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https://www.readbyqxmd.com/read/28938018/genome-wide-identification-of-bacterial-plant-colonization-genes
#1
Benjamin J Cole, Meghan E Feltcher, Robert J Waters, Kelly M Wetmore, Tatiana S Mucyn, Elizabeth M Ryan, Gaoyan Wang, Sabah Ul-Hasan, Meredith McDonald, Yasuo Yoshikuni, Rex R Malmstrom, Adam M Deutschbauer, Jeffery L Dangl, Axel Visel
Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system...
September 22, 2017: PLoS Biology
https://www.readbyqxmd.com/read/28937985/interference-with-plastome-gene-expression-and-clp-protease-activity-in-arabidopsis-triggers-a-chloroplast-unfolded-protein-response-to-restore-protein-homeostasis
#2
Ernesto Llamas, Pablo Pulido, Manuel Rodriguez-Concepcion
Disruption of protein homeostasis in chloroplasts impairs the correct functioning of essential metabolic pathways, including the methylerythritol 4-phosphate (MEP) pathway for the production of plastidial isoprenoids involved in photosynthesis and growth. We previously found that misfolded and aggregated forms of the first enzyme of the MEP pathway are degraded by the Clp protease with the involvement of Hsp70 and Hsp100/ClpC1 chaperones in Arabidopsis thaliana. By contrast, the combined unfolding and disaggregating actions of Hsp70 and Hsp100/ClpB3 chaperones allow solubilization and hence reactivation of the enzyme...
September 22, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28937840/supraoptimal-activity-of-chlorophyll-dephytylase1-results-in-an-increase-in-tocopherol-level-in-mature-arabidopsis-seeds
#3
Yao-Pin Lin, Yee-Yung Charng
Tocopherols are synthesized in photosynthetic organisms, playing a role in plant stress tolerance. Recent studies showed that the phytol moiety of tocopherols comes from the salvaged phytol chain during chlorophyll degradation. However, the enzyme(s) responsible for chlorophyll dephytylation remains unclear. Recently, we reported the identification and characterization of CHLOROPHYLL DEPHYTYLASE1 (CLD1) of Arabidopsis, suggesting its role in chlorophyll turnover at steady state. In this addendum to the report, we presented and discussed the results related to the function of CLD1 in tocopherol biosynthesis...
September 22, 2017: Plant Signaling & Behavior
https://www.readbyqxmd.com/read/28936671/use-of-the-cas9-orthologs-from-streptococcus-thermophilus-and-staphylococcus-aureus-for-non-homologous-end-joining-mediated-site-specific-mutagenesis-in-arabidopsis-thaliana
#4
Jeannette Steinert, Carla Schmidt, Holger Puchta
Since the discovery of the CRISPR/Cas system and its in vivo application for site-specific targeted mutagenesis, this technique is wildly used in a great variety of organisms, such as many plant species. Commonly used for this application is the Cas9 enzyme from Streptococcus pyogenes. Here, we describe the application of two Cas9 orthologs from Streptococcus thermophilus and Staphylococcus aureus for targeted non-homologous end-joining mediated mutagenesis in Arabidopsis thaliana. With both orthologs, we could show efficient inheritance of the induced mutations...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936664/analysis-of-proteins-enriched-in-rice-gamete
#5
Takashi Okamoto
In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. Proteins enriched in each cell type are thought to reflect the biological function of the cells, since the composition of cellular proteins generally differs depending on cell type...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936662/manual-isolation-of-living-cells-from-the-arabidopsis-thaliana-female-gametophyte-by-micromanipulation
#6
Maria Englhart, Lucija Šoljić, Stefanie Sprunck
The few-celled female gametophyte, or embryo sac, of flowering plants is not easily accessible as it is buried within the sporophytic tissues of the ovule. Nevertheless, it has become an attractive model system to study the molecular mechanisms underlying patterning and cell type specification, as well as fertilization of the two female gametes, the egg and the central cell. While female gametes, zygotes, and early embryos can be manually isolated from the embryo sacs in maize, wheat, tobacco, and rice by micromanipulation, this approach had been considered impossible for the much smaller embryo sac of the model plant Arabidopsis thaliana...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936661/isolation-of-rice-sperm-cells-for-transcriptional-profiling
#7
Scott D Russell, Daniel S Jones, Sarah Anderson, Xinkun Wang, Venkatesan Sundaresan, Xiaoping Gou
The male germline of flowering plants displays unexpectedly divergent transcriptional profiles compared to other cell types and tissues of plants. As these are among the smallest cells, and are harbored within pollen, isolating a pure collection of germline RNA presents unusual challenges. The sperm cells of rice represent a particularly challenging subject for study as the pollen are unusually short lived upon release from the anther, and the marker gene sequences that make FACS possible in Arabidopsis have not yet been introduced into rice...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936660/isolation-of-arabidopsis-pollen-sperm-cells-and-vegetative-nuclei-by-fluorescence-activated-cell-sorting-facs
