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Tandem Purification of His6-3x FLAG Tagged Proteins for Mass Spectrometry from Arabidopsis

Featured protocol,  Authors: He Huang
He Huang Affiliation: Donald Danforth Plant Science Center, St. Louis, USA
Bio-protocol author page: a3873
 and Dmitri Anton Nusinow
Dmitri Anton NusinowAffiliation: Donald Danforth Plant Science Center, St. Louis, USA
For correspondence: meter@danforthcenter.org
Bio-protocol author page: a3874
date: 12/5/2016, 192 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2060.

Brief version appeared in eLife, Feb 2016
Tandem affinity purification is a powerful method to identify protein complexes that function in association with a known gene of interest. This protocol describes a methodology to capture proteins tagged with His6-3x FLAG explicitly for the purpose of on-bead digestion and identification by mass spectrometry. The high sensitivity and specificity of our methods allow for purification of proteins expressed at native levels from endogenous promoters to enable uncovering the functional roles of plant protein complexes.

Fusarium graminearum Maize Stalk Infection Assay and Associated Microscopic Observation Protocol

Featured protocol,  Authors: Juan He
Juan HeAffiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Bio-protocol author page: a3791
Tinglu Yuan
Tinglu Yuan Affiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Bio-protocol author page: a3792
 and Wei-Hua Tang
Wei-Hua TangAffiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
For correspondence: whtang@sibs.ac.cn
Bio-protocol author page: a2037
date: 12/5/2016, 32 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2034.

Brief version appeared in PLOS Pathogens, Mar 2016
The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae) causes Gibberella stalk rot in maize (Zea mays) and results in lodging and serious yield reduction. To develop methods to assess the fungal growth and symptom development in maize stalks, we present here a protocol of maize stalk inoculation with conidiospores of fluorescent protein-tagged F. graminearumand microscopic observation of the stalk infection process. The inoculation protocol provides repeatable results in stalk rot symptom development, and allows tracking of fungal hyphal growth inside maize stalks at cellular scale.

Measurement of ATP Hydrolytic Activity of Plasma Membrane H+-ATPase from Arabidopsis thaliana Leaves

Featured protocol,  Authors: Masaki Okumura
Masaki OkumuraAffiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
Bio-protocol author page: a3819
 and Toshinori Kinoshita
Toshinori KinoshitaAffiliation 1: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
Affiliation 2: Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan
For correspondence: kinoshita@bio.nagoya-u.ac.jp
Bio-protocol author page: a3820
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2044.

Brief version appeared in Plant Physiol, May 2016
Plant plasma membrane H+-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H+ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for measurement of the ATP hydrolytic activity of the plasma membrane H+-ATPase from Arabidopsis thaliana leaves.

A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9

Featured protocol,  Authors: Johan Vad-Nielsen*
Johan Vad-NielsenAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
For correspondence: johanvn@biomed.au.dk
Bio-protocol author page: a3868
Lin Lin *
Lin Lin Affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
For correspondence: lin.lin@biomed.au.dk
Bio-protocol author page: a3869
Kristopher Torp Jensen
Kristopher Torp JensenAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3870
Anders Lade Nielsen
Anders Lade Nielsen Affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3871
 and Yonglun Luo
Yonglun LuoAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3872
 (*contributed equally to this work) date: 12/5/2016, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2059.

Brief version appeared in Cell Mol Life Sci, Nov 2016
The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional co-transfection of single gRNA expression vectors, the likelihood of delivering several gRNAs into the same cell decreases in accordance with the number of gRNAs. Thus, we have developed a method to efficiently assemble gRNA expression cassettes (2-30 gRNAs) into one single vector using a Golden-Gate assembly method (Vad-Nielsen et al., 2016). In this protocol, we describe the detailed step-by-step instructions for assembly of the multiplexed gRNA expression array. The gRNA scaffold used in our expression array is the gRNA 1.0 system for the Cas9 protein from Streptococcus pyogenes driven by the human U6 promoter. 

