Chapter 20

Using Clear Nail Polish to Make Arabidopsis Epidermal

Impressions for Measuring the Change of Stomatal
Aperture Size in Immune Response
Shuchi Wu and Bingyu Zhao

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

Healthy plants can effectively prevent most microbes to get inside

of plant tissue, and limit the colonization of the microbes on leaf
surface as epiphytes [1, 2]. While the plant surface, poor with
nutrient, is not a “hospitable” growth environment for most
microbes [2]. The epiphytes on leaf surface can be easily washed
off by heavy rain or killed by intensive UV light [3]. And the dra-
matic fluctuation of temperature and humidity on leaf surface also
cast severe stress to most microbial cells [2]. Comparing to the
“barren” surface, the inside plant mesophyll tissue is a much “fer-
tile” land for microbial pathogens [4]. Therefore, microbial patho-
gens developed a number of distinct strategies to entry host plant
tissue. For example, fungal pathogens (e.g., Hyaloperonospora
parasitica) evolved a set of elaborate piercing structures such as
penetration peg and haustorium to break through plant surface,
and form infection structures inside of mesophyll tissues [5].

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

Unlike fungal pathogens, bacterial pathogens do not equip such

elaborate piercing structures. Therefore, they usually entry through
plant surface wounds and natural openings, such as stomata, lenti-
cels and hydathode [6, 7]. The stomata are the most common
natural openings on leaf surface and therefore become one of the
most important routes for bacterial invasion.
For most plant species, stomatal aperture size is controlled by
two guard cells [8, 9]. It can reduce the aperture size to inhibit
water loss, and increase the aperture size to promote gas exchange
[8, 9]. During microbe pathogen invasion, some pathogen-­
associated molecular patterns (PAMPs) such as flg22, a conserved
domain of bacterial flagellin can be recognized by host plant
immune receptor FLS2, and trigger defense responses called
PAMP-triggered immunity (PTI). The defense signal involves in
salicylic acid (SA) and abscisic acid (ABA) signaling pathways that
regulate guard cells to close the stomatal aperture [10]. On the
other hand, some plant bacterial pathogens evolved ability to
induce the reopening of stomata [10–12]. For example, phyto-
toxin coronatine produced by Psuedomonas syringae (P. syringae)
strains can reopen stomata closed by the PTI [10]. More recently,
Xanthomonas campestris was reported to secrete a small unidenti-
fied molecular that can enforce stomatal reopening [13]. Fungal
toxins, fusicoccin, produced by Fusicoccum amygdali has the ability
of stomatal regulation [14]. Oxalate produced by fungal pathogen
Sclerotinia sclerotiorum can also enhance stomatal opening [15].
Therefore, measurement of stomatal aperture size is an essential and
necessary indicator for studying the stomata related immunity.
Several methods have been developed to prepare microscope
slides of leaf epidermal impressions for measuring the stomatal
aperture size, including directly peeling the epidermis of leaf [10,
16, 17], and using nail polish, silicone adhesive, or stamp pad to
make leaf surface imprint [18, 19]. Because Arabidopsis leaves are
fragile and not easy to make leaf surface imprint, the epidermal
peeling method becomes the most popular approach in the study
of Arabidopsis stomata related immunity. However, in the process
of bacterial invasion, as well as the influence of humidity and circa-
dian, the stomatal guard cells are always in a dynamic status of
opening, closing and reopening [17, 20, 21]. Thus the epidermal
peeling method has a significant deficient that it cannot fix the
stomata opening status, and makes the stomata counting and sto-
matal aperture size measurement unrepeatable.
Here, we are described an optimized protocol of using clear
nail polish to make Arabidopsis epidermal impressions, and mon-
itor the change of stomatal aperture size in plant immune
response. Suitable clear nail polish can instantly fix the status of
stomatal guard cells and provides clear, stable, and almost perma-
nent slides of epidermal impressions for measurement of stomatal
aperture size.
 Stomatal Aperture Size in Plant Immunity Response 245

2 Materials

1. Plant material: Four-week-old Arabidopsis Co-0 plants grown

in growth chamber (50% relative humidity, 100 μE/m2/s
light, 10 h light/22 °C and 14 h dark/20 °C).
2. Stomata opening buffer: 10 mM KCl, 10 mM MES, pH 5.7.
3. Stomata closure elicitors (PAMP): 5 μM flg22 peptide
(GenScript, NJ).
4. Stomata closure elicitors (plant hormone): 10 μM Abscisic acid
(ABA) (Sigma-Aldrich, MO).
5. Nail polish: Sally Hansen® Hard As Nails® Xtreme wear® Nail
Color Invisible (see Note 1).
6. Scissors, forceps, slides, and scotch tape.
7. Light microscope: Zeiss Axio Observer A1 (Carl Zeiss
MicroImaging, Inc., NY).
8. Software: AxioVision LE (Carl Zeiss MicroImaging, Inc., NY).