#8
Mário R Santos, Cláudia Bispo, Jörg D Becker
Efficient methods to isolate highly purified Arabidopsis thaliana pollen and the subcellular components of the male gametophyte (the vegetative nucleus and two sperm cells) have enabled genome-scale studies revealing a highly dynamic reprogramming of the transcriptome and epigenome during pollen development. Here, we describe the isolation of uninucleate microspores, mature pollen, as well as sperm cells and vegetative nuclei by Fluorescence-Activated Cell Sorting.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936659/transmission-electron-microscopy-tem-to-study-histology-of-pollen-and-pollen-tubes
#9
Peng-Fei Jia, Hong-Ju Li, Wei-Cai Yang
Here, we describe methods of transmission electron microscopy (TEM) based on conventional chemical fixation and high-pressure freezing (HPF) and freeze-substitution (FS) to examine the ultrastructure of Arabidopsis pollen grains and pollen tubes. Compared to other plant samples, such as root or leaf, pollen grains have thick pollen coat and cell wall which limit the permeation of fixative. Thus, it is difficult to obtain high-quality ultrastructural images of pollen. Moreover, pollen tube is very soft and the traditional procedure is too harsh to get an intact pollen tube sample...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936658/analysis-of-peroxisome-biogenesis-in-pollen-by-confocal-microscopy-and-transmission-electron-microscopy
#10
Peng-Fei Jia, Hong-Ju Li, Wei-Cai Yang
Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936657/whole-mount-rna-fish-on-ovules-and-developing-seeds
#11
Andrea Bleckmann, Thomas Dresselhaus
A key element to understand developmental and reproductive processes like germline development, double fertilization, and embryogenesis is the study of cell-specific gene expression patterns which is best analyzed by RNA in situ hybridization. Different visualization techniques have been established to mark either the region of mRNA production (using the classical chromogenic detection system) or the specific localization of mRNAs (using fluorescent labeled probes). In this chapter, we describe and compare whole mount RNA in situ hybridization techniques on ovules and young developing seeds from Arabidopsis thaliana using three different detection systems...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936654/imaging-ca-2-dynamics-in-wild-type-and-nadph-oxidase-deficient-mutant-pollen-tubes-with-yellow-cameleon-and-confocal-laser-scanning-microscopy
#12
Christina Maria Franck, Jens Westermann, Aurélien Boisson-Dernier
While cytosolic calcium (Ca(2+)) plays a central role in a myriad of signaling pathways as a secondary messenger, how dynamic changes of cytosolic calcium relate to cell growth control remains poorly understood. The engineering and continuous improvements of genetically encoded calcium sensors such as the Yellow Cameleon (YC) sensors combined with advances in microscopy have allowed imaging with great resolution of the spatiotemporal characteristics of cytosolic [Ca(2+)]cyt in individual cells. An exciting new step consists therefore in cautiously studying calcium dynamics in mutant backgrounds that display disturbed cellular growth behavior to further enhance our understanding on growth-related processes...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936652/staining-and-clearing-of-arabidopsis-reproductive-tissue-for-imaging-of-fluorescent-proteins
#13
Daniel Slane, Patrick Bürgel, Martin Bayer
Imaging of fluorescent proteins in whole-mount tissue is a powerful tool to understand growth and developmental processes, not only in plants. With the advent of genetically encoded fluorescent reporters, which specifically label reproductive cells in Arabidopsis, deep tissue imaging has become increasingly important for the study of plant reproduction. To penetrate the surrounding layers of maternal tissue, however, the tissue has to be cleared by homogenizing the refractive index of the sample, often leading to inactivation of fluorescent proteins...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936651/in-vivo-ploidy-determination-of-arabidopsis-thaliana-male-and-female-gametophytes
#14
Burcu Nur Keçeli, Nico De Storme, Danny Geelen
Organ- or tissue-specific ploidy level determination is often used for answering biological, molecular, genetic, or evolutionary questions in plant sciences. However, current techniques for ploidy determination either cannot provide information on single cell level, require destructive sample preparation, or are laborious and time-consuming. Here, we present a new approach developed in Arabidopsis thaliana, which is not only less labor intensive but also allows in vivo ploidy determination on single cell level...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936649/development-and-observation-of-mature-megagametophyte-cell-specific-fluorescent-markers