Shoot Apical Meristem Size Measurement

Featured protocol,  Authors: Hsuan Chou
Hsuan ChouAffiliation: Department of Biological Sciences, North Carolina State University, Raleigh, USA
For correspondence: hchou2@ncsu.edu
Bio-protocol author page: a3847
Huanzhong Wang
Huanzhong WangAffiliation: Department of Plant Science and Landscape Architecture, Agricultural Biotechnology Laboratory, University of Connecticut, Storrs, USA
Bio-protocol author page: a3848
 and Gerald A. Berkowitz
Gerald A. BerkowitzAffiliation: Department of Plant Science and Landscape Architecture, Agricultural Biotechnology Laboratory, University of Connecticut, Storrs, USA
Bio-protocol author page: a3849
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2055.

Brief version appeared in Plant J, Feb 2016
The shoot apical meristem (SAM) is a collection of cells that continuously renew themselves by cell division and also provide cells to newly developing organs. It has been known that CLAVATA (CLV) 3 peptide regulates a transcription factor WUSCHEL (WUS) to keep numbers of undifferentiated cells constant and maintain the size of the SAM. The interactive feedback control of CLV3 and WUS in a non-cell autonomous signaling cascade determines stem cell fate (maintenance of pluripotency or, alternatively, differentiation into daughter cells) in the SAM. Ca2+ is a secondary messenger that plays a significant role in numerous signaling pathways. The signaling system connecting CLV3 binding to its receptor and WUS expression is not well delineated. We showed that Ca2+ is involved in CLV3 regulation of the SAM size. One of the approaches we used was measuring the size of the SAM. Here we provide a detailed protocol on how to measure Arabidopsis SAM size with Nomarski microscopy. The area of the two-dimensional dome representing the maximal ‘face’ of the SAM was used as a proxy for SAM size. Studies were done on wild type (WT) Arabidopsis in the presence and absence of a Ca2+ channel blocker Gd3+ and the CLV3 peptide, as well on genotypes that lack functional CLV3 (clv3) or a gene encoding a Ca2+-conducting ion channel (‘dnd1’).

Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae

Featured protocol,  Authors: Yuki Kobayashi
Yuki KobayashiAffiliation: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Bio-protocol author page: a3789
 and Kan Tanaka
Kan TanakaAffiliation 1: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Affiliation 2: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Saitama, Japan
For correspondence: kntanaka@res.titech.ac.jp
Bio-protocol author page: a3790
date: 12/5/2016, 35 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2033.

Brief version appeared in Plant Cell Physiol, May 2016
Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced salt stress tolerance (Kobayashi et al., 2016). This report describes the detailed method for the extraction and quantification of ABA in the model red alga C. merolae.

Isolating and Measuring the Growth and Morphology of Pro-Embryogenic Masses in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

Featured protocol,  Authors: Jackellinne Caetano Douétts-Peres
Jackellinne Caetano Douétts-PeresAffiliation: Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil
Bio-protocol author page: a3776
Vanildo Silveira
Vanildo SilveiraAffiliation 1: Laboratório de Biotecnologia, CBB, UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil
Affiliation 2: Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil
Bio-protocol author page: a3777
Marco Antonio Lopes Cruz
Marco Antonio Lopes CruzAffiliation: Laboratório de Biotecnologia Vegetal, Núcleo em Ecologia e Desenvolvimento Sócio-ambiental de Macaé, Universidade Federal do Rio de Janeiro, Macaé, Rio de Janeiro, Brazil
Bio-protocol author page: a3778
 and Claudete Santa-Catarina
Claudete Santa-CatarinaAffiliation: Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil
For correspondence: claudete@uenf.br
Bio-protocol author page: a3779
date: 12/5/2016, 43 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2031.

