1. Research article
A novel hydrogen sulfide donor causes stomatal opening and reduces
nitric oxide accumulation
M. Lisjak a
, N. Srivastava b
, T. Teklic a
, L. Civale b
, K. Lewandowski b
, I. Wilson b
, M.E. Wood c
,
M. Whiteman d
, J.T. Hancock b,*
a
Department of Agroecology, University of J. J. Strossmayer, Osijek, Croatia
b
Faculty of Health and Life Sciences, University of the West of England, Coldharbour Lane, Bristol, BS16 1QY, UK
c
School of Biosciences, University of Exeter, UK
d
Peninsula Medical School, University of Exeter, Exeter, UK
a r t i c l e i n f o
Article history:
Received 17 June 2010
Accepted 22 September 2010
Available online 1 October 2010
Keywords:
GYY4137
Hydrogen sulfide
NaSH
Nitric oxide
Stomata
a b s t r a c t
Effects of hydrogen sulfide (H2S) on plant physiology have been previously studied, but such studies have
relied on the use of NaSH as a method for supplying H2S to tissues. Now new compounds which give
a less severe H2S shock and a more prolonged exposure to H2S have been developed. Here the effects of
one such compound, GYY4137, has been investigated to determine its effects on stomatal closure in
Arabidopsis thaliana. It was found that both NaSH and GYY4137 caused stomatal opening in the light and
prevented stomatal closure in the dark. Nitric oxide (NO) has been well established as a mediator of
stomatal movements and here it was found that both NaSH and GYY4137 reduced the accumulation of
NO in guard cells, perhaps suggesting a mode of action for H2S in this system. GYY4137, and future
related compounds, will be important tools to unravel the effects of plant exposure to H2S and to
determine how H2S may fit into plant cell signalling pathways.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
It is now well established that a variety of reactive chemicals are
involved in the control of cellular events in plants. These chemicals
include reactive oxygen species [1] such as hydrogen peroxide and
reactive nitrogen species such as nitric oxide [2]. Such chemicals
may be produced endogenously by the cell itself or may in fact
arrive at a plant cell from the outside, perhaps from another cell or
tissue. Many of the downstream events modulated by ROS and NO
are now well established and include the alteration of the activity of
proteins such as kinases, phosphatases and transcription factors
[1]. However, other compounds such as carbon monoxide and
hydrogen sulfide have also been suggested to have effects on cell
signalling pathways [3].
Hydrogen sulfide is often thought to be a phytotoxin, being
harmful to the growth and development of plants. It was found to
inhibit oxygen release from young seedlings of six rice cultivars [4],
but it was also noted that although in some cultivars nutrient
uptake was reduced in other cultivars it was increased. Phosphorus
uptake was inhibited in this plant species. Thompson and Kats [5]
treated a variety of plants with continuous fumigation of H2S. In
Medicago, grapes, lettuce, sugar beets, pine and fir 3000 parts per
billion (ppb) H2S caused lesions on leaves, defoliation and reduced
growth of the plants supporting the role of H2S as a phytotoxin.
However lower levels of fumigation, 100ppb, caused a significant
increase in the growth of Medicago, lettuce and sugar beets [5].
Quite recently, Zhang et al. [6], showed that the H2S donor NaSH
would alleviate the osmotic-induced decrease in chlorophyll
concentration in sweetpotato. Furthermore, spraying NaSH
increased the activity of the antioxidant enzymes superoxide dis-
mutase, catalase, ascorbate peroxidase while decreasing the
concentration of hydrogen peroxide and lipoxygenase, and it was
suggested that H2S has a role in protection against oxidative stress
in plants. Supporting this hypothesis are the findings that fumiga-
tion of spinach increased glutathione levels [7], and it was esti-
mated that approximately 40% of the H2S was converted to
glutathione in the leaves. On cessation of fumigation glutathione
levels once again fell, with the levels being comparable to control
levels after 48 h of no H2S treatment. Therefore, clearly H2S can
have intracellular effects which impinge on cell signalling events in
the cells.