#15
Mark A Chamberlin, Shai J Lawit
Visualization of the intact embryo sac within the ovular/gynoecial tissues and clear identification of cell types can be logistically difficult and subject to interpretation. Cellular marker technologies have been available for the embryo sac, but have typically labeled only one cell type in a particular line. Here, we describe techniques for simultaneous labeling each cell type in the embryo sac and visualization methods for such in Arabidopsis, soybean, maize, and sorghum.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936648/live-cell-imaging-of-f-actin-dynamics-during-fertilization-in-arabidopsis-thaliana
#16
Daichi Susaki, Daisuke Maruyama, Ramesh Yelagandula, Frederic Berger, Tomokazu Kawashima
Fertilization comprises a complex series of cellular processes leading to the fusion of a male and female gamete. Many studies have been carried out to investigate each step of fertilization in plants; however, our comprehensive understanding of all the sequential events during fertilization is still limited. This is largely due to difficulty in investigating events in the female gametophyte, which is deeply embedded in the maternal tissue. Recent advances in confocal microcopy assisted by fluorescent marker lines have contributed to visualizing subcellular dynamics in real time during fertilization in vivo...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936647/3d-imaging-of-whole-mount-ovules-at-cellular-resolution-to-study-female-germline-development-in-rice
#17
Ethel Mendocilla-Sato, Wenjing She, Célia Baroux
Recent advances in fluorescence-based staining of cellular compartments coupled with confocal microscopy imaging have allowed the visualization of three-dimensional (3D) structures with cellular resolution in various intact plant tissues and species. Such approaches are of particular interest for the analysis of the reproductive lineage in plants including the meiotic precursor cells deeply embedded within the ovary of the gynoecium enclosed in the flower. Yet, their relative inaccessibility and the lack of optical clarity of plant tissues prevent robust staining and imaging across several cell layers...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936645/an-introduction-to-male-germline-development
#18
Hannes Vogler, Ueli Grossniklaus, Afif Hedhly
In this introductory chapter, we describe male germline development in plants taking Arabidopsis thaliana as a reference species. We first describe the transition from sporophytic to germline development, then microsporogenesis including meiosis, followed by male gametophyte development prior to pollination, and finally the progamic phase culminating in double fertilization, which leads to the formation of the embryo and the endosperm. For detailed information on some of these processes or on the molecular underpinning of certain fate transitions, we refer the reader to recent reviews...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28936218/atoma1-affects-the-oxphos-system-and-plant-growth-in-contrast-to-other-newly-identified-atp-independent-proteases-in-arabidopsis-mitochondria
#19
Iwona Migdal, Renata Skibior-Blaszczyk, Malgorzata Heidorn-Czarna, Marta Kolodziejczak, Arnold Garbiec, Hanna Janska
Compared with yeast, our knowledge on members of the ATP-independent plant mitochondrial proteolytic machinery is rather poor. In the present study, using confocal microscopy and immunoblotting, we proved that homologs of yeast Oma1, Atp23, Imp1, Imp2, and Oct1 proteases are localized in Arabidopsis mitochondria. We characterized these components of the ATP-independent proteolytic system as well as the earlier identified protease, AtICP55, with an emphasis on their significance in plant growth and functionality in the OXPHOS system...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28935922/arabidopsis-r1r2r3-myb-proteins-are-essential-for-inhibiting-cell-division-in-response-to-dna-damage
#20
Poyu Chen, Hirotomo Takatsuka, Naoki Takahashi, Rie Kurata, Yoichiro Fukao, Kosuke Kobayashi, Masaki Ito, Masaaki Umeda
Inhibition of cell division is an active response to DNA damage that enables cells to maintain genome integrity. However, how DNA damage arrests the plant cell cycle is largely unknown. Here, we show that the repressor-type R1R2R3-Myb transcription factors (Rep-MYBs), which suppress G2/M-specific genes, are required to inhibit cell division in response to DNA damage. Knockout mutants are resistant to agents that cause DNA double-strand breaks and replication stress. Cyclin-dependent kinases (CDKs) can phosphorylate Rep-MYBs in vitro and are involved in their proteasomal degradation...
September 21, 2017: Nature Communications
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