Brief version appeared in PLoS One, Apr 2016
Embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) can be used as a model to test the effects of compounds added to the culture medium on the cellular growth and morphology of Pro-Embryogenic Masses (PEMs). PEMs are formed by embryogenic and suspensor-type cells. To measure changes in the cellular growth of embryogenic cultures, we performed sedimented cell volume (SCV) quantification, which is a non-destructive method. Morphological analysis by microscopy allowed for the observation of growth and development of PEMs and the alterations in embryogenic and suspensor-type cells. The methods used here provide an efficient means for monitoring the cellular growth of PEMs and identifying morphological changes during the development of embryogenic cultures. These studies can also be combined with biochemical and molecular analyses, such as proteomics, to further investigate embryo growth and morphology.

Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes

Featured protocol,  Authors: Mariana Edith Tellechea*
Mariana Edith TellecheaAffiliation 1: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari,Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Affiliation 2: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3780
Javier Garcia-Pardo*
Javier Garcia-PardoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3781
Juliana Cotabarren
Juliana CotabarrenAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3782
Daniela Lufrano
Daniela LufranoAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3783
Laura Bakas
Laura BakasAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3784
Francesc Xavier Avilés
Francesc Xavier AvilésAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3785
Walter David Obregon
Walter David ObregonAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3786
Julia Lorenzo
Julia LorenzoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
For correspondence: julia.lorenzo@uab.es
Bio-protocol author page: a3787
 and Sebastián Tanco
Sebastián TancoAffiliation 1: Medical Biotechnology Center, VIB, Ghent, Belgium
Affiliation 2: Department of Biochemistry, Ghent University, Ghent, Belgium
For correspondence: sebastian.tanco@vib-ugent.be
Bio-protocol author page: a3788
 (*contributed equally to this work) date: 12/5/2016, 40 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2032.

Brief version appeared in Phytochemistry Dec 2015
Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et al., 2007). In this context, the study and discovery of new MCP inhibitors from plants constitute a valuable approach for the development of new therapeutic strategies. Herein we describe a simple and accessible microplate method for the study of the specific and dose-response carboxypeptidase A inhibitory activities present in Andean potato tubers. Our protocol combines an extraction method optimized for small protein inhibitors in plant tissues, with the measurement of enzyme kinetics using a microplate reader. These instruments are capable of reading small sample volumes, for many samples in a very short time-frame, therefore reducing the time and costs of high-throughput screening experiments. Although this protocol describes the study of Andean potatoes, our approach is also applicable to the analysis other plant samples.

Visualising Differential Growth of Arabidopsis Epidermal Pavement Cells Using Thin Plate Spline Analysis

Featured protocol,  Authors: William Jonathan Armour
William Jonathan ArmourAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
For correspondence: armour.william@gmail.com
Bio-protocol author page: a3748
Deborah Anne Barton
Deborah Anne BartonAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Bio-protocol author page: a3749
 and Robyn Lynette Overall
Robyn Lynette OverallAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Bio-protocol author page: a3750
date: 11/20/2016, 181 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2022.

Brief version appeared in Plant Cell, Sep 2015
Epidermal pavement cells in Arabidopsis leaves and cotyledons develop from relatively simple shapes to form complex cells that have multiple undulations of varying sizes. Analyzing the growth of individual parts of the cell wall boundaries over time is essential to understanding how pavement cells develop their complex shapes. Thin plate spline analysis is a method for visualizing the change of size and shape of objects through warping or deformation of a regular mesh and can be applied to understand cell wall growth. This protocol describes the application of thin plate spline analysis to visualize the development of individual pavement cells over time.

Arabidopsis Seed Germination Assay with Gibberellic Acid

Featured protocol,  Authors: Chunmei Zhong
Chunmei ZhongAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Bio-protocol author page: a3705
Hao Xu
Hao XuAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Bio-protocol author page: a3706
Siting Ye
Siting YeAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Bio-protocol author page: a3707
Shengchun Zhang
Shengchun ZhangAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Bio-protocol author page: a3708
 and Xiaojing Wang
Xiaojing WangAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
For correspondence: wangxj@scnu.edu.cn
Bio-protocol author page: a3709
date: 11/20/2016, 152 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2005.

