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Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination

Featured protocol,  Authors: Jason Bosch
Jason BoschAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Bio-protocol author page: a4828
 and Armin Djamei
Armin DjameiAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
For correspondence: armin.djamei@gmi.oeaw.ac.at
Bio-protocol author page: a4829
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2392.

Brief version appeared in Elife, Nov 2016
Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black fungal spores. While it is possible to directly use this spore material to infect Brachypodium seeds, in many cases it is more useful to isolate individual strains of U. bromivora for a genetically homogenous population. This protocol describes how to collect and germinate the spores of U. bromivora on plate in order to obtain strains derived from a single cell.

Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC

Featured protocol,  Authors: Sheo Shankar Pandey
Sheo Shankar PandeyAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4903
Prashantee Singh
Prashantee SinghAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4904
Biswajit Samal
Biswajit SamalAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4905
Raj Kumar Verma
Raj Kumar VermaAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4906
 and Subhadeep Chatterjee
Subhadeep ChatterjeeAffiliation: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
For correspondence: subhadeep@cdfd.org.in
Bio-protocol author page: a4907
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2410.

Brief version appeared in PLoS Pathog, Nov 2016
Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces less siderophores than Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). Siderophore production can be measured by HPLC and with the CAS (Chrome azurol S)-agar plate assay, however HPLC is a more accurate method over CAS-agar plate assay for siderophore quantification in Xanthomonads. Here we describe how to quantify siderophores from xanthomonads using HPLC.

GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression

Featured protocol,  Authors: Zhao Liu
Zhao LiuAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4858
Wei Wang
Wei WangAffiliation: Basic Forestry and Proteomics Research Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, China
Bio-protocol author page: a4859
Chun-Guang Zhang
Chun-Guang ZhangAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4860
Jun-Feng Zhao
Jun-Feng ZhaoAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4861
 and Yu-Ling Chen
Yu-Ling Chen Affiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
For correspondence: yulingchen@mail.hebtu.edu.cn
Bio-protocol author page: a4862
date: 7/20/2017, 12 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2446.

Brief version appeared in Plant Physiol, Jun 2017
Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both mesophyll and guard cells, it is hard to exhibit a clear expression of the gene in guard cells by a GUS staining image from leaf. To gain a clear guard cell GUS image of small G protein ROP7, a gene expressed in both mesophyll and guard cells, we peeled the epidermal strips from the leaf of 3-4 week-old plants. After removing the mesophyll cells, the epidermal strips were used for GUS staining. We compared the GUS staining images from epidermal strips or leaf of small G protein ROP7 and RopGEF4, a gene specifically expressed in guard cells, and found that GUS staining of epidermal strips provided a good method to show the guard cell expression of a gene expressed in both mesophyll and guard cells. This protocol is applicable for any genes that are expressed in guard cells of Arabidopsis, or other plants that epidermal strips can be easily peeled from the leaf.

Rice Lamina Joint Inclination Assay

Featured protocol,  Authors: Hsing-Yi Li*
Hsing-Yi LiAffiliation: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Bio-protocol author page: a4901
Hsin-Mei Wang*
Hsin-Mei WangAffiliation: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Bio-protocol author page: a4902
 and Seonghoe Jang
Seonghoe JangAffiliation 1: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Affiliation 2: Institute of Tropical Plant Science, National Cheng Kung University, Tainan, Taiwan
For correspondence: florigen@gate.sinica.edu.tw
Bio-protocol author page: a4900
 (*contributed equally to this work) date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2409.

Brief version appeared in Plant Physiol, Jan 2017
Brassinosteroids (BRs) promote rice lamina inclination. Recently, we showed that OsBUL1 knockout mutant rice (osbul1) is defective in brassinosteroid signaling (Jang et al., 2017). To show that lamina joint inclination of osbul1 is less-sensitive than WT to exogenous brassinolide (BL) treatment in the lamina joint inclination bioassays, we applied the protocol presented below. The protocol focuses on: (1) how to prepare rice samples for the assay, and (2) how to treat BL exogenously. Finally, we have added a result showing lamina inclination between WT and osbul1 in BL solutions of various concentrations.