As well as effects on plants, many species of plant have been
found to generate H2S, suggesting that it may be an endogenous
chemical and suitable to be acting as signalling molecule. Using
* Corresponding author.
E-mail address: john.hancock@uwe.ac.uk (J.T. Hancock).
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage: www.elsevier.com/locate/plaphy
0981-9428/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2010.09.016
Plant Physiology and Biochemistry 48 (2010) 931e935

2. a sulfur-specific flame photometric detector, Wilson et al. [8]
showed that cucumber, squash, pumpkin, soybean and cotton,
amongst other plants, were able to generate volatile sulfur
compounds such as H2S using a light-dependent activity. If the
roots were supplied sulfate and if the plants were illuminated the
emissions lasted for several hours. Furthermore, if leaves were fed
sulfate through their petioles, or the roots of plants were
mechanically damaged, the rate of H2S emissions were significantly
increased. Sekiya et al. [9] reported that young leaves emit much
more H2S than mature leaves, using cucumber as their model
species. H2S release from plants was further confirmed by Ren-
nenberg [10]. It was found that leaves from pumpkins emitted H2S
if supplied with sulfate, sulfite, cysteine or SO2, but different
metabolic routes were used for different sulfur sources.
However, H2S can also be generated and accumulate outside the
plant, but still have effects on plant growth and survival. For
example H2S may be released into the atmosphere [11], and so be
present at the surface of plants.
Both ROS and NO can cause stomatal closure [12,13], and clearly
leaves are exposed to both exogenous H2S from the atmosphere
[11], and endogenous H2S from its cellular synthesis [8], and
therefore the effects of H2S on the closure of stomata were studied
using Arabidopsis thaliana as a model plant.
2. Materials and methods
2.1. Plant growth and synthesis of GYY4137
Wild-type, both Landsberg erecta and Columbia ecotypes, A.
thaliana were sown in Levington’s F2 compost (Avoncrop, Bristol,
UK) and grown under a 12-h photo-period in plant growth cham-
bers (Sanyo Gallenkamp,Loughborough, UK). Fully expanded wild-
type leaves were harvested at 4e5 weeks and used immediately.
To generate H2S sodium hydrosulfide (NaSH) or a novel water
soluble donor morpholin-4-ium 4 methoxyphenyl(morpholino)
phosphinodithioate (GYY4137) were used. When added to aqueous
solution, NaSH generates H2S instantaneously [14e16]. In contrast,
H2S release from GYY4137 is slow and sustained and more readily
models enzymatically derived synthesis of H2S [14e16]. GYY4137
was synthesised as previously described [14e16].
2.2. Stomatal assays
Stomatal bioassays were carried out as described previously by
Desikan et al. [17]. Whole leaves were incubated in MES-KCl buffer
[10 mM 2-morpholino ethane sulfonic acid (MES), 5 mM KCl, 50 mM
CaCl2, pH 6.15] for 2.5 h under light conditions (eg 20 mE mÀ2
sÀ1
)
before the addition of various compounds, or left in the dark (see
appropriate figure legends). Thewholeleaves were usedand placedin
the Petri dishes (lower epidermis downwards) containing 1, 10 or
100 mM NaSH or GYY4137 in 3 mL MES, pH 6.15. Stomatal apertures
were observed in epidermal fragments after further 2 h incubation.
Apertures were measured using a light microscope (40Â) and
imaging camera with LEICA QWIN image processing and analysis
software (LeicaMicrosystems and Imaging Solutions, Cambridge, UK).
2.3. Measurement of nitric oxide using DAF2-DA
Nitric oxide accumulation was estimated using the specific NO
dye DAF2-DA (Calbiochem, Nottingham, UK), using the method
described previously by Desikan et al. [17]. Epidermal fragments in
MES-KCl buffer (10 mM MES, 5 mM KCl, 50 mM CaCl2, pH 6.15) were
loaded with 15 mM DAF2-DA for 15 min before washing with MES-
KCl buffer for 20 min. Fragments were subsequently incubated for
a further 25 min in the presence of various compounds (as indicated
in figure legends) before images were visualized. Images were
visualized using CLSM (excitation 488 nm, emission 515 nm; Nikon
PCM2000, Kingston-upon-Thames, UK). Images acquired were
analysed using SCION IMAGE software (Scion, Frederick, MD, USA).