Brief version appeared in Plant Physiol, Nov 2015
This assay analyzes Arabidopsis seed germination in response to gibberellic acid (GA). During seed imbibition, visible physiological changes allow precise determination of germination rate. This protocol utilizes a stereoscopic microscope to improve characterization of seed germination process.

Documentation of floral secretory glands in Pleurothallidinae (Orchidaceae) using Scanning Electron Microscopy (SEM)

Featured protocol,  Authors: Adam P. Karremans
Adam P. KarremansAffiliation 1: Lankester Botanical Garden, University of Costa Rica, Cartago, Costa Rica
Affiliation 2: Naturalis Biodiversity Center, Leiden, The Netherlands
For correspondence: adam.karremans@ucr.ac.cr
Bio-protocol author page: a3744
Bertie Joan van Heuven
Bertie Joan van HeuvenAffiliation: Naturalis Biodiversity Center, Leiden, The Netherlands
Bio-protocol author page: a3745
Rob Langelaan
Rob LangelaanAffiliation: Naturalis Biodiversity Center, Leiden, The Netherlands
Bio-protocol author page: a3746
 and Barbara Gravendeel
Barbara GravendeelAffiliation: Naturalis Biodiversity Center, Leiden, The Netherlands
Bio-protocol author page: a3747
date: 11/20/2016, 105 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2021.

Brief version appeared in Ann Bot, Sep 2015
A clear, step by step description of the treatment of orchid flowers, subtribe Pleurothallidinae, with Critical Point Drying for SEM is presented. It shows that a simple, short fixation and dehydration method prior to Critical Point Drying is sufficient to obtain good results.

Cryopreservation Protocol for Chlamydomonas reinhardtii

Featured protocol,  Authors: Duanpeng Yang
Duanpeng YangAffiliation 1: Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
Affiliation 2: University of Chinese Academy of Sciences, Beijing, China
Affiliation 3: Institute of Crop Sciences, Fujian Academy of Agricultural Sciences, Fuzhou, China
Affiliation 4: Fujian Engineering Research Center for Characteristic Floriculture, Fuzhou, China
Bio-protocol author page: a3753
 and Weiqi Li
Weiqi LiAffiliation 1: Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
Affiliation 2: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
For correspondence: weiqili@mail.kib.ac.cn
Bio-protocol author page: a3754
date: 11/20/2016, 112 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2024.

Brief version appeared in PLoS One, Jan 2016
Cryopreservation is commonly used for storing viable cells, tissues, organs or organisms at ultralow temperatures, and usually involves immersion in liquid nitrogen at -196 °C. Here we provide a detailed cryopreservation protocol for C. reinhardtii based on Crutchfield’s work (Crutchfield et al., 1999), with minor changes (Yang and Li, 2016). In this study, we compared the cryoprotection effect of two common cryopreservation agents (CPAs), methanol and DMSO. Furthermore, the two-step cryopreservation process was divided into five stages to study the factors affecting the survival rate at each stage. We found that the use of methanol as the CPA, combined with the cooling process outlined here (cooling from 25 °C to -55 °C at a rate of 1 °C/min), were indispensable for cell survival after cryopreservation. The thawing process described here (thawing at 35 °C for 5 min) was also important for increasing the survival rate.

Tandem Purification of His6-3x FLAG Tagged Proteins for Mass Spectrometry from Arabidopsis

Authors: He Huang
He Huang Affiliation: Donald Danforth Plant Science Center, St. Louis, USA
Bio-protocol author page: a3873
 and Dmitri Anton Nusinow
Dmitri Anton NusinowAffiliation: Donald Danforth Plant Science Center, St. Louis, USA
For correspondence: meter@danforthcenter.org
Bio-protocol author page: a3874
date: 12/5/2016, 192 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2060.

