Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2015 Mar 27;168(1):343–356. doi: 10.1104/pp.15.00030

Salt Stress Reduces Root Meristem Size by Nitric Oxide-Mediated Modulation of Auxin Accumulation and Signaling in Arabidopsis1,[OPEN]

Wen Liu 1, Rong-Jun Li 1, Tong-Tong Han 1, Wei Cai 1, Zheng-Wei Fu 1, Ying-Tang Lu 1,*
PMCID: PMC4424022  PMID: 25818700

Nitric oxide functions downstream of salt stress to modulate auxin response for salt-mediated inhibition of root meristem development.

Abstract

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 Nω-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.

RESULTS

Salt-Mediated Inhibition of Root Meristem Development Is Due to Reduced Auxin Accumulation in Roots

To begin to elucidate how salt stress reduces root meristem size, we transferred 5-d-old seedlings germinated on one-half-strength Murashige and Skoog (MS) plates to new plates supplemented with or without 100 mm NaCl and measured primary root growth 2 d after transfer. We chose to move the seedlings after germination because salt stress inhibits seed germination in Arabidopsis (Park et al., 2011). We found that primary root elongation was inhibited and root meristem size was reduced in 100 mm NaCl-treated seedlings (Supplemental Fig. S1), similar to the results of West et al. (2004). Based on these results, 100 mm NaCl was used in subsequent experiments.

Auxin plays an essential role in root meristem maintenance (Swarup et al., 2002; Overvoorde et al., 2010). The defective root meristem patterning observed under salt stress raised the question of whether auxin content or auxin signaling is affected by salt stress. Hence, we looked for changes in auxin signaling in salt-treated roots using the auxin-responsive DR5::GFP marker line, which reports auxin accumulation and distribution (Friml et al., 2003). For this purpose, 5-d-old seedlings were treated with or without 100 mm NaCl for 24 h and the expression of DR5::GFP was monitored. The fluorescence intensity in salt-treated DR5::GFP roots was significantly lower than that in untreated roots (Fig. 1, A and B). We directly measured endogenous IAA in roots using gas chromatography (GC)-mass spectrometry and found that IAA levels were significantly lower in salt-treated roots than in the untreated control (Fig. 1C), suggesting that decreased auxin accumulation may be responsible for the reduced root meristem size under salt stress. We tested this notion by experiments employing exogenous application of auxin. We transferred 5-d-old seedlings germinated on one-half-strength MS plates to new plates containing 100 mm NaCl supplemented with various concentrations of IAA and measured the root meristem length and cell number 2 d after transfer. Whereas the root meristem length and cell number were significantly reduced upon 100 mm NaCl treatment, application of IAA led to a longer root meristem and increased root meristem cell number in roots subjected to 100 mm NaCl treatment (Fig. 1, D and E), indicating that exogenous auxin partially rescues the salt-related inhibition of root meristem size.

Figure 1.

Figure 1.

Open in a new tab

Salt stress reduces auxin accumulation in roots, inhibiting root meristem growth. A, GFP fluorescence in the roots of DR5::GFP seedlings treated without or with 100 mm NaCl for 24 h. B, Quantification of DR5::GFP fluorescence intensities in plants treated as in A. The fluorescence intensity of untreated roots was set to 1. At least 15 seedlings were imaged per treatment for each of three replicates. C, IAA contents in the roots of wild-type seedlings treated without or with 100 mm NaCl for 24 h. D and E, Root meristem length (D) and root meristem cell number (E) of wild-type seedlings treated without or with 100 mm NaCl plus 0 nm IAA, 0.1 nm IAA, or 0.5 nm IAA for 2 d. F and G, Root meristem length (F) and root meristem cell number (G) of wild-type seedlings treated without or with 100 mm NaCl in the presence or absence of 5 μm NPA for 2 d. Error bars represent sd. Asterisks (***) indicate significant differences with respect to the corresponding control (Student’s t test, P < 0.001), and different letters indicate significantly different values (P < 0.05 by Tukey’s test). FW, Fresh weight. Bars = 50 µm.

Auxin accumulation in root tips is modulated by PAT though auxin carriers (Blilou et al., 2005; Overvoorde et al., 2010). The changes in auxin levels in roots subjected to salt stress may be due to changes in PAT. Thus, we treated wild-type plants with NaCl in the presence or absence of naphthylphthalamic acid (NPA), an auxin transport inhibitor, and examined both root meristem length and cell number. Whereas treatment with NaCl alone decreased root meristem size, root meristem length and cell number were not further reduced by the presence of NPA (Fig. 1, F and G), suggesting that PAT is required for the modulation of root meristem size by salt stress.

PIN1, PIN3, and PIN7 Are Involved in Regulating Root Meristem Development under Salt Stress

An auxin gradient and maximum in the root apex is established by PAT via auxin carriers, such as PIN1, PIN3, and PIN7 (Blilou et al., 2005; Dello Ioio et al., 2008; Hong et al., 2014). The reduced auxin accumulation observed in roots subjected to salt stress may have been due to the suppression of auxin carriers. We found that the mRNA levels of PIN1, PIN3, and PIN7 were significantly reduced in roots subjected to 6 h of salt stress (Fig. 2A). This conclusion was further confirmed by analyzing PIN1::PIN1-GFP, PIN3::PIN3-GFP, and PIN7::PIN7-GFP lines. As visualized by PIN-GFP fluorescence, the protein levels of PIN1, PIN3, and PIN7 were reduced in salt-treated roots (Fig. 2, B and C), although their distribution patterns were not altered under salt stress.