3. Results
To assess whether H2S, like NO or ROS [12,13] could cause
stomatal closure, leaves were exposed to light to open the stomata,
and then treated with the plant hormone abscisic acid (ABA) or the
H2S donor NaSH. As can be seen in Fig. 1, ABA caused a significant
closure of the stomata following a 2.5 h treatment. However, no
closure was seen on NaSH treatment, and in fact the stomatal
opened more in a dose-dependent manner. Even with 1 mM NaSH
treatment a significant amount of opening compared to the control
was seen.
New compounds have been developed which release H2S more
slowly than NaSH, and over a longer period of time [14e16], which
would be more representative of the physiological generation of
H2S. Therefore the ability for H2S to affect stomatal movements was
assessed using the compound GYY4137. Once again, no closure was
seen and the stomata were more open than the control, albeit to
a lesser extent than seen with NaSH (Fig. 2). Maximal opening was
seen with 10 mM GYY4137, and interesting as the concentration was
increased the effects were annulled, with the stomata opening
being comparable to that of controls.
As H2S appeared to cause opening the experiment was repeated
without the previous exposure to high light to cause opening prior to
treatment with the H2S releasing compound GYY4137. As expected
the control aperture size was smaller and ABA caused closure, but
once again treatment with GYY4137 resulted in stomatal opening,
with the effect being dose dependent (Fig. 3). High concentrations
had a larger effect than seen with pre-light treatment (Fig. 2), with
opening increasing up to 200 mM GYY4137 (Fig. 3).
Data in Figs. 2 and 3 show that in the light GYY4137 caused
stomatal opening so the effects of both NaSH and GYY4137 were
assessed following dark treatment, a condition which would cause
0
1
2
3
4
5
Aperture(µm)
Fig. 1. NaSH treatment causes stomatal opening. Leaf samples of Arabidopsis thaliana
ecotype Columbia were placed in MES buffer (pH 6.15) and exposed to direct lighting
for 2.5 h. Samples were sheltered from direct lighting and treated with ABA or NaSH
for next 2.5 h, and stomata apertures were analysed as in Section 2.
M. Lisjak et al. / Plant Physiology and Biochemistry 48 (2010) 931e935932

3. a physiological closure of the stomata. After 2 h in the dark control
stomata were seen to close (Fig. 4), but if treated simultaneously
with either NaSH or GYY4137 the stomata did not close as much,
with 100 mM GYY4137 treatment showing stomata open to the
same extent as the samples which had been treated with light. A
similar trend was seen if the treatments were carried out over 4 h,
although the effects of both GYY4137 and NaSH were less.
The treatment of leaves with either ABA or darkness will cause
the production of NO, and therefore DAF2-DA mediated fluores-
cence was used in conjunction with confocal microscopy to assess
the accumulation of NO in samples which had been treated with
H2S, either administered as NaSH or GYY4137. To assess the ability
of the compounds to interfere with the DAF-based assays, the levels
of DAF2-based fluorescence which was caused by NO release from
SNP was assessed in a fluorimeter in the presence and absence of
NaSH or GYY4137. No reduction in DAF2 fluorescence was seen
(data not shown), suggesting that these compounds do no interfere.
Therefore in the presence of these compounds DAF2 would be
a representative measure of NO accumulation as reported by others
[18]. As can be seen in Fig. 5 control untreated samples showed no
increase in DAF2-mediated fluorescence, whereas ABA treatment
caused a large increase in the NO-mediated signal seen in the guard
cells. Treatment with either NaSH or GYY4137 on their own did
cause a small rise in fluorescence, suggesting that these compounds
may stimulate NO production, or reduce NO scavenging in the
stomata. However, more significantly, when GYY4137 or NaSH were
added at the same time as ABA, the fluorescence seen was
considerably less than when ABA was added on it own, suggesting
that the GYY4137 or NaSH had reduced the NO accumulation which
usually results from ABA treatment (Fig. 5).
