Professional Documents
Culture Documents
Nail Polish - Stomata - Method
Nail Polish - Stomata - Method
Nail Polish - Stomata - Method
Abstract
Plant stomata are an essential route for bacterial pathogens to entry inside host tissue and cause diseases.
As an important defense mechanism, plant stomata can actively restrict bacterial invasion by dynamically
regulating the opening, closing, and reopening of stomatal guard cells. Therefore, accurately measuring
the stomatal aperture size during the bacterial pathogenesis is an important approach to study the stomata
related immunity. Several methods have been developed for stomatal aperture measurement. Here, we
described a detailed protocol of using clear nail polish to make Arabidopsis epidermal impressions for
investigate the change of stomatal aperture size in plant immune response. The application of this approach
can instantly fix the status of stomatal guard cells, and provides clear, stable, and almost permanent slides
of epidermal impressions for measurement of stomatal aperture size.
Key words Nail polish, Arabidopsis thaliana, Epidermal impressions, Stomatal guard cell
1 Introduction
Libo Shan and Ping He (eds.), Plant Pattern Recognition Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 1578,
DOI 10.1007/978-1-4939-6859-6_20, © Springer Science+Business Media LLC 2017
243
244 Shuchi Wu and Bingyu Zhao
2 Materials
3 Methods
3.1 Prepare Slides 1. Move Arabidopsis plants out of growth chamber, cover the
of Epidermal whole flat with deep dome-shape lid (Fig. 1a), and incubate in
Impressions dark at room temperature for 3 h.
2. Cut off similar sized matured leaves from Arabidopsis plants
(Fig. 1b) (see Note 2).
3. Paint a thin layer of nail polish (see Note 1) on the lower epi-
dermis (Fig. 1c).
4. Incubate the leaves at room temperature to air dry the nail pol-
ish for about 10 min (see Notes 3 and 4).
5. Use the tip of forceps to punch a small wound at the edge of
the nail polished area (Fig. 1d) (see Note 5).
6. Carefully start to peel off dried nail polish film with forceps
from the wound site.
7. Stop peeling when about 0.5 cm nail polish film has been
peeled off, and use scissors to cut off the peeled off nail polish
film.
8. Transfer the nail polish film onto the microscope object slide
and covered it with another object slide (Fig. 1e) (see Note 6).
9. Carefully press tight the slide and use scotch tape to seal the
edge of the slides.
10. Keep the slides in a box container in the room temperature
(see Note 7).
246 Shuchi Wu and Bingyu Zhao
Fig. 1 The process of making Arabidopsis epidermal impressions. (1) Four-week-old Arabidopsis plants were
maintained in higher humidity. (2) Detached leaf used for making the leaf reprint. (3) Apply the finger nail pol-
ish. (4) Peel the thin layer of film with a sharp forceps. (5) Mount the imprinted film on slide for microscope
observation
4 Notes
Fig. 2 The stomata on the nail polish films under 400× amplification. The left one is under 3 h dark treatment,
and the right three are under another 3 h light treatment, with or without 5 μM flg22 peptide or 10 μM ABA.
The legend bar represents 20 μm
Acknowledgments
References
1. Jones JD, Dangl JL (2006) The plant immune 5. Wilson M, Hirano S, Lindow S (1999)
system. Nature 444(7117):323–329 Location and survival of leaf-associated bacte-
2. Lindow SE, Brandl MT (2003) Microbiology ria in relation to pathogenicity and potential
of the phyllosphere. Appl Environ Microbiol for growth within the leaf. Appl Environ
69(4):1875–1883 Microbiol 65(4):1435–1443
3. Andrews JH, Harris RF (2000) The ecology 6. Bunster L, Fokkema NJ, Schippers B (1989)
and biogeography of microorganisms on plant Effect of surface-active Pseudomonas spp. on
surfaces. Annu Rev Phytopathol leaf wettability. Appl Environ Microbiol
38(1):145–180 55(6):1340–1345
4. Beattie GA, Lindow SE (1995) The secret life 7. Quigley NB, Gross D (1994) Syringomycin
of foliar bacterial pathogens on leaves. Annu production among strains of Pseudomonas
Rev Phytopathol 33(1):145–172 syringae pv. syringae: conservation of the syrB
248 Shuchi Wu and Bingyu Zhao
and syrD genes and activation of phytotoxin of Sclerotinia sclerotiorum on Phaseolus vul-
production by plant signal molecules. Mol garis. Physiol Mol Plant Pathol 37(3):
Plant Microbe Interact 7:78–78 179–191
8. Fan LM, Zhao Z, Assmann SM (2004) Guard 16. Desikan R et al (2002) A new role for an old
cells: a dynamic signaling model. Curr Opin enzyme: nitrate reductase-mediated nitric
Plant Biol 7(5):537–546 oxide generation is required for abscisic acid-
9. Schroeder JI et al (2001) Guard cell signal induced stomatal closure in Arabidopsis thali-
transduction. Annu Rev Plant Biol 52(1): ana. Proc Natl Acad Sci U S A 99(25):
627–658 16314–16318
10. Melotto M et al (2006) Plant stomata function 17. Zeng W, He SY (2010) A prominent role of
in innate immunity against bacterial invasion. the flagellin receptor FLAGELLIN-
Cell 126(5):969–980 SENSING2 in mediating stomatal response to
11. Mittal S, Davis KR (1995) Role of the phyto- Pseudomonas syringae pv tomato DC3000 in
toxin coronatine in the infection of Arabidopsis Arabidopsis. Plant Physiol 153(3):1188–1198
thaliana by Pseudomonas syringae pv. tomato. 18. Hilu KW, Randall JL (1984) Convenient
Mol Plant Microbe Interactions 8(1):165–171 method for studying grass leaf epidermis.
12. Schulze-Lefert P, Robatzek S (2006) Plant Taxon 33:413–415
pathogens trick guard cells into opening the 19. Behera S, Kudla J (2013) Live cell imaging of
gates. Cell 126(5):831–834 cytoplasmic Ca2+ dynamics in Arabidopsis
13. Gudesblat GE, Torres PS, Vojnov AA (2009) guard cells. Cold Spring Harb Protoc
Xanthomonas campestris overcomes 2013(7):pdb.prot072983
Arabidopsis stomatal innate immunity through 20. Mao J et al (2005) A role for Arabidopsis
a DSF cell-to-cell signal-regulated virulence cryptochromes and COP1 in the regulation of
factor. Plant Physiol 149(2):1017–1027 stomatal opening. Proc Natl Acad Sci U S A
14. Turner NC, Graniti A (1969) Fusicoccin: a 102(34):12270–12275
fungal toxin that opens stomata. Nature 21. Mustilli A-C et al (2002) Arabidopsis OST1
223(5210):1070–1071 protein kinase mediates the regulation of sto-
15. Godoy G et al (1990) Use of mutants to dem- matal aperture by abscisic acid and acts
onstrate the role of oxalic acid in pathogenicity upstream of reactive oxygen species produc-
tion. Plant Cell 14(12):3089–3099