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Cell Jun 2021Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a...
Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.
Topics: Animals; Arabidopsis; Arabidopsis Proteins; Calcium; Carrier Proteins; Cell Death; Cell Membrane; Cell Membrane Permeability; Disease Resistance; Glutamic Acid; Lipid Bilayers; Oocytes; Plant Cells; Plant Immunity; Protein Multimerization; Protoplasts; Reactive Oxygen Species; Signal Transduction; Single Molecule Imaging; Vacuoles; Xenopus
PubMed: 33984278
DOI: 10.1016/j.cell.2021.05.003 -
Molecular Plant Jan 2021The rapid and enthusiastic adoption of single-cell RNA sequencing (scRNA-seq) has demonstrated that this technology is far more than just another way to perform... (Review)
Review
The rapid and enthusiastic adoption of single-cell RNA sequencing (scRNA-seq) has demonstrated that this technology is far more than just another way to perform transcriptome analysis. It is not an exaggeration to say that the advent of scRNA-seq is revolutionizing the details of whole-transcriptome snapshots from a tissue to a cell. With this disruptive technology, it is now possible to mine heterogeneity between tissue types and within cells like never before. This enables more rapid identification of rare and novel cell types, simultaneous characterization of multiple different cell types and states, more accurate and integrated understanding of their roles in life processes, and more. However, we are only at the beginning of unlocking the full potential of scRNA-seq applications. This is particularly true for plant sciences, where single-cell transcriptome profiling is in its early stage and has many exciting challenges to overcome. In this review, we compare and evaluate recent pioneering studies using the Arabidopsis root model, which has established new paradigms for scRNA-seq studies in plants. We also explore several new and promising single-cell analysis tools that are available to those wishing to study plant development and physiology at unprecedented resolution and scale. In addition, we propose some future directions on the use of scRNA-seq technology to tackle some of the critical challenges in plant research and breeding.
Topics: Cell Size; Gene Expression Profiling; Genomics; Plants; Protoplasts; Single-Cell Analysis
PubMed: 33152518
DOI: 10.1016/j.molp.2020.10.012 -
Molecules (Basel, Switzerland) May 2020Plants contain abundant autofluorescent molecules that can be used for biochemical, physiological, or imaging studies. The two most studied molecules are chlorophyll... (Review)
Review
Plants contain abundant autofluorescent molecules that can be used for biochemical, physiological, or imaging studies. The two most studied molecules are chlorophyll (orange/red fluorescence) and lignin (blue/green fluorescence). Chlorophyll fluorescence is used to measure the physiological state of plants using handheld devices that can measure photosynthesis, linear electron flux, and CO assimilation by directly scanning leaves, or by using reconnaissance imaging from a drone, an aircraft or a satellite. Lignin fluorescence can be used in imaging studies of wood for phenotyping of genetic variants in order to evaluate reaction wood formation, assess chemical modification of wood, and study fundamental cell wall properties using Förster Resonant Energy Transfer (FRET) and other methods. Many other fluorescent molecules have been characterized both within the protoplast and as components of cell walls. Such molecules have fluorescence emissions across the visible spectrum and can potentially be differentiated by spectral imaging or by evaluating their response to change in pH (ferulates) or chemicals such as Naturstoff reagent (flavonoids). Induced autofluorescence using glutaraldehyde fixation has been used to enable imaging of proteins/organelles in the cell protoplast and to allow fluorescence imaging of fungal mycelium.
Topics: Cell Wall; Chlorophyll; Fluorescence; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; Lignin; Luminescent Proteins; Plant Leaves; Plants
PubMed: 32455605
DOI: 10.3390/molecules25102393 -
Developmental Cell Feb 2021Crop productivity depends on activity of meristems that produce optimized plant architectures, including that of the maize ear. A comprehensive understanding of...
Crop productivity depends on activity of meristems that produce optimized plant architectures, including that of the maize ear. A comprehensive understanding of development requires insight into the full diversity of cell types and developmental domains and the gene networks required to specify them. Until now, these were identified primarily by morphology and insights from classical genetics, which are limited by genetic redundancy and pleiotropy. Here, we investigated the transcriptional profiles of 12,525 single cells from developing maize ears. The resulting developmental atlas provides a single-cell RNA sequencing (scRNA-seq) map of an inflorescence. We validated our results by mRNA in situ hybridization and by fluorescence-activated cell sorting (FACS) RNA-seq, and we show how these data may facilitate genetic studies by predicting genetic redundancy, integrating transcriptional networks, and identifying candidate genes associated with crop yield traits.