4. Discussion
It is well established that reactive gaseous compounds such as
NO can have profound effects on plant physiology, and in fact the
effects of H2S have been studied previously by several other groups
[5,6]. However, new compounds are now being developed which
will release H2S in a more gradual manner [14e16]. This is more
representative of long term exposure to H2S, rather than a sudden
0
1
2
3
4Aperture(µm)
Fig. 2. Treatment with GYY4137 causes stomatal opening. Leaf samples of Arabidopsis
thaliana ecotype Columbia were placed in MES buffer (pH 6.15) and exposed to direct
lighting for 2.5 h. Samples were sheltered from direct lighting and treated with ABA or
GYY4137 for next 2.5 h, and stomata apertures were analysed as in Section 2.
0
1
2
3
4
Aperture(µm)
Fig. 3. Treatment with GYY4137 causes stomatal opening when not previously treated
with light. Leaf samples of Arabidopsis thaliana ecotype Landsberg erecta were placed
in MES buffer (pH 6.15). Leaf samples were not exposed to high lighting which would
encourage stomatal opening, but treated directly with ABA or GYY. Stomata apertures
were analysed 2.5 h after the exposure to day light while being treated.
0
1
2
3
4
Aperture(µm)
Fig. 4. Treatment with H2S donors causes stomata to open in the dark. Leaf samples
were put into the MES buffer, pH 6.15, treated with GYY4137 and NaSH and put into the
darkness or day light for 2 or 4 h with appropriate treatments: L, light; D, darkness.
M. Lisjak et al. / Plant Physiology and Biochemistry 48 (2010) 931e935 933

4. and not prolonged exposure which would result from the use of
NaSH, which previous studies have used [6]. This is the first report
of the effects of such compounds on plant tissues, and shows that
the effects of GYY4137 mimic well the effects seen with NaSH, and
could be used more extensively to study the effects of H2S on plant
function. Here the focus was on stomatal function, and to deter-
mine if like NO, H2S caused stomatal closure. Previously it has been
reported that transpiration rates of several species of plants
including maize, pumpkins and spinach were unaffected by short-
term exposure to atmospheric H2S [19], but here it was found that
H2S donors caused increased stomatal opening, both after opening
in the high light, or during exposure to ambient light. Furthermore,
H2S donors appeared to stop the stomatal closure due to dark
exposure. This would increase the rate of transpiration in the dark
at a time when plants would normally close their stomata and
reduce water loss.
NO is well established as a mediator of stomatal movements,
and interacts with other signalling compounds involved in that
system. It is known to be downstream of ABA [20] and to be
involved in hydrogen peroxide mediated stomatal movements [21].
Therefore the effects of NaSH and GYY4137 on NO accumulation
were investigated. As expected, ABA caused a significant accumu-
lation of NO as estimated by DAF2-DA fluorescence, but both NaSH
and GYY4137 reduced this accumulation to a large extent (Fig. 5).
Neither concentrations used completely removed the NO, but the
presence of NO was considerably less. In vitro studies of DAF2-
mediated NO fluorescence using NO donors showed no indication
that either NaSH or GYY4137 interfered with the DAF2-mediated
estimation of NO accumulation (not shown). As NO is involved in
the signalling pathways which cause stomatal closure it is tempting
to speculate that the fact that H2S removes NO could account for
why these H2S donors did not cause closure in the dark, and in fact
caused opening in the light. It is not just here that an interaction
between NO and H2S is being proposed. In animal systems such
interactions have already been reported [22]. Quenching of NO by
H2S has been previously observed in vivo in isolated tissues and in
mammalian cell culture [23,24] with the formation of a ‘nitro-
sothiol-like’ intermediate. It has also been suggested that in animal
systems the H2S inhibits NO generation through inhibition of nitric
oxide synthase [25]; although such an enzyme has yet to be iden-
tified in plants [26]. However, it will be important to establish the
effects both in vitro and in vivo of H2S donors on enzymes likely to
be involved in NO signalling, such as nitrate reductase [17]. It will
also be important to measure the ability of H2S donors to reduce NO
accumulation in other plants systems such as roots [27], and to use
other measurements of NO such as EPR [28] to confirm DAF2 data.