Assessing Plant Tolerance to Acute Heat Stress

Featured protocol,  Authors: Minsoo Kim
Minsoo KimAffiliation: Department of Biochemistry & Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA
Bio-protocol author page: a4885
Fionn McLoughlin
Fionn McLoughlinAffiliation: Department of Biochemistry & Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA
Present address: Washington University in St. Louis, St. Louis, MO, USA
Bio-protocol author page: a4887
Eman Basha
Eman BashaAffiliation: Department of Botany, Tanta University, Tanta, Egypt
Bio-protocol author page: a4888
 and Elizabeth Vierling
Elizabeth VierlingAffiliation: Department of Biochemistry & Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA
For correspondence: vierling@biochem.umass.edu
Bio-protocol author page: a4886
date: 7/20/2017, 19 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2405.

Brief version appeared in Plant Physiol, Oct 2016
It is well-established that plants are able to acclimate to temperatures above or below the optimal temperature for their growth. Here, we provide protocols for assays that can be used quantitatively or qualitatively to assess the relative ability of plants to acquire tolerance to high temperature stress. The hypocotyl elongation assay described was developed to screen for mutants defective in the acquisition of tolerance to extreme temperature stress, and other assays were developed to further characterize mutant and transgenic plants for heat tolerance of other processes or at other growth stages. Although the protocols provide details for application to Arabidopsis thaliana, the same basic methods can be adopted to assay heat tolerance in other plant species.

Isolation of Fucus serratus Gametes and Cultivation of the Zygotes

Featured protocol,  Authors: Amandine Siméon
Amandine SiméonAffiliation: CNRS–UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, France
For correspondence: amandine.simeon@sb-roscoff.fr
Bio-protocol author page: a4898
 and Cécile Hervé
Cécile HervéAffiliation: CNRS–UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, France
For correspondence: cecile.herve@sb-roscoff.fr
Bio-protocol author page: a4899
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2408.

Brief version appeared in J Exp Bot, Nov 2016
Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from sexually mature fronds in the laboratory and thousands of synchronously developing zygotes are easily obtained. In contrast to other eukaryotic models, such as land plants (Brownlee and Berger, 1995), the embryo is free of maternal tissues and therefore readily amenable to pharmacological approaches. The zygotes are relatively large (up to 100 µm in diameter), facilitating manipulations and imaging studies. During the first hours of zygote development, the alignment of the axis to external cues such as light is labile and can be reversed by light gradients from different directions. A few hours before rhizoid emergence, the alignment of the axis and the polarity are fixed and the cells germinate accordingly. At this stage the zygotes are naturally attached to the substratum through the secretion of cell wall adhesive materials (Kropf et al., 1988; Hervé et al., 2016). The first cell division occurs about 24 h after fertilisation and the early embryo is composed of only two cell types that differ in size, shape and developmental fates (i.e., thallus cells and rhizoid cells) (Bouget et al., 1998). The embryo can be successfully cultivated in the laboratory for a few more days (4 weeks maximum) and has an invariant division pattern during the early stages, which allows cell lineages to be traced histologically.

Non-invasive Protocol for Kinematic Monitoring of Root Growth under Infrared Light

Featured protocol,  Authors: François Bizet
François BizetAffiliation: EEF, INRA, Université de Lorraine, Champenoux, France
Bio-protocol author page: a4822
Lionel X. Dupuy
Lionel X. DupuyAffiliation: James Hutton Institute, Dundee, UK
Bio-protocol author page: a2070
Anthony Glyn Bengough
Anthony Glyn BengoughAffiliation 1: James Hutton Institute, Dundee, UK
Affiliation 2: School of Science and Engineering, University of Dundee, Dundee, UK
For correspondence: glyn.bengough@hutton.ac.uk
Bio-protocol author page: a4823
Alexis Peaucelle
Alexis PeaucelleAffiliation: Institut Jean-Pierre Bourgin, INRA, CNRS, AgroParisTech, Université Paris-Saclay, Versailles, France
Bio-protocol author page: a4824
Irène Hummel*
Irène HummelAffiliation: EEF, INRA, Université de Lorraine, Champenoux, France
Bio-protocol author page: a4825
 and Marie-Béatrice Bogeat-Triboulot*
Marie-Béatrice Bogeat-TriboulotAffiliation: EEF, INRA, Université de Lorraine, Champenoux, France
For correspondence: marie-beatrice.bogeat@inra.fr
Bio-protocol author page: a4826
 (*contributed equally to this work) date: 7/20/2017, 20 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2390.