[Abstract] Tandem affinity purification is a powerful method to identify protein complexes that function in association with a known gene of interest. This protocol describes a methodology to capture proteins tagged with His6-3x FLAG explicitly for the purpose of on-bead digestion and identification by mass spectrometry. The high sensitivity and specificity of ...

Fusarium graminearum Maize Stalk Infection Assay and Associated Microscopic Observation Protocol

Authors: Juan He
Juan HeAffiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Bio-protocol author page: a3791
Tinglu Yuan
Tinglu Yuan Affiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Bio-protocol author page: a3792
 and Wei-Hua Tang
Wei-Hua TangAffiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
For correspondence: whtang@sibs.ac.cn
Bio-protocol author page: a2037
date: 12/5/2016, 32 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2034.

[Abstract] The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae) causes Gibberella stalk rot in maize (Zea mays) and results in lodging and serious yield reduction. To develop methods to assess the fungal growth and symptom development in maize stalks, we present here a protocol of maize stalk inoculation with conidiospores of fluorescent ...

Measurement of ATP Hydrolytic Activity of Plasma Membrane H+-ATPase from Arabidopsis thaliana Leaves

Authors: Masaki Okumura
Masaki OkumuraAffiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
Bio-protocol author page: a3819
 and Toshinori Kinoshita
Toshinori KinoshitaAffiliation 1: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
Affiliation 2: Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan
For correspondence: kinoshita@bio.nagoya-u.ac.jp
Bio-protocol author page: a3820
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2044.

[Abstract] Plant plasma membrane H+-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H+ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for measurement of the ATP hydrolytic ...

A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9

Authors: Johan Vad-Nielsen*
Johan Vad-NielsenAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
For correspondence: johanvn@biomed.au.dk
Bio-protocol author page: a3868
Lin Lin *
Lin Lin Affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
For correspondence: lin.lin@biomed.au.dk
Bio-protocol author page: a3869
Kristopher Torp Jensen
Kristopher Torp JensenAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3870
Anders Lade Nielsen
Anders Lade Nielsen Affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3871
 and Yonglun Luo
Yonglun LuoAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3872
 (*contributed equally to this work) date: 12/5/2016, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2059.

[Abstract] The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional ...

Shoot Apical Meristem Size Measurement

Authors: Hsuan Chou
Hsuan ChouAffiliation: Department of Biological Sciences, North Carolina State University, Raleigh, USA
For correspondence: hchou2@ncsu.edu
Bio-protocol author page: a3847
Huanzhong Wang
Huanzhong WangAffiliation: Department of Plant Science and Landscape Architecture, Agricultural Biotechnology Laboratory, University of Connecticut, Storrs, USA
Bio-protocol author page: a3848
 and Gerald A. Berkowitz
Gerald A. BerkowitzAffiliation: Department of Plant Science and Landscape Architecture, Agricultural Biotechnology Laboratory, University of Connecticut, Storrs, USA
Bio-protocol author page: a3849
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2055.

[Abstract] The shoot apical meristem (SAM) is a collection of cells that continuously renew themselves by cell division and also provide cells to newly developing organs. It has been known that CLAVATA (CLV) 3 peptide regulates a transcription factor WUSCHEL (WUS) to keep numbers of undifferentiated cells constant and maintain the size of the SAM. The interactive ...

Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae

Authors: Yuki Kobayashi
Yuki KobayashiAffiliation: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Bio-protocol author page: a3789
 and Kan Tanaka
Kan TanakaAffiliation 1: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Affiliation 2: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Saitama, Japan
For correspondence: kntanaka@res.titech.ac.jp
Bio-protocol author page: a3790
date: 12/5/2016, 35 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2033.

[Abstract] Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced salt stress tolerance ...

