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Preparation of Epidermal Peels and Guard Cell Protoplasts for Cellular, Electrophysiological, and -Omics Assays of Guard Cell Function

  • Mengmeng Zhu
  • Byeong Wook Jeon
  • Sisi Geng
  • Yunqing Yu
  • Kelly Balmant
  • Sixue Chen
  • Sarah M. AssmannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1363)

Abstract

Bioassays are commonly used to study stomatal phenotypes. There are multiple options in the choice of plant materials and species used for observation of stomatal and guard cell responses in vivo. Here, detailed procedures for bioassays of stomatal responses to abscisic acid (ABA) in Arabidopsis thaliana are described, including ABA promotion of stomatal closure, ABA inhibition of stomatal opening, and ABA promotion of reaction oxygen species (ROS) production in guard cells. We also include an example of a stomatal bioassay for the guard cell CO2 response using guard cell-enriched epidermal peels from Brassica napus. Highly pure preparations of guard cell protoplasts can be produced, which are also suitable for studies on guard cell signaling, as well as for studies on guard cell ion transport. Small-scale and large-scale guard cell protoplast preparations are commonly used for electrophysiological and -omics studies, respectively. We provide a procedure for small-scale guard cell protoplasting from A. thaliana. Additionally, a general protocol for large-scale preparation of guard cell protoplasts, with specifications for three different species, A. thaliana, B. napus, and Vicia faba is also provided.

Key words

Electrophysiology Epidermal peels Guard cells Guard cell-enriched epidermal peels Protoplasting Reactive oxygen species (ROS) Stomatal movement 

Notes

Acknowledgements

Research on guard cell signaling in the Assmann laboratory is supported by BARD grant IS-4541-12 and by NSF grants IOS-1025837, MCB-1121612, MCB-1157921, and MCB-1412644 to S.M.A. Research on guard cell signaling in the Chen laboratory is supported by NSF grants MCB- 0818051, MCB-1158000, and MCB-1412547 to S.C.

