A Signaling Pathway Linking Nitric Oxide Production to Heterotrimeric G Protein and Hydrogen Peroxide Regulates Extracellular Calmodulin Induction of Stomatal Closure in Arabidopsis  

Abstract

Extracellular calmodulin (ExtCaM) regulates stomatal movement by eliciting a cascade of intracellular signaling events including heterotrimeric G protein, hydrogen peroxide (H 2 O 2 ), and Ca 2+ . However, the ExtCaM-mediated guard cell signaling pathway remains poorly understood. In this report, we show that Arabidopsis (Arabidopsis thaliana) NITRIC OXIDE ASSOCIATED1 (AtNOA1)-dependent nitric oxide (NO) accumulation plays a crucial role in ExtCaM-induced stomatal closure. ExtCaM triggered a significant increase in NO levels associated with stomatal closure in the wild type, but both effects were abolished in the Atnoa1 mutant. Furthermore, we found that ExtCaM-mediated NO generation is regulated by GPA1, the Ga-subunit of heterotrimeric G protein. The ExtCaM-dependent NO accumulation was nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGa). In addition, cGa Atnoa1 and gpa1-2 Atnoa1 double mutants exhibited a similar response as did Atnoa1. The defect in gpa1 was rescued by overexpression of AtNOA1. Finally, we demonstrated that G protein activation of NO production depends on H 2 O 2 . Reduced H 2 O 2 levels in guard cells blocked the stomatal response of cGa lines, whereas exogenously applied H 2 O 2 rescued the defect in ExtCaM-mediated stomatal closure in gpa1 mutants. Moreover, the atrbohD/F mutant, which lacks the NADPH oxidase activity in guard cells, had impaired NO generation in response to ExtCaM, and H 2 O 2 -induced stomatal closure and NO accumulation were greatly impaired in Atnoa1. These findings have established a signaling pathway leading to ExtCaM-induced stomatal closure, which involves GPA1-dependent activation of H 2 O 2 production and subsequent AtNOA1-dependent NO accumulation.Plant guard cells control opening and closure of the stomata in response to phytohormones (e.g. abscisic acid [ABA]) and various environmental signals such as light and temperature, thereby regulating gas exchange for photosynthesis and water status via transpiration . Cytosolic calcium ([Ca 2+ ] i ) has been shown to be a key second messenger that changes in response to multiple stimuli in guard cells (McAinsh et al., 1995;Grabov and Blatt, 1998;Wood et al., 2000). A large proportion of Ca 2+ is localized in extracellular space. It has been shown that external Ca 2+ concentration ([Ca 2+ ] o ) promotes stomatal closure and induces oscillation in [Ca 2+ ] i in guard cells (MacRobbie, 1992;McAinsh et al., 1995;Allen et al., 2001 Calmodulin is a well-known Ca 2+ sensor that is activated upon binding of Ca 2+ . It has been shown that calmodulin exists not only intracellularly but also extracellularly in many plant species

Role and Interrelationship of Gα Protein, Hydrogen Peroxide, and Nitric Oxide in Ultraviolet B-Induced Stomatal Closure in Arabidopsis Leaves

“…Similarly, UV-B also failed to induce NO production ( As both H 2 O 2 and NO act downstream of Ga in UV-Binduced stomatal closure (Figs. 5 and 6) and reports from several groups imply that H 2 O 2 induces NO generation in guard cells (He et al, 2005;Bright et al, 2006;Li et al, 2009), we finally investigated the relationship between H 2 O 2 and NO in the Ga mediation of UV-B-induced stomatal closure. First, it is proposed that if NO acts downstream of H 2 O 2 , then the deficiency of H 2 O 2 could be bypassed by NO, but the deficiency of NO could not be compensated by H 2 O 2 , and vice versa.…”

“…Although our results here show an important role of Ga in UV-B guard cell signaling, whether Gbg and Ga can act additively or synergically to mediate UV-B signaling in guard cells and other plant cells is still an interesting question to be addressed in the future. H 2 O 2 is one of the extensively studied signaling components involved in guard cell signaling (Neill et al, 2002;Wang and Song, 2008), and its origin in guard cells has been proposed to be multiple sources, including cell wall peroxidases (Khokon et al, 2010), NADPH oxidases (Kwak et al, 2003;Chen et al, 2004;Joo et al, 2005;Bright et al, 2006;Sagi and Fluhr, 2006;Li et al, 2009), and amine oxidase-type enzymes . With regard to the enzymatic source of H 2 O 2 mediating UV-B-induced stomatal closure, our previous study has provided pharmacological evidence that 0.8 W m 22 UV-B-induced H 2 O 2 generation in broad bean guard cells depends on peroxidases but not NADPH oxidases (He et al, 2011a).…”

“…Thus, the regulation of stomatal apertures is extremely important for the survival of plants. Phenotypic analyses of Arabidopsis mutants lacking the Ga-or Gb-subunit show that these G proteins are involved in stomatal movement regulated by abscisic acid (ABA; Wang et al, 2001;Pandey and Assmann, 2004;Liu et al, 2007;Fan et al, 2008;Zhang et al, 2011), sphingosine-1-P (Coursol et al, 2003(Coursol et al, , 2005, phosphatidic acid (PA; Mishra et al, 2006), extracellular calmodulin (ExtCaM; Chen et al, 2004;Li et al, 2009), extracellular ATP (Hao et al, 2012), and the pathogenassociated molecular pattern flg22 , suggesting that plant G proteins respond to various stimuli as key regulators of stomatal movement. On exposure to UV-B radiation, many plant species exhibit decreases in stomatal conductance and/or aperture under growth chamber, greenhouse, and field conditions (Musil and Wand, 1993;Nogués et al, 1999;Jansen and Noort, 2000).…”

“…The heterotrimeric G proteins also participate in ROS metabolism in plant cells (Wei et al, 2008;Zhao et al, 2010). During stomatal movement, G proteins mediate H 2 O 2 production induced by ABA (Zhang et al, 2011), ExtCaM (Chen et al, 2004;Li et al, 2009), and extracellular ATP (Hao et al, 2012) as well as NO production induced by ExtCaM in guard cells ). In addition, phospholipase Da and its product PA, which interact with GPA1 during ABA inhibition of stomatal opening (Mishra et al, 2006), also promote ABAinduced ROS production ).…”

“…For example, both H 2 O 2 and NO have been implicated in ABA-, salicylic acid (SA)-, ethylene-, ExtCaM-, and darkness-induced stomatal closure. Furthermore, several main cellular players in stomatal movement, such as mitogen-activated protein kinases, protein phosphatases, cytoskeleton, and ion channels, have already been identified as likely targets downstream of H 2 O 2 or NO Wang and Song, 2008;Huang et al, 2009;Li et al, 2009;Wilkins et al, 2011;Yemets et al, 2011). G protein signaling to the membrane-bound H 2 O 2 synthetic enzyme, NADPH oxidase, has been implicated in the development of disease resistance and the apoptotic hypersensitive response in rice (Oryza sativa; Suharsono et al, 2002).…”