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

3.2 Induce 1. Prepare stomata opening buffer with or without 5 μM flg22

the Stomata Opening peptide or 10 μM ABA.
and Closure 2. Cut off similar sized matured leaves from Arabidopsis plants
that have been treated for 3 h at dark, and float the leaves in
stomata opening solutions.
3. Incubate the leaves floating in stomata opening buffer at room
temperature under light for 3 h.
4. Take out the leaves and wipe off the buffer residue on the leaf
surface.
5. Follow the protocol from steps 3 to 10 above to make slides
of epidermal impressions.
6. Take pictures of prepared slides under 400× amplification light
microscope (Fig. 2).
7. Use the “ruler tools” to measure the width of 30 stomatal
apertures for each treatment.

4 Notes

1. Be aware of other “clear nail polish” like makeup, such as nail

strengthener or nail base. These functional “nail polishes” are
not easy to be peeled off.
 Stomatal Aperture Size in Plant Immunity Response 247

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

2. Do not use very young leaves, which are too fragile.

3. Do not use chemical hood to air dry the nail polish paint,
otherwise the leaves will also become dried and stick with the
nail polish film.
4. Use the tips of forceps to test if the nail polish is dried.
5. Do not try to peel off the entire nail polish film, otherwise
the film will get stretched. Use scissors to cut off 0.5 cm nail
polish film, which is enough for stomata observation and
measurement.
6. To use another object slide instead of a cover slide that can bet-
ter protect the nail polish films.
7. The nail polish films will shrink slowly in low humidity.

Acknowledgments

This work was supported by the National Science Foundation.

References
1. Jones JD, Dangl JL (2006) The plant immune
system. Nature 444(7117):323–329
2. Lindow SE, Brandl MT (2003) Microbiology
of the phyllosphere. Appl Environ Microbiol
69(4):1875–1883
3. Andrews JH, Harris RF (2000) The ecology
and biogeography of microorganisms on plant
surfaces. Annu Rev Phytopathol
38(1):145–180
4. Beattie GA, Lindow SE (1995) The secret life
of foliar bacterial pathogens on leaves. Annu
Rev Phytopathol 33(1):145–172
5. Wilson M, Hirano S, Lindow S (1999)
Location and survival of leaf-associated bacte-
ria in relation to pathogenicity and potential
for growth within the leaf. Appl Environ
Microbiol 65(4):1435–1443
6. Bunster L, Fokkema NJ, Schippers B (1989)
Effect of surface-active Pseudomonas spp. on
leaf wettability. Appl Environ Microbiol
55(6):1340–1345
7. Quigley NB, Gross D (1994) Syringomycin
production among strains of Pseudomonas
syringae pv. syringae: conservation of the syrB
248 Shuchi Wu and Bingyu Zhao
and syrD genes and activation of phytotoxin
production by plant signal molecules. Mol
Plant Microbe Interact 7:78–78
8. Fan LM, Zhao Z, Assmann SM (2004) Guard
cells: a dynamic signaling model. Curr Opin
Plant Biol 7(5):537–546
9. Schroeder JI et al (2001) Guard cell signal
transduction. Annu Rev Plant Biol 52(1):
627–658
10. Melotto M et al (2006) Plant stomata function
in innate immunity against bacterial invasion.
Cell 126(5):969–980
11. Mittal S, Davis KR (1995) Role of the phyto-
toxin coronatine in the infection of Arabidopsis
thaliana by Pseudomonas syringae pv. tomato.
Mol Plant Microbe Interactions 8(1):165–171
12. Schulze-Lefert P, Robatzek S (2006) Plant
pathogens trick guard cells into opening the
gates. Cell 126(5):831–834
13. Gudesblat GE, Torres PS, Vojnov AA (2009)
Xanthomonas campestris overcomes
Arabidopsis stomatal innate immunity through
a DSF cell-to-cell signal-regulated virulence
factor. Plant Physiol 149(2):1017–1027
14. Turner NC, Graniti A (1969) Fusicoccin: a
fungal toxin that opens stomata. Nature
223(5210):1070–1071
15. Godoy G et al (1990) Use of mutants to dem-
onstrate the role of oxalic acid in pathogenicity
of Sclerotinia sclerotiorum on Phaseolus vul-
garis. Physiol Mol Plant Pathol 37(3):
179–191
16. Desikan R et al (2002) A new role for an old
enzyme: nitrate reductase-mediated nitric
oxide generation is required for abscisic acid-­
induced stomatal closure in Arabidopsis thali-
ana. Proc Natl Acad Sci U S A 99(25):
16314–16318
17. Zeng W, He SY (2010) A prominent role of
the flagellin receptor FLAGELLIN-­
SENSING2 in mediating stomatal response to
Pseudomonas syringae pv tomato DC3000 in
Arabidopsis. Plant Physiol 153(3):1188–1198
18. Hilu KW, Randall JL (1984) Convenient
method for studying grass leaf epidermis.
Taxon 33:413–415
19. Behera S, Kudla J (2013) Live cell imaging of
cytoplasmic Ca2+ dynamics in Arabidopsis
guard cells. Cold Spring Harb Protoc
2013(7):pdb.prot072983
20. Mao J et al (2005) A role for Arabidopsis
cryptochromes and COP1 in the regulation of
stomatal opening. Proc Natl Acad Sci U S A
102(34):12270–12275
21. Mustilli A-C et al (2002) Arabidopsis OST1
protein kinase mediates the regulation of sto-
matal aperture by abscisic acid and acts
upstream of reactive oxygen species produc-
tion. Plant Cell 14(12):3089–3099