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    Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana
    Authors:  Susumu Uehara, Yasuko Ito-Inaba and Takehito Inaba, date: 02/05/2018, view: 123, Q&A: 0
    [Abstract] In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide ...
    In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6
    Authors:  Kyungmin Baeg, Yukihide Tomari and Hiro-oki Iwakawa, date: 01/05/2018, view: 314, Q&A: 0
    [Abstract] RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant ...
    Fatty Acid Content and Composition of Triacylglycerols of Chlorella kessleri
    Authors:  Motohide Aoki and Norihiro Sato, date: 01/05/2018, view: 329, Q&A: 0
    [Abstract] Triacylglycerols (TAGs) are esters formed from one glycerol and three fatty acids. TAGs are induced to accumulate in algal cells under environmental stress conditions including nutrient-limitation, hyperosmosis, and low temperature, for the storage of metabolic energy and carbon, and also for the consumption of excess energy (e.g., Hirai ...
    Determination of H+-ATPase Activity in Arabidopsis Guard Cell Protoplasts through H+-pumping Measurement and H+-ATPase Quantification
    Authors:  Shota Yamauchi and Ken-ichiro Shimazaki, date: 12/20/2017, view: 542, Q&A: 0
    [Abstract] The opening of stomata in plants in response to blue light is driven by the plasma membrane H+-ATPase in guard cells. To evaluate the activation of the H+-ATPase in vivo, we can use H+-pumping by guard cells in response to blue light and fusicoccin. To do this, it is required to prepare a large amount of ...
    Estimation of Silica Cell Silicification Level in Grass Leaves Using in situ Charring Method
    Authors:  Santosh Kumar and Rivka Elbaum, date: 11/20/2017, view: 500, Q&A: 0
    [Abstract] Silica cells are specialized leaf epidermal cells in grasses with almost the whole cell volume filled with solid silica. In sorghum, silica deposition in silica cells takes place in young, elongating leaves around the mid-length of the leaf. We developed a protocol for estimating the level of silica cell silicification in Sorghum bicolor ...
    Analysis of Xyloglucan Composition in Arabidopsis Leaves
    Authors:  Javier Sampedro, Cristina Gianzo, Esteban Guitián, Gloria Revilla and Ignacio Zarra, date: 10/05/2017, view: 767, Q&A: 0
    [Abstract] Xyloglucan is one of the main components of the primary cell wall in most species of plants. This protocol describes a method to analyze the composition of the enzyme-accessible and enzyme-inaccessible fractions of xyloglucan in the model species Arabidopsis thaliana. It is based on digestion with an endoglucanase that attacks ...
    Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts
    Authors:  Mareike Hauenstein and Stefan Hörtensteiner, date: 09/20/2017, view: 1221, Q&A: 0
    [Abstract] Hydroxylation of chlorophyll catabolites at the so-called C32 position (Hauenstein et al., 2016) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the ...
    Detection of Protein S-nitrosothiols (SNOs) in Plant Samples on Diaminofluorescein (DAF) Gels
    Authors:  Marta Rodríguez-Ruiz, Paulo T. Mioto, José M. Palma and Francisco J. Corpas , date: 09/20/2017, view: 992, Q&A: 0
    [Abstract] In plant cells, the analysis of protein S-nitrosothiols (SNOs) under physiological and adverse stress conditions is essential to understand the mechanisms of Nitric oxide (NO)-based signaling. We adapted a previously reported protocol for detecting protein SNOs in animal systems (King et al., 2005) for plant samples. Briefly, ...
    Ubiquitin Proteasome Activity Measurement in Total Plant Extracts
    Authors:  Suayib Üstün and Frederik Börnke, date: 09/05/2017, view: 1208, Q&A: 0
    [Abstract] The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to ...
    Extraction and Molybdenum Blue-based Quantification of Total Phosphate and Polyphosphate in Parachlorella
    Authors:  Shuhei Ota and Shigeyuki Kawano, date: 09/05/2017, view: 1064, Q&A: 0
    [Abstract] Inorganic phosphorus is a non-renewable resource and an essential element for life on Earth. Organisms such as algae, protists, and animals can store phosphate (Pi) through uptake of Pi as polyphosphate (poly-P), which is a linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds. Here, we describe procedures for ...