NO and Ca²⁺

Abstract

Nitric oxide (NO) is a secondary messenger involved in a wide range of signal transduction pathways, including plant immune responses. NO production can be triggered by various microbial pathogens and endogenous defense signaling molecules. Some phyllosphere pathogens invade plant leaves through the leaf surface stomatal pore formed by a pair of guard cells. To prevent the first-line attack, a series of innate immune signaling cascades occur in the guard cells that trigger stomatal closure. NO is one of the critical components involved in stomatal closure, evidenced by using pharmacological reagents and genetic resources. NO biosynthesis in plant cells is thought to occur by two pathways. One is the l-arginine-dependent pathway and the other is the nitrate reduction pathway. However, enzymes responsible for NO synthesis in the l-arginine-dependent pathway are still unidentified. Therefore the currently available NO synthesis related mutants are nia1, nia2 (the two genes encoding nitrate reductase) and noa1, an NO-associated gene mistakenly characterized as the NO synthase previously. Studies on stomatal defense signaling have demonstrated an interdependency between NO and several other secondary messengers, such as Ca²⁺, reactive oxygen species and cGMP and the gasotransmitter H₂S. These findings indicate that NO is downstream from those signaling molecules. However, recent studies have shown a distinct function of NO that NO-derived moieties bind to certain molecules, resulting in S-nitrosylation of cysteine and tyrosine nitration. NO-associated modification has been identified in several molecules involved in guard cell signaling, indicating a feed-forward role that NO could play in the guard cell. Perspectives and insights on how NO contributes to stomatal immunity through its interplay with other signaling molecules using genetic approaches are proposed and discussed.

Introduction

Nitric oxide (NO) is a free radical gas identified as a ubiquitous secondary messenger involved in a plethora of physiological processes in plants, such as seed germination, flowering, pollen tube growth, root growth, stomatal closure, fruit ripening and senescence (Besson-Bard et al., 2008, Simontacchi et al., 2013). In the past two decades, an extensive body of evidence has supported the essential role of NO in plant defense responses to invading pathogens by triggering hypersensitive response (HR) or upregulating defense gene expression (Dangl, 1998, Durner et al., 1998, Jeandroz et al., 2013, Ma, 2011, Ma et al., 2013, Polverari et al., 2003, Romero-Puertas et al., 2004, Trapet et al., 2014, Wendehenne et al., 2001, Zottini et al., 2009). Although NO generation contributing to HR development is due to l-Arginine (L-Arg)-dependent nitric oxide synthase (NOS) activity (Delledonne et al., 1998, Zhang et al., 2003), the exact NO enzymatic biosynthesis pathway is still a controversial topic (Moreau, Lindermayr, Durner, & Klessig, 2010). Nevertheless, the application of a mammalian NOS inhibitors diminishes HR in Arabidopsis and Nicotiana spp. (Delledonne et al., 1998; Huang & Knopp, 1998). Inoculation with an avirulent pathogen elicits an NO burst in Arabidopsis leaf tissue (Zeier et al., 2004, Zhang et al., 2003) and this NO generation is blocked by an NOS inhibitor (Zhang et al., 2003).

Another critical and early component in plant immune signaling cascades is Ca²⁺, whose cytosolic concentration is transiently elevated causing a Ca²⁺ burst. The perception of evolutionarily conserved components of microbial invaders known as microbe-associated molecular patterns (MAMPs), such as flagellin, elongation factor-Tu (EF-Tu), lipopolysaccharide (LPS), chitin or damage-associated molecular patterns (DAMP) by plant cells triggers a rapid cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation, which is required to activate downstream molecular and physiological processes (Ali et al., 2007, Aslam et al., 2009, Jeworutzki et al., 2010, Kwaaitaal et al., 2011, Ma et al., 2012, Ma et al., 2013). It has been well established that there is a connection between [Ca²⁺]_cyt elevation and NO synthesis during plant innate immune response (Ali et al., 2007, Lamotte et al., 2004, Lecourieux et al., 2006). NO also forms an interaction network with other defense signaling molecules, such as reactive oxygen species (ROS), cyclic nucleotides, mitogen-activated protein kinases (MAPKs; MPK is used when describing specific genes) and phosphatidic acid, to regulate the defense responses in plant cells (Gaupels, Kuruthukulangarakoola, & Durner, 2011). Thus, NO may play a role in immune signaling upstream from the Ca²⁺ burst in post-translational modification (PTM) of signaling proteins, and also may be involved in the immune cascade downstream from the Ca²⁺ burst which leads to NO generation.

Stomata are minute pores formed by a pair of guard cells located at the leaf epidermis. They function like ‘mouths’ in plants for regulating gas exchange and transpiratory water loss between the exterior environment and leaf surface. Additionally, stomata act as gateways for the entry of pathogens. The entry of some pathogenic microbes into the plant interior is controlled by stomata movement. Stomatal opening and closing occurs through turgor pressure changes in the pairs of guard cells forming the stomatal pore. Changes in guard cell turgor pressure can be regulated by the plant hormone abscisic acid (ABA) and cytosolic secondary messengers such as NO (García-Mata and Lamattina, 2013, Gayatri et al., 2013, Hancock et al., 2011, Yoshioka et al., 2011).

This chapter discusses and updates knowledge on recent findings of the interdependent relationship of NO and Ca²⁺ generation and how these signaling molecules contribute to stomatal innate immune responses to pathogens and danger signals.