Isolating and Measuring the Growth and Morphology of Pro-Embryogenic Masses in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

Authors: Jackellinne Caetano Douétts-Peres
Jackellinne Caetano Douétts-PeresAffiliation: Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil
Bio-protocol author page: a3776
Vanildo Silveira
Vanildo SilveiraAffiliation 1: Laboratório de Biotecnologia, CBB, UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil
Affiliation 2: Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil
Bio-protocol author page: a3777
Marco Antonio Lopes Cruz
Marco Antonio Lopes CruzAffiliation: Laboratório de Biotecnologia Vegetal, Núcleo em Ecologia e Desenvolvimento Sócio-ambiental de Macaé, Universidade Federal do Rio de Janeiro, Macaé, Rio de Janeiro, Brazil
Bio-protocol author page: a3778
 and Claudete Santa-Catarina
Claudete Santa-CatarinaAffiliation: Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil
For correspondence: claudete@uenf.br
Bio-protocol author page: a3779
date: 12/5/2016, 43 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2031.

[Abstract] Embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) can be used as a model to test the effects of compounds added to the culture medium on the cellular growth and morphology of Pro-Embryogenic Masses (PEMs). PEMs are formed by embryogenic and suspensor-type cells. To measure changes in the cellular growth of embryogenic ...

Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes

Authors: Mariana Edith Tellechea*
Mariana Edith TellecheaAffiliation 1: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari,Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Affiliation 2: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3780
Javier Garcia-Pardo*
Javier Garcia-PardoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3781
Juliana Cotabarren
Juliana CotabarrenAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3782
Daniela Lufrano
Daniela LufranoAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3783
Laura Bakas
Laura BakasAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3784
Francesc Xavier Avilés
Francesc Xavier AvilésAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3785
Walter David Obregon
Walter David ObregonAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3786
Julia Lorenzo
Julia LorenzoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
For correspondence: julia.lorenzo@uab.es
Bio-protocol author page: a3787
 and Sebastián Tanco
Sebastián TancoAffiliation 1: Medical Biotechnology Center, VIB, Ghent, Belgium
Affiliation 2: Department of Biochemistry, Ghent University, Ghent, Belgium
For correspondence: sebastian.tanco@vib-ugent.be
Bio-protocol author page: a3788
 (*contributed equally to this work) date: 12/5/2016, 40 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2032.

[Abstract] Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et al., 2007). In this context, the study and discovery of new MCP inhibitors ...

Visualising Differential Growth of Arabidopsis Epidermal Pavement Cells Using Thin Plate Spline Analysis

Authors: William Jonathan Armour
William Jonathan ArmourAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
For correspondence: armour.william@gmail.com
Bio-protocol author page: a3748
Deborah Anne Barton
Deborah Anne BartonAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Bio-protocol author page: a3749
 and Robyn Lynette Overall
Robyn Lynette OverallAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Bio-protocol author page: a3750
date: 11/20/2016, 181 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2022.

[Abstract] Epidermal pavement cells in Arabidopsis leaves and cotyledons develop from relatively simple shapes to form complex cells that have multiple undulations of varying sizes. Analyzing the growth of individual parts of the cell wall boundaries over time is essential to understanding how pavement cells develop their complex shapes. Thin plate spline analysis ...

Arabidopsis Seed Germination Assay with Gibberellic Acid

Authors: Chunmei Zhong
Chunmei ZhongAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Bio-protocol author page: a3705
Hao Xu
Hao XuAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Bio-protocol author page: a3706
Siting Ye
Siting YeAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Bio-protocol author page: a3707
Shengchun Zhang
Shengchun ZhangAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Bio-protocol author page: a3708
 and Xiaojing Wang
Xiaojing WangAffiliation: Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
For correspondence: wangxj@scnu.edu.cn
Bio-protocol author page: a3709
date: 11/20/2016, 152 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2005.

[Abstract] This assay analyzes Arabidopsis seed germination in response to gibberellic acid (GA). During seed imbibition, visible physiological changes allow precise determination of germination rate. This protocol utilizes a stereoscopic microscope to improve characterization of seed germination process.

