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Stable Transformation of Arabidopsis thaliana Cell Suspension Cultures: A Case Study for the Overexpression of The COI1 Receptor

Authors: Eva K. Díaz-Sánchez, José A. O’Brien, Imma Pérez-Salamó, Jovaras Krasauskas, Moritz Bömer and Alessandra Devoto, date: 01/05/2021, view: 3287, Q&A: 1

[Abstract] Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value chemicals reproducibly and in elevated quantities.

In this protocol, a method to stably transform and maintain Arabidopsis cell ...

Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis

Authors: Yu Jin and Sebastian Marquardt, date: 10/20/2020, view: 3801, Q&A: 0

[Abstract] CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an ...

Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple

Authors: Aparecida L. Silva, Keini Dressano, Paulo H. O. Ceciliato, Juan Carlos Guerrero-Abad and Daniel S. Moura, date: 04/05/2018, view: 7475, Q&A: 1

[Abstract] The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol ...

Electron Tomography to Study the Three-dimensional Structure of Plasmodesmata in Plant Tissues–from High Pressure Freezing Preparation to Ultrathin Section Collection

Authors: William J. Nicolas, Emmanuelle Bayer and Lysiane Brocard, date: 01/05/2018, view: 7783, Q&A: 0

[Abstract] Plasmodesmata (PD) are nanometric (~20 nm wide) membrane lined pores encased in the cell walls of the adjacent plant cells. They allow the cells to exchange all types of molecules ranging from nutrients like sugar, hormones, to RNAs and various proteins. Unfortunately, they are also hijacked by phyto-viruses, enabling them to spread from ...

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: 7579, 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 ...

Polyamine and Paraquat Transport Assays in Arabidopsis Seedling and Callus

Authors: Haoxi Chai, Yun Shen and Huazhong Shi, date: 08/05/2017, view: 6095, Q&A: 0

[Abstract] Polyamines (PAs) are polycationic compounds found in all living organisms and play crucial roles in growth and survival. We here show the ‘Polyamine and paraquat (PQ) transport assay’ protocol, which can be used to examine the uptake activity of PA/PQ transporters. We have used this protocol to demonstrate that PUT3 in Arabidopsis is a ...

A Ribosome Footprinting Protocol for Plants

Authors: Catharina Merchante, Qiwen Hu, Steffen Heber, Jose Alonso and Anna N. Stepanova, date: 11/05/2016, view: 12373, Q&A: 0

[Abstract] Ribosome footprinting, or Ribo-seq, has revolutionized the studies of translation. It was originally developed for yeast and mammalian cells in culture (Ingolia et al., 2009). Herein, we describe a plant-optimized hands-on ribosome footprinting protocol derived from previously published procedures of polysome isolation (Ingolia et al ...

LC/MS-based Detection of Hydroxyproline O-galactosyltransferase Activity

Authors: Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi, date: 01/20/2016, view: 6592, Q&A: 0

[Abstract] Arabinogalactan proteins (AGPs) are plant-specific extracellular glycoproteins regulating a variety of processes during growth and development. AGP biosynthesis involves O-galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. The initial Hyp O-galactosylation is mediated ...

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