Brief version appeared in J Exp Bot, Oct 2016
Phenotyping the dynamics of root responses to environmental cues is necessary to understand plant acclimation to their environment. Continuous monitoring of root growth is challenging because roots normally grow belowground and are very sensitive to their growth environment. This protocol combines infrared imaging with hydroponic cultivation for kinematic analyses. It allows continuous imaging at fine spatiotemporal resolution and disturbs roots minimally. Examples are provided of how the procedure and materials can be adapted for 3D monitoring and of how environmental stress may be manipulated for experimental purposes.

Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis

Featured protocol,  Authors: Francisco J Corpas
Francisco J CorpasAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
For correspondence: javier.corpas@eez.csic.es
Bio-protocol author page: a4857
Larisse de Freitas-Silva
Larisse de Freitas-SilvaAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4864
Nuria García-Carbonero
Nuria García-CarboneroAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4865
Alba Contreras
Alba ContrerasAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4866
Fátima Terán
Fátima TeránAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4867
Carmelo Ruíz-Torres
Carmelo Ruíz-TorresAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4868
 and José M. Palma
José M. PalmaAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4869
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2399.

Brief version appeared in Physiol Plant, Feb 2009
6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, chloroplast, mitochondria and peroxisomes (Corpas et al., 1998; Krepinsky et al., 2001; Mateos et al., 2009; Fernández-Fernández and Corpas, 2016; Hölscher et al., 2016). Using Arabidopsis thaliana as plant model and sweet pepper (Capsicum annuum L.) fruits as a plant with agronomical interest, this protocol illustrates how to prepare the plant extracts for the separation of the potential 6PGDH isoforms by electrophoresis on 6% polyacrylamide non-denaturing gels. Thus, this method allows detecting three 6PGDH isoforms in Arabidopsis seedlings and two 6PGDH isoforms in sweet pepper fruits.

Determination of the Effects of Local and Systemic Iron Excess on Lateral Root Initiation in Arabidopsis thaliana

Featured protocol,  Authors: Guangjie Li
Guangjie LiAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4793
Lin Zhang
Lin ZhangAffiliation 1: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Affiliation 2: University of the Chinese Academy of Sciences, No.19(A) Yuquan Road, Shijingshan District, Beijing 100049, China
Bio-protocol author page: a384
 and Weiming Shi
Weiming ShiAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
For correspondence: wmshi@issas.ac.cn
Bio-protocol author page: a1896
date: 7/5/2017, 159 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2387.

Brief version appeared in Plant Physiol, Dec 2015
Root system architecture depends on nutrient availability. A symptom of iron (Fe) toxicity in plants is stunted root growth, yet little is known about the effects of excess Fe on lateral root (LR) development. To better understand how nutrient signals are integrated into root developmental programs, we investigated the morphological response of Arabidopsis thaliana root systems to Fe by testing homogeneous supply and localized Fe supply treatment.

Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea

Featured protocol,  Authors: Giovanna Salbitani
Giovanna SalbitaniAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
Bio-protocol author page: a4677
Claudia Bottone
Claudia BottoneAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
Bio-protocol author page: a4765
 and Simona Carfagna
Simona CarfagnaAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
For correspondence: simcarfa@unina.it
Bio-protocol author page: a4678
date: 7/5/2017, 146 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2372.

Brief version appeared in Plant Cell Physiol, Sep 2016
Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.

Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9

Featured protocol,  Authors: Florian Hahn
Florian HahnAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4806
Marion Eisenhut
Marion EisenhutAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4807
Otho Mantegazza
Otho MantegazzaAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4808
 and Andreas P. M. Weber
Andreas P. M. WeberAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
For correspondence: Andreas.Weber@uni-duesseldorf.de
Bio-protocol author page: a4809
date: 7/5/2017, 224 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2384.

Brief version appeared in Front Plant Sci, Jan 2017
The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. The procedure can likely be adapted for other transformable plant species.

A Simple and Rapid Assay for Measuring Phytoalexin Pisatin, an Indicator of Plant Defense Response in Pea (Pisum sativum L.)

Featured protocol,  Authors: Lee A. Hadwiger
Lee A. HadwigerAffiliation: Department of Plant Pathology, Washington State University, Pullman, WA, USA
For correspondence: chitosan@wsu.edu
Bio-protocol author page: a4744
 and Kiwamu Tanaka
Kiwamu TanakaAffiliation: Department of Plant Pathology, Washington State University, Pullman, WA, USA
Bio-protocol author page: a4745
date: 7/5/2017, 167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2362.

