Plant MAP Kinases, Softcover reprint of the original 1st ed. 2014
Methods and Protocols
Methods in Molecular Biology Series, Vol. 1171

Mitogen-activated protein kinases (MAPKs) are versatile phosphorylating enzymes which regulate multiple proteins involved in gene expression, cell architecture, plant development and reaction to diverse abiotic and biotic factors. The main aim of Plant MAP Kinases: Methods and Protocols is to provide established and new MAPK protocols adapted to the challenges posed by working with plants. The book contains 19 chapters which encompass a wide array of methods progressively scaling from the single gene, protein or cell level to large-scale arrays of proteomic, phosphoproteomic and interactomic data in order to uncover previously unidentified plant MAPK signaling pathways and to tackle with the challenging task of substrate identification. Techniques for MAPK sequence analysis and subcellular localization helping to identify their substrates and subcellular compartmentalization are also provided. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and key tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, Plant MAP Kinases: Methods and Protocols represents a collection of useful plant MAPK protocols written by experts in the field for researchers and students.

PART I: Expression and Activity of Plant MAPKs

1. Transient Expression in Arabidopsis Leaf Mesophyll Protoplast System for Cell-based Functional Analysis of MAPK Cascades Signaling

Jong Hee Im and Sang-Dong Yoo

2. Quantification of Stress Induced Mitogen-activated Protein Kinase Expressional Dynamic using Quantitative Real-time PCR

Pavel Krenek and Veronika Smékalová

3. Analysis of MAPK Activities using MAPK-specific Antibodies

Roland Willmann, Daniel J. Haischer, and Andrea A. Gust

4. Detection of Protein Phosphorylation and Charge Isoforms using Vertical one-Dimensional Isoelectric Focusing Gels

Jeffrey C. Anderson and Scott C. Peck

5. Affinity-based SDS PAGE Identification of Phosphorylated Arabidopsis MAPKs and Substrates by Acrylamide Pendant Phos-Tag™

George Komis, Tomáš Takáč, Slávka Bekešová, Pavol Vadovič, and Jozef Šamaj

PART II: Genetic Manipulation of MAPK Function

6. Identification of Constitutively Active AtMPK6 Mutants using a Functional Screen in Saccharomyces Cerevisiae

7. Virus-induced Gene Silencing in Plant MAPK Research

Christian Hettenhausen, Ian T. Baldwin, and Jianqiang Wu

8. RNA Interference of Plant MAPK Cascades for Functional Studies

Juan Xu and Shuqun Zhang

PART III: Localization Studies of MAPKs and Their Interactions

9. Immunofluorescent Localization of MAPKs and Colocalization with Microtubules in Arabidopsis Seedling Whole Mount Probes

Olga Šamajová, George Komis, and Jozef Šamaj

10. Immunofluorescent Localization of MAPKs in Steedman’s Wax Sections

Miroslav Ovečka, Olga Šamajová, František Baluška, and Jozef Šamaj

11. Fluorescent Protein Tagging of Arabidopsis MAPKs for In Vivo Localization Studies

Anna Doskočilová, Ivan Luptovčiak, Veronika Smékalová, and Jozef Šamaj

12. Bimolecular Fluorescent Complementation (BiFC) by MAP Kinases and MAPK Phosphatases

Alois Schweighofer, Volodymyr Shubchynskyy, Vaiva Kazanaviciute, Armin Djamei, and Irute Meskiene

Part IV: Analysis of MAPK Substrates

13. Determination of Phosphorylation Sites in Microtubule Associated Protein MAP65-1

Andrei Smertenko

14. In vivo Phosphorylation of WRKY Transcription Factor by MAPK

Nobuaki Ishihama, Hiroaki Adachi, Miki Yoshioka,  and Hirofumi Yoshioka

15. Rapid Mutagenesis-based Analysis of Phosphorylation Sites in Mitogen-activated Protein Kinase Substrates

Lennart Eschen-Lippold, Nicole Bauer, Julia Löhr, Mieder A.T. Palm-Forster, and Justin Lee

PART V: Large Scale Studies on MAPKs, Substrates, and Interactions

16. Yeast Two-hybrid System for Dissecting the Rice MAPK Interactome

Raksha Singh, Sarmina Dangol, and Nam-Soo Jwa

17. Experimental and Analytical Approaches to Characterize Plant Kinases using Protein

Microarrays

Elizabeth K. Brauer, Sorina C. Popescu, and George V. Popescu 

18 Protein Complexes Characterization in Arabidopsis thaliana by Tandem Affinity Purification Coupled to Mass Spectrometry Analysis

Jean Bigeard, Delphine Pflieger, Jean Colcombet, Loïc Gérard, Hakim Mireau and Heribert Hirt

19 Quantitative Phosphoproteomic Analysis using iTRAQ Method

Asano T and Nishiuchi T

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts

Includes supplementary material: sn.pub/extras