In summary, H2S donors, including a new one never before used
in plants, cause stomata to open in the light and the dark, and
furthermore they reduce ABA-mediated accumulation of NO in
these cells. Clearly such new H2S donors as GYY4137 will be
important in future work to unravel the impact of H2S exposure to
plant tissues and to determine how H2S fits with other cell sig-
nalling compounds.
Acknowledgements
We would like to thank the British Scholarship Trust and Croa-
tian Ministry of Science, Education and Sports (research project
Physiological mechanisms of plant tolerance to abiotic stress e
079-0790494-0559) for financial support.
References
[1] J.T. Hancock, The role of redox mechanisms in cell signalling, Mol. Biotechnol.
43 (2009) 162e166.
[2] I. Wilson, S.J. Neill, J.T. Hancock, Nitric oxide signalling in plants, Plant Cell
Environ. 31 (2008) 622e631.
[3] A.K. Mustafa, M.M. Gadalla, S.H. Snyder, Signaling by gasotransmitters, Sci.
Signall. 2 (2009) re2.
[4] M.M. Joshi, I.K.A. Ibrahium, J.P. Hollis, Hydrogen sulphide: effects on the
physiology of rice plants and relation to straighthead disease, Phytophatology
65 (1975) 1170e1175.
[5] C.R. Thompson, G. Kats, Effects of continuous hydrogen sulphide fumigation
on crop and forest plants, Environ. Sci. Technol. 12 (1978) 550e553.
[6] H. Zhang, Y.-K. Ye, S.-H. Wang, J.-P. Luo, J. Tang, D.-F. Ma, Hydrogen sulphide
counteracts chlorophyll loss in sweetpotato seedling leaves and alleviates
oxidative damage against osmotic stress, Plant Growth Regul. 58 (2009)
243e250.
[7] L.J. De Kok, W. Bosma, F.M. Maas, P.J.C. Kuiper, The effect of short-term H2S
fumigation on water-soluble sulphydryl and glutathione levels in spinach,
Plant Cell Environ. 8 (1985) 189e194.
[8] L.G. Wilson, R.A. Bressan, P. Filner, Light-dependent emission of hydrogen
sulphide from plants, Plant Physiol. 61 (1978) 184e189.
[9] J. Sekiya, L.G. Wilson, P. Filner, Resistance to injury by sulfur dioxide: corre-
lation with its reduction to, and emission of, hydrogen sulfide in Cucurbita-
ceae, Plant Physiol. 70 (1982) 437e441.
[10] H. Rennenberg, Role of O-acetylserine in hydrogen sulphide emission from
pumpkin leaves in response to sulphate, Plant Physiol. 73 (1983) 560e565.
[11] V. Aneja, W.H. Schlesinger, J.W. Erisman, Farming pollution, Nat. Geosci. 1
(2008) 409e411.
[12] L. Lamattina, C. García-Mata, Nitric oxide induces stomatal closure and
enhances the adaptive plant responses against drought stress, Plant Physiol.
126 (2001) 1196e1204.
Fig. 5. H2S donors reduce nitric oxide accumulation in guard cells. NO accumulation
was estimated by use of the DAF2-DA based method in conjunction with confocal
microscopy as described in Section 2. (A) Control with no treatment. (B) ABA (50 mM)
treatment. (C) GYY4137 (100 mM) treatment alone. (D) ABA treatment in the presence
of GYY4137. (E) NaSH (100 mM) treatment alone. (F) ABA treatment in the presence of
NaSH.
M. Lisjak et al. / Plant Physiology and Biochemistry 48 (2010) 931e935934

5. [13] X. Zhang, L. Zhang, F. Dong, J. Gao, D.W. Galbraith, C.-P. Song, Hydrogen
peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba,
Plant Physiol. 126 (2001) 1438e1448.
