Salt Stress Reduces Root Meristem Size by Nitric Oxide-Mediated Modulation of Auxin Accumulation and Signaling in Arabidopsis

The development of the plant root system is highly plastic, which allows the plant to adapt to various environmental stresses. Salt stress inhibits root elongation by reducing the size of the root meristem. However, the mechanism underlying this process remains unclear. In this study, we explored whether and how auxin and nitric oxide (NO) are involved in salt-mediated inhibition of root meristem growth in Arabidopsis (Arabidopsis thaliana) using physiological, pharmacological, and genetic approaches. We found that salt stress significantly reduced root meristem size by down-regulating the expression of PINFORMED (PIN) genes, thereby reducing auxin levels. In addition, salt stress promoted AUXIN RESISTANT3 (AXR3)/INDOLE-3-ACETIC ACID17 (IAA17) stabilization, which repressed auxin signaling during this process. Furthermore, salt stress stimulated NO accumulation, whereas blocking NO production with the inhibitor N^ω-nitro-l-arginine-methylester compromised the salt-mediated reduction of root meristem size, PIN down-regulation, and stabilization of AXR3/IAA17, indicating that NO is involved in salt-mediated inhibition of root meristem growth. Taken together, these findings suggest that salt stress inhibits root meristem growth by repressing PIN expression (thereby reducing auxin levels) and stabilizing IAA17 (thereby repressing auxin signaling) via increasing NO levels.Due to agricultural practices and climate change, soil salinity has become a serious factor limiting the productivity and quality of agricultural crops (Zhu, 2007). Worldwide, high salinity in the soil damages approximately 20% of total irrigated lands and takes 1.5 million ha out of production each year (Munns and Tester, 2008). In general, high salinity affects plant growth and development by reducing plant water potential, altering nutrient uptake, and increasing the accumulation of toxic ions (Hasegawa et al., 2000; Munns, 2002; Zhang and Shi, 2013). Together, these effects severely reduce plant growth and survival.Because the root is the first organ to sense high salinity, salt stress plays a direct, important role in modulating root system architecture (Wang et al., 2009). For instance, salt stress negatively regulates root hair formation and gravitropism (Sun et al., 2008; Wang et al., 2008). The role of salt in lateral root formation depends on the NaCl concentration. While high NaCl levels inhibit lateral root formation, lower NaCl levels stimulate lateral root formation in an auxin-dependent manner (Zolla et al., 2010; Ji et al., 2013). The root meristem plays an essential role in sustaining root growth (Perilli et al., 2012). Salt stress inhibits primary root elongation by suppressing root meristem activity (West et al., 2004). However, how this inhibition occurs remains largely unclear.Plant hormones are important intermediary signaling compounds that function downstream of environmental stimuli. Among plant hormones, indole-3-acetic acid (IAA) is thought to play a fundamental role in root system architecture by regulating cell division, expansion, and differentiation. In Arabidopsis (Arabidopsis thaliana) root tips, a distal auxin maximum is formed and maintained by polar auxin transport (PAT), which determines the orientation and extent of cell division in the root meristem as well as root pattern formation (Sabatini et al., 1999). PINFORMED (PIN) proteins, which are components of the auxin efflux machinery, regulate primary root elongation and root meristem size (Blilou et al., 2005; Dello Ioio et al., 2008; Yuan et al., 2013, 2014). The auxin signal transduction pathway is activated by direct binding of auxin to its receptor protein, TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB), promoting the degradation of Aux/IAA proteins, releasing auxin response factors (ARFs), and activating the expression of auxin-responsive genes (Gray et al., 2001; Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). Aux/IAA proteins are short-lived, nuclear-localized proteins that play key roles in auxin signal activation and root growth modulation (Rouse et al., 1998). Other hormones and stresses often regulate auxin signaling by affecting Aux/IAA protein stability (Lim and Kunkel, 2004; Nemhauser et al., 2004; Wang et al., 2007; Kushwah and Laxmi, 2014).Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants (He et al., 2004; Fernández-Marcos et al., 2011; Shi et al., 2012), including important roles in the regulation of root growth and development. NO functions downstream of auxin during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002). Exogenous auxin-induced NO biosynthesis is associated with nitrate reductase activity during lateral root formation, and NO is necessary for auxin-induced lateral root and root hair development (Pagnussat et al., 2002; Lombardo et al., 2006). Pharmacological and genetic analyses in Arabidopsis indicate that NO suppresses primary root growth and root meristem activity (Fernández-Marcos et al., 2011). Additionally, both exogenous application of the NO donor sodium nitroprusside (SNP) and overaccumulation of NO in the mutant chlorophyll a/b binding protein underexpressed1 (cue1)/nitric oxide overproducer1 (nox1) result in reduced PIN1 expression and auxin accumulation in root tips. The auxin receptors protein TIR1 is S-nitrosylated by NO, suggesting that this protein is a direct target of NO in the regulation of root development (Terrile et al., 2012).Because NO is a free radical, NO levels are dynamically regulated by endogenous and environmental cues. Many phytohormones, including abscisic acid, auxin, cytokinin, salicylic acid, jasmonic acid, and ethylene, induce NO biosynthesis (Zottini et al., 2007; Kolbert et al., 2008; Tun et al., 2008; García et al., 2011). In addition, many abiotic and biotic stresses or stimuli, such as cold, heat, salt, drought, heavy metals, and pathogens/elicitors, also stimulate NO biosynthesis (Zhao et al., 2009; Mandal et al., 2012). Salt stress stimulates NO and ONOO^– accumulation in roots (Corpas et al., 2009), but the contribution of NO to root meristem growth under salinity stress has yet to be examined in detail.In this study, we found that salt stress significantly down-regulated the expression of PIN genes and promoted AUXIN RESISTANT3 (AXR3)/IAA17 stabilization. Furthermore, salt stress stimulated NO accumulation, and pharmacological inhibition of NO biosynthesis compromised the salt-mediated reduction in root meristem size. Our results support a model in which salt stress reduces root meristem size by increasing NO accumulation, which represses PIN expression and stabilizes IAA17, thereby reducing auxin levels and repressing auxin signaling.