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      Probing the physiological role of the plastid outer-envelope membrane using the oemiR plasmid collection

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          Abstract

          Plastids are the site of complex biochemical pathways, most prominently photosynthesis. The organelle evolved through endosymbiosis with a cyanobacterium, which is exemplified by the outer envelope membrane that harbors more than 40 proteins in Arabidopsis. Their evolutionary conservation indicates high significance for plant cell function. While a few proteins are well-studied as part of the protein translocon complex the majority of outer envelope protein functions is unclear. Gaining a deeper functional understanding has been complicated by the lack of observable loss-of-function mutant phenotypes, which is often rooted in functional genetic redundancy. Therefore, we designed outer envelope-specific artificial micro RNAs ( oemiRs) capable of downregulating transcripts from several loci simultaneously. We successfully tested oemiR function by performing a proof-of-concept screen for pale and cold-sensitive mutants. An in-depth analysis of pale mutant alleles deficient in the translocon component TOC75 using proteomics provided new insights into putative compensatory import pathways. The cold stress screen not only recapitulated 3 previously known phenotypes of cold-sensitive mutants but also identified 4 mutants of additional oemiR outer envelope loci. Altogether our study revealed a role of the outer envelope to tolerate cold conditions and showcasts the power of the oemiR collection to research the significance of outer envelope proteins.

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          MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.

          Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
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            The Perseus computational platform for comprehensive analysis of (prote)omics data.

            A main bottleneck in proteomics is the downstream biological analysis of highly multivariate quantitative protein abundance data generated using mass-spectrometry-based analysis. We developed the Perseus software platform (http://www.perseus-framework.org) to support biological and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data analysis covering normalization, pattern recognition, time-series analysis, cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary analysis of complex large data sets.
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              Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana

              The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                G3 (Bethesda)
                Genetics
                g3journal
                G3: Genes|Genomes|Genetics
                Oxford University Press (US )
                2160-1836
                October 2023
                12 August 2023
                12 August 2023
                : 13
                : 10
                : jkad187
                Affiliations
                Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-Universität Munich , 82152 Planegg-Martinsried, Germany
                Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-Universität Munich , 82152 Planegg-Martinsried, Germany
                Plant Biochemistry, Faculty of Biology, Ludwig-Maximilians-Universität Munich , 82152 Planegg-Martinsried, Germany
                Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-Universität Munich , 82152 Planegg-Martinsried, Germany
                School of Biological Sciences, Washington State University , PO Box 644236, Pullman, WA 99164-4236, USA
                Independent Researcher
                Plant Biochemistry, Faculty of Biology, Ludwig-Maximilians-Universität Munich , 82152 Planegg-Martinsried, Germany
                Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-Universität Munich , 82152 Planegg-Martinsried, Germany
                Plant Biochemistry, Faculty of Biology, Ludwig-Maximilians-Universität Munich , 82152 Planegg-Martinsried, Germany
                School of Biological Sciences, Washington State University , PO Box 644236, Pullman, WA 99164-4236, USA
                Author notes
                Corresponding author: Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-Universität Munich, 82152 Planegg-Martinsried, German. Email: serena.schwenkert@ 123456lmu.de
                Corresponding author: Plant Biochemistry, Faculty of Biology, Ludwig-Maximilians-Universität Munich, 82152 Planegg-Martinsried, Germany. Email: kunz@ 123456lmu.de

                Conflict of interest The authors declare no conflict of interest.

                Author information
                https://orcid.org/0000-0003-4301-5176
                Article
                jkad187
                10.1093/g3journal/jkad187
                10542568
                37572358
                0dc9b2c4-fa11-4aa8-bf70-e47756f7584b
                © The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 20 July 2023
                : 04 August 2023
                : 05 September 2023
                Page count
                Pages: 12
                Funding
                Funded by: National Science Foundation, DOI 10.13039/501100008982;
                Funded by: NSF, DOI 10.13039/100000001;
                Award ID: Award IOS-1553506
                Categories
                Mutant Screen Report
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140

                Genetics
                arabidopsis thaliana,artificial micro rna,chloroplast outer envelope,cold acclimation,toc75,plant proteome

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