[14] L. Li, M. Whiteman, C.H. Tan, Y.Y. Guan, K.L. Neo, Y. Cheng, S.H. Lee, Y. Zhao,
R. Baskar, P.K. Moore, Characterization of a novel, water-soluble hydrogen
sulfide releasing molecule (GYY4137): new insights into the biology of
hydrogen sulfide, Circulation 117 (2008) 2351e2360.
[15] L. Li, M. Whiteman, M. Salto-Tellez, C.H. Tan, P.K. Moore, GYY4137, a novel
hydrogen sulfide-releasing molecule, protects against endotoxic shock in the
rat, Free Radic. Biol. Med 47 (2009) 103e113.
[16] M. Whiteman, L. Ling, P. Rose, H. C-Tan, D.B. Parkinson, P.K. Moore, The effect
of hydrogen sulfide donors on lipopolysaccharide-induced formation of
inflammatory mediators in macrophages. Antiox, Redox Signal. 12 (2010)
1147e1154.
[17] R. Desikan, R. Griffiths, J.T. Hancock, S.J. Neill, A new role for an old enzyme:
nitrate reductase-mediated nitric oxide generation is required for abscisic
acid-induced stomatal closure in Arabidopsis thaliana, Proc. Natl. Acad. Sci.,
USA 99 (2002) 16319e16324.
[18] C. García-Mata, L. Lamattina, Nitric oxide and abscisic acid cross talk in guard
cells, Plant Physiol. 128 (2002) 790e792.
[19] L.J. De Kok, K. Stahl, H. Rennenberg, Fluxes of atmospheric hydrogen sulphide
to plant shoots, New Phytol. 112 (1989) 533e542.
[20] S.J. Neill, R. Desikan, A. Clarke, J.T. Hancock, Nitric oxide is a novel component
of abscisic acid signaling in stomatal guard cells, Plant Physiol. 128 (2002)
13e16.
[21] J. Bright, R. Desikan, J.T. Hancock, S.J. Neill, ABA-induced NO generation and
stomatal closure in Arabidopsis are dependent on H2O2 synthesis, Plant J. 45
(2006) 113e122.
[22] Y.-F. Wang, P. Mainali, C.-S. Tang, L. Shi, C.-Y. Zhang, H. Yan, X.-Q. Liu, J.-B. Du,
Effects of nitric oxide and hydrogen sulfide on the relaxation of pulmonary
arteries in rats, Chin. Med. J. 121 (2008) 420e423.
[23] M.Y. Ali, Y.P. Cheong, Y.P. Y-Mok, M. Whiteman, M. Bhatia, P.K. Moore, Effect
of hydrogen sulfide and nitric oxide alone and together on rat aortic
contractility and blood pressure, Br. J. Pharmacol. 149 (2006) 625e634.
[24] M. Whiteman, L. Li, I. Kostetski, S.H. Chu, J.-L. Siau, M. Bhatia, P.K. Moore,
Evidence for the formation of a novel nitrosothiol from the gaseous mediators
nitric oxide and hydrogen sulfide, Biochem. Biophys. Res. Commun. 343
(2006) 303e310.
[25] S. Kubo, Y. Kurokawa, I. Doe, T. Masuko, F. Sekiguchi, A. Kawabata, Hydrogen
sulfide inhibits activity of three isoforms of recombinant nitric oxide synthase,
Toxicology 241 (2007) 92e97.
[26] T. Zemojtel, A. Fröhlich, M.C. Palmieri, M. Kolanczyk, I. Mikula, L.S. Wyrwicz,
E.E. Wanker, S. Mundlos, M. Vingron, P. Martasek, J. Durner, Plant nitric oxide
synthase: a never-ending story? Trends Plant Sci. 11 (2006) 524e525.
[27] N. Correa-Aragunde, M. Graziano, L. Lamattina, Nitric oxide plays a central
role in determining lateral root development in tomato, Planta 218 (2004)
900e905.
[28] J. Bright, S. Hiscock, P.E. James, J.T. Hancock, Pollen generates nitric oxide and
nitrite: a possible link to pollen-induced allergic responses, Plant Physiol.
Biochem. 47 (2009) 49e55.
M. Lisjak et al. / Plant Physiology and Biochemistry 48 (2010) 931e935 935