Brief version appeared in Molecules, Dec 2014
Phytoalexins are antimicrobial substance synthesized in plants upon pathogen infection. Pisatin (Pisum sativum phytoalexin) is the major phytoalexin in pea, while it is also a valuable indicator of plant defense response. Pisatin can be quantitated in various methods from classical organic chemistry to Mass-spectrometry analysis. Here we describe a procedure with high reproducibility and simplicity that can easily handle large numbers of treatments. The method only requires a spectrophotometer as laboratory equipment, does not require any special analytical instruments (e.g., HPLC, mass spectrometers, etc.) to measure the phytoalexin molecule quantitatively, i.e., most scientific laboratories can perform the experiment.

Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination

Authors: Jason Bosch
Jason BoschAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Bio-protocol author page: a4828
 and Armin Djamei
Armin DjameiAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
For correspondence: armin.djamei@gmi.oeaw.ac.at
Bio-protocol author page: a4829
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2392.

[Abstract] Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black fungal spores. While it is possible to directly ...

Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC

Authors: Sheo Shankar Pandey
Sheo Shankar PandeyAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4903
Prashantee Singh
Prashantee SinghAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4904
Biswajit Samal
Biswajit SamalAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4905
Raj Kumar Verma
Raj Kumar VermaAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4906
 and Subhadeep Chatterjee
Subhadeep ChatterjeeAffiliation: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
For correspondence: subhadeep@cdfd.org.in
Bio-protocol author page: a4907
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2410.

[Abstract] Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces ...

GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression

Authors: Zhao Liu
Zhao LiuAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4858
Wei Wang
Wei WangAffiliation: Basic Forestry and Proteomics Research Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, China
Bio-protocol author page: a4859
Chun-Guang Zhang
Chun-Guang ZhangAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4860
Jun-Feng Zhao
Jun-Feng ZhaoAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4861
 and Yu-Ling Chen
Yu-Ling Chen Affiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
For correspondence: yulingchen@mail.hebtu.edu.cn
Bio-protocol author page: a4862
date: 7/20/2017, 12 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2446.

[Abstract] Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both mesophyll ...

Rice Lamina Joint Inclination Assay

Authors: Hsing-Yi Li*
Hsing-Yi LiAffiliation: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Bio-protocol author page: a4901
Hsin-Mei Wang*
Hsin-Mei WangAffiliation: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Bio-protocol author page: a4902
 and Seonghoe Jang
Seonghoe JangAffiliation 1: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Affiliation 2: Institute of Tropical Plant Science, National Cheng Kung University, Tainan, Taiwan
For correspondence: florigen@gate.sinica.edu.tw
Bio-protocol author page: a4900
 (*contributed equally to this work) date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2409.

[Abstract] Brassinosteroids (BRs) promote rice lamina inclination. Recently, we showed that OsBUL1 knockout mutant rice (osbul1) is defective in brassinosteroid signaling (Jang et al., 2017). To show that lamina joint inclination of osbul1 is less-sensitive than WT to exogenous brassinolide (BL) treatment in the lamina joint inclination bioassays, we applied ...

Assessing Plant Tolerance to Acute Heat Stress

Authors: Minsoo Kim
Minsoo KimAffiliation: Department of Biochemistry & Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA
Bio-protocol author page: a4885
Fionn McLoughlin
Fionn McLoughlinAffiliation: Department of Biochemistry & Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA
Present address: Washington University in St. Louis, St. Louis, MO, USA
Bio-protocol author page: a4887
Eman Basha
Eman BashaAffiliation: Department of Botany, Tanta University, Tanta, Egypt
Bio-protocol author page: a4888
 and Elizabeth Vierling
Elizabeth VierlingAffiliation: Department of Biochemistry & Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA
For correspondence: vierling@biochem.umass.edu
Bio-protocol author page: a4886
date: 7/20/2017, 19 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2405.

[Abstract] It is well-established that plants are able to acclimate to temperatures above or below the optimal temperature for their growth. Here, we provide protocols for assays that can be used quantitatively or qualitatively to assess the relative ability of plants to acquire tolerance to high temperature stress. The hypocotyl elongation assay described was ...