[Background] Seed germination is a critical process of ...

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Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves Updates
The author made some updates (highlighted in blue) to the protocol on 09/19/2016.

Authors: Arsalan Daudi
Arsalan DaudiAffiliation 1: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Affiliation 2: Department of Plant Pathology, University of California, Davis, CA, USA
For correspondence: aadaudi@ucdavis.edu
Bio-protocol author page: a107
 and Jose A. O’Brien
Jose A. O’BrienAffiliation: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Bio-protocol author page: a108
date: 9/20/2012, 29506 views, 15 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.263.

[Abstract] In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi ...

[Bio101] Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/20/2011, 29068 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.95.

[Abstract] Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, ...

[Bio101] Pollen Fertility/viability Assay Using FDA Staining

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 5/20/2011, 15307 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.75.

[Abstract] Pollen grains can be fertile or sterile by nature. This method stains pollen grains for an enzyme as the vital indicator of membrane integrity. Only fertile grains fluoresce under microscopic examination....

[Bio101] Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/5/2011, 14668 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.93.

[Abstract] Promoter-driven GUS (beta-glucuronidase) activity is the most commonly used technique for tissue-specific expression patterns in Arabidopsis. In this procedure, GUS enzyme converts 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) to a blue product. The staining is very sensitive. Processed samples can ...

[Bio101] Arabidopsis Pollen Tube Aniline Blue Staining

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Scienc, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 6/20/2011, 14469 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.88.

[Abstract] The aim of this experiment is to track pollen tube growth in vivo in the female tissues after pollination. This can be used to phenotype pollen germination, tube growth and guidance, and reception....

[Bio101] Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well)

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/5/2011, 13529 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.90.

[Abstract] This is a simple protocol for isolating genomic DNA from fresh plant tissues. DNA from this experiment can be used for all kinds of genetics studies, including genotyping and mapping. This protocol uses Edward’s extraction buffer to isolate DNA....

In vitro Protein Ubiquitination Assays

Authors: Qingzhen Zhao
Qingzhen ZhaoAffiliation: College of Life Science, Liaocheng University, Liaocheng, China
Bio-protocol author page: a887
 and Qi Xie
Qi XieAffiliation: State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology Chinese Academy of Sciences, Beijing, China
For correspondence: qxie@genetics.ac.cn
Bio-protocol author page: a888
date: 10/5/2013, 12834 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.928.

[Abstract] Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity ...

[Bio101] Arabidopsis Growing Protocol – A General Guide

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 9/5/2011, 12187 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.126.

[Abstract] Arabidopsis as the model organism for higher plants is widely studied among plant biology labs around the world. However, taking care of this tiny plant may not be trivial. Here is a general guide used for the Heven Sze lab at the University of Maryland, College Park. A lot of efforts have been taken ...

[Bio101] A Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 5/20/2011, 10616 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.70.

[Abstract] This method can be used to free and separate the mesophyll cells from Arabidopsis leaves. The protoplasts that are generated in this way can be used for transient expression for protein activity and subcellular localization assays....

Seed Germination and Viability Test in Tetrazolium (TZ) Assay

Authors: Pooja Verma
Pooja VermaAffiliation: NIPGR, National Institute of Plant Genome Research, New Delhi, India
Bio-protocol author page: a799
 and Manoj Majee
Manoj MajeeAffiliation: NIPGR, National Institute of Plant Genome Research, New Delhi, India
For correspondence: manojmajee@nipgr.ac.in
Bio-protocol author page: a800
date: 9/5/2013, 10516 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.884.

[Abstract] Tetrazolium (TZ) assay is the fast evaluation for seed viability and alternative quick method for seed’s germinability (Porter et al., 1947; Wharton, 1955). All respiring tissues are capable of converting a colourless compound, TZ (2,3,5 triphenyl tetrazolium chloride) to a carmine red coloured water-insoluble ...
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