmata from meristematic cells to end walls in mature leptoids. Controlled drying resulted in a significant increase in label density around plasmodesmata and pores over counts in hydrated plants. Phylogenetic analysis of the CalS protein family recovered main clades (A, B, and C). Different from tracheophytes, where the greatest diversity of homologs is found in clade A, the majority of gene duplication in bryophytes is in clade B. Discussion: This work identifies callose as a crucial cell wall polymer around plasmodesmata from their inception to functioning in leptoids, and during water stress similar to sieve elements of tracheophytes. Among bryophytes, mosses exhibit the greatest number of multiple duplication events, while only two duplications are revealed in hornwort and none in liverworts. The absence in bryophytes of the CalS 7 gene that is essential for sieve pore development in angiosperms, reveals that a different gene is responsible for synthesizing the callose associated with leptoids in mosses.

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.

Traveling with purpose: cell-to-cell transport of plant mRNAs.

Messenger RNAs (mRNAs) in multicellular organisms can act as signals transported cell-to-cell and over long distances. In plants, mRNAs traffic cell-to-cell via plasmodesmata (PDs) and over long distances via the phloem vascular system to control diverse biological processes - such as cell fate and tissue patterning - in destination organs. Research on long-distance transport of mRNAs in plants has made remarkable progress, including the cataloguing of many mobile mRNAs, characterization of mRNA features important for transport, identification of mRNA-binding proteins involved in their transport, and understanding of the physiological roles of mRNA transport. However, information on short-range mRNA cell-to-cell transport is still limited. This review discusses the regulatory mechanisms and physiological functions of mRNA transport at the cellular and whole plant levels.

C4 rice engineering, beyond installing a C4 cycle.

C4 photosynthesis in higher plants is carried out by two distinct cell types: mesophyll cells and bundle sheath cells, as a result highly concentrated carbon dioxide is released surrounding RuBisCo in chloroplasts of bundle sheath cells and the photosynthetic efficiency is significantly higher than that of C3 plants. The evolution of the dual-cell C4 cycle involved complex modifications to leaf anatomy and cell ultra-structures. These include an increase in leaf venation, the formation of Kranz anatomy, changes in chloroplast morphology in bundle sheath cells, and increases in the density of plasmodesmata at interfaces between the bundle sheath and mesophyll cells. It is predicted that cereals will be in severe worldwide shortage at the mid-term of this century. Rice is a staple food that feeds more than half of the world's population. If rice can be engineered to perform C4 photosynthesis, it is estimated that rice yield will be increased by at least 50% due to enhanced photosynthesis. Thus, the Second Green Revolution has been launched on this principle by genetically installing C4 photosynthesis into C3 crops. The studies on molecular mechanisms underlying the changes in leaf morphoanatomy involved in C4 photosynthesis have made great progress in recent years. As there are plenty of reviews discussing the installment of the C4 cycle, we focus on the current progress and challenges posed to the research regarding leaf anatomy and cell ultra-structure modifications made towards the development of C4 rice.

Assaying Effector Cell-to-Cell Mobility in Plant Tissues Identifies Hypermobility and Indirect Manipulation of Plasmodesmata.

In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. While plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To establish a methodology for identification and characterization of the cell-to-cell mobility of effectors, we performed a quantitative live imaging-based screen of candidate effectors of the fungal pathogen Colletotrichum higginsianum. We predicted C. higginsianum effectors by their expression profiles, the presence of a secretion signal, and their predicted and in planta localization when fused to green fluorescent protein. We assayed for cell-to-cell mobility of nucleocytosolic effectors and identified 14 that are cell-to-cell mobile. We identified that three of these effectors are "hypermobile," showing cell-to-cell mobility greater than expected for a protein of that size. To explore the mechanism of hypermobility, we chose two hypermobile effectors and measured their impact on plasmodesmata function and found that even though they show no direct association with plasmodesmata, each increases the transport capacity of plasmodesmata. Thus, our methods for quantitative analysis of cell-to-cell mobility of candidate microbe-derived effectors, or any suite of host proteins, can identify cell-to-cell hypermobility and offer greater understanding of how proteins affect plasmodesmal function and intercellular connectivity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.