Gasotransmitters and Stomatal Closure: Is There Redundancy, Concerted Action, or Both?

The epidermis of the aerial part of land plants is pierced by pores through which plants perform gas exchange with environment. The guard cells (GCs), the specialized cells that surround the pore, have the capacity to sense diverse environmental and endogenous stimuli and integrate them into a single output which is the regulation of the stomatal pore width. The stomatal pore size is modulated by changes of the guard cell volume, driven by variations in the osmotic potential of the GCs. The stress hormone abscisic acid (ABA), the master regulator of stomatal movement, induces stomatal closure by the inhibition of H+-ATPases and activation of rapid and slow anion channels, producing the depolarization of the plasma membrane (PM) in GCs, and by an increase in the cytosolic Ca2+ concentratrion [Ca2+]cyt. While the rise of the [Ca2+]cyt blocks the influx of K+ by the inactivation of the inward rectifying K+ channels (Kin+), the depolarization of the PM, in turn promotes K+ efflux driven by outward rectifying K+ channels (Kout+; Blatt, 2000). This process is closely regulated by a complex signaling network that involves the participation of numerous ubiquitous signaling components like ROS, protein kinases, phospholipases, and protein phosphatases (Kim et al., 2010; Song et al., 2014); and by other signaling components that are emerging as active players in this signaling network, such is the case of gasotransmitters (Garcia-Mata and Lamattina, 2013). A gasotransmitter is a small gas molecule that: (i) can freely permeates biological membranes; (ii) it is endogenously generated by specific enzymes; (iii) it has specific functions at physiologically relevant concentrations; (iv) it functions can be mimicked by exogenous application of a donor; and (v) it has specific cellular and molecular targets (Wang, 2002). The group of gasotransmitters is, so far, composed by Nitric Oxide (NO), Carbon Monoxide (CO), and Hydrogen Sulfide (H2S) and the three of them have been reported to participate in the promotion of stomatal closure (Garcia-Mata and Lamattina, 2013), however, the biology of CO in this physiological process is less known than that of NO and H2S. Therefore, this opinion will be focused mainly on the action and interaction of NO and H2S. The two of them are accepted as active players in the regulation of stomatal movement, however there are still obscure points and some of them will be discussed in this opinion article: (i) their specific molecular targets; (ii) the molecular mechanisms underpinning their action; (iii) the interplay between them during the stomatal closure induction; and (iv) the crossed-regulation of their metabolism. All the three gasotransmitters are synthesized during the promotion of stomatal closure. CO is synthesized via the activity of heme oxygenase (Shekhawat and Verma, 2010). In Vicia faba CO induces stomatal closure in a dose-dependent manner and acts upstream of the production of NO during ABA-dependent stomatal closure (Cao et al., 2007; She and Song, 2008). It has been reported that the hormones ABA and ethylene (Eth) require the production of NO for the regulation of stomatal movement (Garcia-Mata and Lamattina, 2002; Neill et al., 2002; He et al., 2011; Song et al., 2011). NO can be synthesized either from NO2 by two genes, NIA1 and NIA2, that code for a nitrate reductase (NR), or from L-arginine in a reaction catalyzed by an enzyme with nitric oxide synthase (NOS)-like activity, even though the involved enzyme named AtNOA1 possesses GTPase activity (Moreau et al., 2008). However, it was reported that the Arabidopsis triple mutant nia1/nia2/atnoa1, which produces very low levels of NO, is hypersensitive to ABA (Lozano-Juste and Leon, 2010), suggesting that NO could have a dual role in ABA-dependent responses. Moreover, it has been found that increased levels of NO are dependent on the NADPHox-dependent production of H2O2 (Bright et al., 2006). NO levels can also be modified by an Alternative Oxidase (AOX). Tobacco plants lacking AOX show high NO levels that impacts in stomatal function (Cvetkovska et al., 2014). Recently Garcia et al. (2010) have shown that Arabidopsis mutant plants in β-Cyanoalanine synthase (Cys-C1), a mitochondrial enzymatic source of H2S, show higher AOX1a transcript levels than wild type, however exogenous application of H2S to rice cell culture induced AOX expression (Xiao et al., 2010). Further physiological studies are needed to clarify this interaction. H2S is produced during the passage of L-cysteine to pyruvate and ammonia in a reaction catalyzed by L-cysteine desulfhydrase (DES1; Alvarez et al., 2010). In Arabidopsis there are three different genes involved in this reaction: the DES1 gene (Alvarez et al., 2010; Scuffi et al., 2014), the At-LCDES gene (Jin et al., 2011), and L-CDes gene (Hou et al., 2013). Recently, it has been shown that the expression of these genes is upregulated in response to ABA, Eth, JA, and SA, all hormones that modulate stomatal movement (Hou et al., 2013). Even if sequence analysis has shown that the promoter region of the DES1 gene contains ABA-responsive elements (Scuffi et al., 2014), further work is needed in order to have a better understanding about the mechanism by which these hormones induce the expression of those genes. Although, DES1 was reported to mediate ABA-dependent stomatal closure (Scuffi et al., 2014), it was recently reported that H2S regulates the activity of Kin+ channel mostly in an ABA- and Ca2+-independent manner, suggesting the existence of ABA-regulated signaling pathways that can be, alternatively, activated in response to other stimuli (Papanatsiou et al., 2015).