References

  1. 1.
    Hetherington AM, Woodward FI (2003) The role of stomata in sensing and driving environmental change. Nature 424:901–908CrossRefPubMedGoogle Scholar
  2. 2.
    Sirichandra C, Wasilewska A, Vlad F, Valon C, Leung J (2009) The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action. J Exp Bot 60:1439–1463CrossRefPubMedGoogle Scholar
  3. 3.
    Gudesblat GE, Torres PS, Vojnov AA (2009) Stomata and pathogens. Plant Signal Behav 4:1114–1116PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Kollist H, Nuhkat M, Roelfsema MR (2014) Closing gaps: linking elements that control stomatal movement. New Phytol 203:44–62CrossRefPubMedGoogle Scholar
  5. 5.
    Yin Y, Adachi Y, Ye W, Hayashi M, Nakamura Y, Kinoshita T et al (2013) Difference in abscisic acid perception mechanisms between closure induction and opening inhibition of stomata. Plant Physiol 163:600–610PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Zhao Z, Zhang W, Stanley BA, Assmann SM (2008) Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways. Plant Cell 20:3210–3226PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Acharya BR, Jeon BW, Zhang W, Assmann SM (2013) Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis guard cells. New Phytol 200:1049–1063CrossRefPubMedGoogle Scholar
  8. 8.
    Turner NC, Schulze ED, Gollan T (1984) The responses of stomata and leaf gas exchange to vapor pressure deficits and soil water content. Oecologia 63:338–342CrossRefGoogle Scholar
  9. 9.
    Afzal M, Matsugo S, Sasai M, Xu B, Aoyama K, Takeuchi T (2003) Method to overcome photoreaction, a serious drawback to the use of dichlorofluorescin in evaluation of reactive oxygen species. Biochem Biophys Res Commun 304:619–624CrossRefPubMedGoogle Scholar
  10. 10.
    Wang P, Song CP (2008) Guard-cell signaling for hydrogen peroxide and abscisic acid. New Phytol 178:703–718CrossRefPubMedGoogle Scholar
  11. 11.
    Hedrich R (2012) Ion channels in plants. Physiol Rev 92:1777–1811CrossRefPubMedGoogle Scholar
  12. 12.
    Zhang W, Nilson SE, Assmann SM (2008) Isolation and whole-cell patch clamping of Arabidopsis guard cell protoplasts. CSH Protoc 2008:pdb.prot5014Google Scholar
  13. 13.
    Jin X, Wang RS, Zhu M, Jeon BW, Albert R, Chen S et al (2013) Abscisic acid-responsive guard cell metabolomes of Arabidopsis wild-type and gpa1 G-protein mutants. Plant Cell 25:4789–4811PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Leonhardt N, Kwak JM, Robert N, Waner D, Leonhardt G, Schroeder JI (2004) Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell 16:596–615PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Zhu M, Dai S, McClung S, Yan X, Chen S (2009) Functional differentiation of Brassica napus guard cells and mesophyll cells revealed by comparative proteomics. Mol Cell Proteomics 8:752–766PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Zhu M, Zhu N, Song W, Harmon A, Assmann SM, Chen S (2014) Thiol-based redox proteins in Brassica napus guard cell abscisic acid and methyl jasmonate signaling. Plant J 78:491–515PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Mott KA, Sibbernsen ED, Shope JC (2008) The role of the mesophyll in stomatal responses to light and CO2. Plant Cell Environ 31:1299–1306CrossRefPubMedGoogle Scholar
  18. 18.
    Yakir E, Hassidim M, Melamed-Book N, Hilman D, Kron I, Green RM (2011) Cell autonomous and cell-type specific circadian rhythms in Arabidopsis. Plant J 68:520–531CrossRefPubMedGoogle Scholar
  19. 19.
    Wang RS, Pandey S, Li S, Gookin TE, Zhao Z, Albert R et al (2011) Common and unique elements of the ABA-regulated transcriptome of Arabidopsis guard cells. BMC Genomics 12:216PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Pandey S, Wang RS, Wilson L, Li S, Zhao Z, Gookin TE et al (2010) Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action. Mol Syst Biol 6:372PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Fan LM, Zhao Z, Assmann SM (2004) Guard cells: a dynamic signaling model. Curr Opin Plant Biol 7:537–546CrossRefPubMedGoogle Scholar
  22. 22.
    Pandey S, Wang X, Coursol SA, Assmann SM (2002) Preparation and applications of Arabidopsis thaliana guard cell protoplasts. New Phytol 153:517–526CrossRefGoogle Scholar
  23. 23.
    Yang Y, Costa A, Leonhardt N, Siegel RS, Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool. Plant Methods 4:6–21PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Zhao Z, Stanley BA, Zhang W, Assmann SM (2010) ABA-regulated G protein signaling in Arabidopsis guard cells: a proteomic perspective. J Proteome Res 9:1637–1647CrossRefPubMedGoogle Scholar
  25. 25.
    Zhu M, Simons B, Zhu N, Oppenheimer DG, Chen S (2010) Analysis of abscisic acid responsive proteins in Brassica napus guard cells by multiplexed isobaric tagging. J Proteomics 73:790–805CrossRefPubMedGoogle Scholar
  26. 26.
    Kruse T, Tallman G, Zeiger E (1989) Isolation of guard cell protoplasts from mechanically prepared epidermis of Vicia faba leaves. Plant Physiol 90:1382–1386PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Davies WJ, Zhang J (1991) Root signals and the regulation of growth and development of plants in drying soil. Annu Rev Plant Biol 42:55–76CrossRefGoogle Scholar
  28. 28.
    Cornish K, Zeevaart JA (1985) Abscisic acid accumulation by roots of Xanthium strumarium L. and Lycopersicon esculentum Mill. in relation to water stress. Plant Physiol 79:653–658PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333CrossRefPubMedGoogle Scholar
  30. 30.
    Gaedeke N (2001) The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement. EMBO J 20:1875–1887PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Hashimoto M, Negi J, Young J, Israelsson M, Schroeder JI, Iba K (2006) Arabidopsis HT1 kinase controls stomatal movements in response to CO2. Nat Cell Biol 8:391–397CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Mengmeng Zhu
    • 1
  • Byeong Wook Jeon
    • 1
  • Sisi Geng
    • 2
  • Yunqing Yu
    • 1
  • Kelly Balmant
    • 2
  • Sixue Chen
    • 2
  • Sarah M. Assmann
    • 1
    Email author
  1. 1.Biology DepartmentPenn State UniversityUniversity ParkUSA
  2. 2.Plant Molecular and Cellular Biology Program, Department of Biology, Genetics InstituteUniversity of FloridaGainesvilleUSA

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