Isolation of Fucus serratus Gametes and Cultivation of the Zygotes

Authors: Amandine Siméon
Amandine SiméonAffiliation: CNRS–UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, France
For correspondence: amandine.simeon@sb-roscoff.fr
Bio-protocol author page: a4898
 and Cécile Hervé
Cécile HervéAffiliation: CNRS–UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, France
For correspondence: cecile.herve@sb-roscoff.fr
Bio-protocol author page: a4899
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2408.

[Abstract] Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from sexually ...

Non-invasive Protocol for Kinematic Monitoring of Root Growth under Infrared Light

Authors: François Bizet
François BizetAffiliation: EEF, INRA, Université de Lorraine, Champenoux, France
Bio-protocol author page: a4822
Lionel X. Dupuy
Lionel X. DupuyAffiliation: James Hutton Institute, Dundee, UK
Bio-protocol author page: a2070
Anthony Glyn Bengough
Anthony Glyn BengoughAffiliation 1: James Hutton Institute, Dundee, UK
Affiliation 2: School of Science and Engineering, University of Dundee, Dundee, UK
For correspondence: glyn.bengough@hutton.ac.uk
Bio-protocol author page: a4823
Alexis Peaucelle
Alexis PeaucelleAffiliation: Institut Jean-Pierre Bourgin, INRA, CNRS, AgroParisTech, Université Paris-Saclay, Versailles, France
Bio-protocol author page: a4824
Irène Hummel*
Irène HummelAffiliation: EEF, INRA, Université de Lorraine, Champenoux, France
Bio-protocol author page: a4825
 and Marie-Béatrice Bogeat-Triboulot*
Marie-Béatrice Bogeat-TriboulotAffiliation: EEF, INRA, Université de Lorraine, Champenoux, France
For correspondence: marie-beatrice.bogeat@inra.fr
Bio-protocol author page: a4826
 (*contributed equally to this work) date: 7/20/2017, 20 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2390.

[Abstract] Phenotyping the dynamics of root responses to environmental cues is necessary to understand plant acclimation to their environment. Continuous monitoring of root growth is challenging because roots normally grow belowground and are very sensitive to their growth environment. This protocol combines infrared imaging with hydroponic cultivation for kinematic ...

Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis

Authors: Francisco J Corpas
Francisco J CorpasAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
For correspondence: javier.corpas@eez.csic.es
Bio-protocol author page: a4857
Larisse de Freitas-Silva
Larisse de Freitas-SilvaAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4864
Nuria García-Carbonero
Nuria García-CarboneroAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4865
Alba Contreras
Alba ContrerasAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4866
Fátima Terán
Fátima TeránAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4867
Carmelo Ruíz-Torres
Carmelo Ruíz-TorresAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4868
 and José M. Palma
José M. PalmaAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4869
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2399.

[Abstract] 6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, chloroplast, ...

Determination of the Effects of Local and Systemic Iron Excess on Lateral Root Initiation in Arabidopsis thaliana

Authors: Guangjie Li
Guangjie LiAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4793
Lin Zhang
Lin ZhangAffiliation 1: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Affiliation 2: University of the Chinese Academy of Sciences, No.19(A) Yuquan Road, Shijingshan District, Beijing 100049, China
Bio-protocol author page: a384
 and Weiming Shi
Weiming ShiAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
For correspondence: wmshi@issas.ac.cn
Bio-protocol author page: a1896
date: 7/5/2017, 159 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2387.

[Abstract] Root system architecture depends on nutrient availability. A symptom of iron (Fe) toxicity in plants is stunted root growth, yet little is known about the effects of excess Fe on lateral root (LR) development. To better understand how nutrient signals are integrated into root developmental programs, we investigated the morphological response of Arabidopsis ...

Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea

Authors: Giovanna Salbitani
Giovanna SalbitaniAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
Bio-protocol author page: a4677
Claudia Bottone
Claudia BottoneAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
Bio-protocol author page: a4765
 and Simona Carfagna
Simona CarfagnaAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
For correspondence: simcarfa@unina.it
Bio-protocol author page: a4678
date: 7/5/2017, 146 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2372.

[Abstract] Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric ...
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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, 34354 views, 16 Q&A
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, 33012 views, 0 Q&A
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, 17343 views, 2 Q&A
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, 16479 views, 4 Q&A
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 ...

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, 16041 views, 0 Q&A
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 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, 15987 views, 4 Q&A
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, 15720 views, 1 Q&A
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....

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, 15290 views, 0 Q&A
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 ...

[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, 14421 views, 1 Q&A
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, 11888 views, 0 Q&A
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....
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