The signaling role of hydrogen gas (
H2) has attracted increasing attention from animals to plants. However, the physiological significance and molecular mechanism of
H2 in drought tolerance are still largely unexplored. In this article, we report that abscisic acid (
ABA) induced stomatal closure in Arabidopsis (
Arabidopsis thaliana) by triggering intracellular signaling events involving
H2, reactive oxygen species (
ROS), nitric oxide (
NO), and the guard cell outward-rectifying K
+ channel (GORK).
ABA elicited a rapid and sustained
H2 release and production in Arabidopsis. Exogenous hydrogen-rich water (
HRW) effectively led to an increase of intracellular
H2 production, a reduction in the stomatal aperture, and enhanced drought tolerance. Subsequent results revealed that
HRW stimulated significant inductions of
NO and
ROS synthesis associated with stomatal closure in the wild type, which were individually abolished in the nitric reductase mutant
nitrate reductase1/2 (
nia1/2) or the NADPH oxidase-deficient mutant
rbohF (for respiratory burst oxidase homolog). Furthermore, we demonstrate that the
HRW-promoted
NO generation is dependent on
ROS production. The
rbohF mutant had impaired
NO synthesis and stomatal closure in response to
HRW, while these changes were rescued by exogenous application of
NO. In addition, both
HRW and hydrogen peroxide failed to induce
NO production or stomatal closure in the
nia1/2 mutant, while
HRW-promoted
ROS accumulation was not impaired. In the
GORK-null mutant, stomatal closure induced by
ABA,
HRW,
NO, or hydrogen peroxide was partially suppressed. Together, these results define a main branch of
H2-regulated stomatal movement involved in the
ABA signaling cascade in which RbohF-dependent
ROS and nitric reductase-associated
NO production, and subsequent GORK activation, were causally involved.Stomata are responsible for leaves of terrestrial plants taking in carbon dioxide for photosynthesis and likewise regulate how much water plants evaporate through the stomatal pores (
Chaerle et al., 2005). When experiencing water-deficient conditions, surviving plants balance photosynthesis with controlling water loss through the stomatal pores, which relies on turgor changes by pairs of highly differentiated epidermal cells surrounding the stomatal pore, called the guard cells (
Haworth et al., 2011;
Loutfy et al., 2012).Besides the characterization of the significant roles of abscisic acid (
ABA) in regulating stomatal movement, the key factors in guard cell signal transduction have been intensively investigated by performing forward and reverse genetics approaches. For example, both reactive oxygen species (
ROS) and nitric oxide (
NO) have been identified as vital intermediates in guard cell
ABA signaling (
Bright et al., 2006;
Yan et al., 2007;
Suzuki et al., 2011;
Hao et al., 2012). The key
ROS-producing enzymes in Arabidopsis (
Arabidopsis thaliana) guard cells are the respiratory burst oxidase homologs (Rboh) D and F (
Kwak et al., 2003;
Bright et al., 2006;
Mazars et al., 2010;
Marino et al., 2012). Current available data suggest that there are at least two distinct pathways responsible for
NO synthesis involved in
ABA signaling in guard cells: the nitrite reductase (
NR)- and
l-Arg-dependent pathways (
Desikan et al., 2002;
Besson-Bard et al., 2008). Genetic evidence further demonstrated that removal of the major known sources of either
ROS or
NO significantly impairs
ABA-induced stomatal closure.
ABA fails to induce
ROS production in the
atrbohD/F double mutant (
Kwak et al., 2003;
Wang et al., 2012) and
NO synthesis in the
NR-deficient mutant
nitrate reductase1/2 (
nia1/2;
Bright et al., 2006;
Neill et al., 2008), both of which lead to impaired stomatal closure in Arabidopsis. Most importantly,
ROS and
NO, which function both synergistically and independently, have been established as ubiquitous signal transduction components to control a diverse range of physiological pathways in higher plants (
Bright et al., 2006;
Tossi et al., 2012).The
guard cell outward-rectifying K+ channel (
GORK) encodes the exclusive voltage-gated outwardly rectifying K
+ channel protein, which was located in the guard cell membrane (
Ache et al., 2000;
Dreyer and Blatt, 2009). Expression profiles revealed that this gene is up-regulated upon the onset of drought, salinity, and cold stress and
ABA exposure (
Becker et al., 2003;
Tran et al., 2013). Reverse genetic evidence further showed that GORK plays an important role in the control of stomatal movements and allows the plant to reduce transpirational water loss significantly (
Hosy et al., 2003) and participates in the regulation of salinity tolerance by preventing salt-induced K
+ loss (
Jayakannan et al., 2013). Due to the high complexity of guard cell signaling cascades, whether and how
ABA-triggered
GORK up-regulation is attributed to the generation of cellular secondary messengers, such as
ROS and
NO, is less clear.Hydrogen gas (
H2) was recently revealed as a signaling modulator with multiple biological functions in clinical trails (
Ohsawa et al., 2007;
Itoh et al., 2009;
Ito et al., 2012). It was previously found that a hydrogenase system could generate
H2 in bacteria and green algae (
Meyer, 2007;
Esquível et al., 2011). Although some earlier studies discovered the evolution of
H2 in several higher plant species (
Renwick et al., 1964;
Torres et al., 1984), it was also proposed that the eukaryotic hydrogenase-like protein does not metabolize
H2 (
Cavazza et al., 2008;
Mondy et al., 2014). Since the explosion limit of
H2 gas is about 4% to 72.4% (v/v, in the air), the direct application of
H2 gas in experiments is flammable and dangerous. Regardless of these problems to be resolved, the methodology, such as using exogenous hydrogen-rich water (
HRW) or hydrogen-rich saline, which is safe, economical, and easily available, provides a valuable approach to investigate the physiological function of
H2 in animal research and clinical trials. For example, hydrogen dissolved in Dulbecco’s modified Eagle’s medium was found to react with cytotoxic
ROS and thus protect against oxidative damage in PC12 cells and rats (
Ohsawa et al., 2007). The neuroprotective effect of
H2-loaded eye drops on retinal ischemia-reperfusion injury was also reported (
Oharazawa et al., 2010). In plants, corresponding results by using
HRW combined with gas chromatography (
GC) revealed that
H2 could act as a novel beneficial gaseous molecule in plant responses against salinity (
Xie et al., 2012;
Xu et al., 2013), cadmium stress (
Cui et al., 2013), and paraquat toxicity (
Jin et al., 2013). More recently, the observation that
HRW could delay the postharvest ripening and senescence of kiwifruit (
Actinidia deliciosa) was reported (
Hu et al., 2014).Considering the fact that the signaling cascades for salt, osmotic, and drought stresses share a common cascade in an
ABA-dependent pathway, it would be noteworthy to identify whether and how
H2 regulates the bioactivity of
ABA-induced downstream components and, thereafter, biological responses, including stomatal closure and drought tolerance. To resolve these scientific questions,
rbohD,
rbohF,
nia1/2,
nitric oxide associated1 (
noa1;
Van Ree et al., 2011),
nia1/2/noa1, and
gork mutants were utilized to investigate the relationship among
H2,
ROS,
NO, and GORK in the guard cell signal transduction network. By the combination of pharmacological and biochemical analyses with this genetics-based approach, we provide comprehensive evidence to show that
H2 might be a newly identified bioeffective modulator involved in
ABA signaling responsible for drought tolerance, that
HRW-promoted stomatal closure was mainly attributed to the modulation of
ROS-dependent
NO generation, and that GORK might be the downstream target protein of
H2 signaling.
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