Topics: Base Sequence; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Gene Regulatory Networks; Genetic Association Studies; Protoplasts; Quantitative Trait Loci; Reproducibility of Results; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome; Zea mays
PubMed: 33400914
DOI: 10.1016/j.devcel.2020.12.015 -
Protist Feb 2022The Vampyrellida (Endomyxa, Rhizaria) is a group of free-living, predatory amoebae, which is most closely related to the Phytomyxea (plasmodiophorids and phagomyxids).... (Review)
Review
The Vampyrellida (Endomyxa, Rhizaria) is a group of free-living, predatory amoebae, which is most closely related to the Phytomyxea (plasmodiophorids and phagomyxids). It encompasses about 50 credibly described species that have a characteristic life history with the regular alternation of trophic amoebae and immobile digestive cysts. All known vampyrellid amoebae are naked and filose, but the different species display a broad morphological variety. Vampyrellids also vary greatly in their feeding habits, and range from generalist predators to specialized 'protoplast feeders' that exclusively feed on the cell contents of eukaryotic prey. They can be found in freshwater, soil and marine habitats, and appear to be globally distributed. Yet, the phenotypic diversity and ecological roles of the Vampyrellida are still poorly explored. Currently, there are eight well-recognized subclades that comprise four families (Vampyrellidae, Leptophryidae, Placopodidae and Sericomyxidae) as well as some lineages without any phenotypic information. Research on vampyrellids is challenging due to their cryptic occurrence in nature, intricate feeding habits that complicate cultivation, and a convoluted taxonomic history. Here, we review available information about cell structure, diversity, ecology, taxonomy and phylogenetics, and provide an up-to-date introduction to the Vampyrellida that may facilitate future research.
Topics: Amoeba; Cercozoa; Ecosystem; Humans; Phylogeny; Rhizaria
PubMed: 35091168
DOI: 10.1016/j.protis.2021.125854 -
Biomolecules Apr 2020Ginsenosides are secondary metabolites that belong to the triterpenoid or saponin group. These occupy a unique place in the pharmaceutical sector, associated with the... (Review)
Review
Ginsenosides are secondary metabolites that belong to the triterpenoid or saponin group. These occupy a unique place in the pharmaceutical sector, associated with the manufacturing of medicines and dietary supplements. These valuable secondary metabolites are predominantly used for the treatment of nervous and cardiac ailments. The conventional approaches for ginsenoside extraction are time-consuming and not feasible, and thus it has paved the way for the development of various biotechnological approaches, which would ameliorate the production and extraction process. This review delineates the biotechnological tools, such as conventional tissue culture, cell suspension culture, protoplast culture, polyploidy, in vitro mutagenesis, hairy root culture, that have been largely implemented for the enhanced production of ginsenosides. The use of bioreactors to scale up ginsenoside yield is also presented. The main aim of this review is to address the unexplored aspects and limitations of these biotechnological tools, so that a platform for the utilization of novel approaches can be established to further increase the production of ginsenosides in the near future.
Topics: Agrobacterium; Biotechnology; Ginsenosides; Transformation, Genetic
PubMed: 32252467
DOI: 10.3390/biom10040538 -
Cytometry. Part a : the Journal of the... Apr 2021
Topics: Flow Cytometry; Protoplasts
PubMed: 33398930
DOI: 10.1002/cyto.a.24295 -
The Plant Journal : For Cell and... Jun 2022Single-cell sequencing approaches reveal the intracellular dynamics of individual cells and answer biological questions with high-dimensional catalogs of millions of... (Review)
Review
Single-cell sequencing approaches reveal the intracellular dynamics of individual cells and answer biological questions with high-dimensional catalogs of millions of cells, including genomics, transcriptomics, chromatin accessibility, epigenomics, and proteomics data across species. These emerging yet thriving technologies have been fully embraced by the field of plant biology, with a constantly expanding portfolio of applications. Here, we introduce the current technical advances used for single-cell omics, especially single-cell genome and transcriptome sequencing. Firstly, we overview methods for protoplast and nucleus isolation and genome and transcriptome amplification. Subsequently, we use well-executed benchmarking studies to highlight advances made through the application of single-cell omics techniques. Looking forward, we offer a glimpse of additional hurdles and future opportunities that will introduce broad adoption of single-cell sequencing with revolutionary perspectives in plant biology.
Topics: Epigenomics; Genome; Genomics; Metabolomics; Plants; Proteomics; Transcriptome
PubMed: 35426954
DOI: 10.1111/tpj.15772 -
Frontiers in Genome Editing 2021In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating... (Review)
Review
In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.
PubMed: 34713263
DOI: 10.3389/fgeed.2021.717017 -
Frontiers in Genome Editing 2021The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global... (Review)
Review
The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.
PubMed: 34713266
DOI: 10.3389/fgeed.2021.734951