Figure 2.

Figure 2.

Open in a new tab

Salt stress represses the expression of PIN genes, resulting in short root meristems. A, qRT-PCR analysis of PIN1, PIN3, and PIN7 expression in the roots of wild-type seedlings treated without or with 100 mm NaCl for 6 h. The expression levels of the indicated genes in untreated roots were set to 1. B, Expression of PIN1-GFP, PIN3-GFP, and PIN7-GFP in the roots of PIN1::PIN1-GFP, PIN3::PIN3-GFP, and PIN7::PIN7-GFP seedlings treated without or with 100 mm NaCl for 12 h. C, Quantification of the fluorescence intensities in plants treated as in B. The fluorescence intensity of the indicated line in untreated roots was set to 1. At least 15 seedlings were imaged per line for each of three replicates. D and E, Relative root meristem length (D) and root meristem cell number (E) of each genotype treated with 100 mm NaCl compared with untreated plants. Error bars represent sd. Asterisks (***) indicate significant differences with respect to the corresponding control (Student’s t test, P < 0.001), and different letters indicate significantly different values (P < 0.05 by Tukey’s test). Col-0, Ecotype Columbia. Bars = 50 µm.

We then explored the role of PIN genes in salt-mediated inhibition of root meristem growth using pin mutants. While both pin3 and pin7 had similar phenotypes to those of wild-type seedlings, pin1 exhibited less of a reduction in root meristem length and cell number under salt stress compared with wild-type seedlings (Fig. 2, D and E), implying that PIN1 plays a role in the response to salt stress. In addition, the pin1pin3pin7 triple mutant was even more tolerant to salt stress than pin1 in terms of root meristem length and cell number (Fig. 2, D and E). Taken together, these results suggest that these PIN genes function additively in salt-mediated root meristem inhibition and that PIN1 plays a major role, whereas PIN3 and/or PIN7 function in a lesser capacity in this process.

Salt Stress Stabilizes IAA17, Leading to Salt-Mediated Inhibition of Root Meristem Growth

Next, we wondered whether auxin signaling was affected by salt stress. Aux/IAA proteins are generally thought to be transcriptional repressors of auxin-responsive reporter gene expression (Rouse et al., 1998; Overvoorde et al., 2005; Wang et al., 2013), and Aux/IAA protein levels are maintained by the E3 ubiquitin ligase complex S-Phase Kinase-Associated Protein1-Cullin1-F-Box (SCF)TIR1 (Tan et al., 2007; Maraschin et al., 2009). Accordingly, we monitored the effect of salt stress on the stability of IAA17 in the reporter line harboring a construct encoding the amino terminus (NT) of AXR3/IAA17 (AXR3NT) and the GUS reporter under the control of a heat shock (HS)-inducible promoter (HS::AXR3NT-GUS; Gray et al., 2001). In this system, GUS activity is reduced upon recognition and degradation of AXR3NT-GUS by the SCFTIR1 complex (Gray et al., 2001). We incubated HS::AXR3NT-GUS seedlings at 37°C for 2 h to enable accumulation of AXR3NT-GUS, and we assayed GUS activity in the roots of seedlings transferred to medium containing 100 mm NaCl and incubated at 23°C for 45 min. GUS activity was significantly higher in salt-treated roots than in untreated roots (Fig. 3, A and B). These results suggest that salt stress stabilizes IAA17, an important component of auxin signaling.

Figure 3.

Figure 3.

Open in a new tab

Salt stress stabilizes IAA17, which functions in salt-mediated inhibition of root meristem growth. A, GUS staining images of HS::AXR3NT-GUS. Seedlings were heat shocked at 37°C for 2 h and treated without or with 100 mm NaCl for 45 min at 23°C, followed by GUS staining. B, Relative GUS activity of HS::AXR3NT-GUS as treated in A. The GUS activity in untreated plants was set to 1. C and D, Root meristem length (C) and root meristem cell number (D) of wild-type, axr3-3, tir1afb2afb3, and axr3/iaa17 seedlings treated without or with 100 mm NaCl for 2 d. E and F, Relative root meristem length (E) and root meristem cell number (F) of each genotype treated with 100 mm NaCl compared with untreated plants. Error bars represent sd. Asterisks (***) indicate significant differences with respect to the corresponding control (Student’s t test, P < 0.001), and different letters indicate significantly different values (P < 0.05 by Tukey’s test). Col-0, Ecotype Columbia. Bars = 50 µm.

To further investigate whether IAA17 is involved in salt-induced inhibition of meristem development, we analyzed the root meristem length and cell number of iaa17 plants upon salt stress. The axr3-3 mutant, in which IAA17 protein is stabilized due to a single point mutation (V89G) in domain II (Rouse et al., 1998), had much shorter root meristems with fewer cells compared with wild-type seedlings (Fig. 3, C and D). However, this repression of root meristem growth was not affected by salt stress (Fig. 3, C and D), perhaps because salt treatment could not further stabilize the mutant AXR3/IAA17 in axr3-3 to further repress root meristem size. We also examined the responsiveness of the tir1afb2afb3 mutant to NaCl treatment, as this mutant also has enhanced AXR3/IAA17 stability (Dharmasiri et al., 2005b). The mutant was more sensitive to NaCl treatment compared with the wild type in terms of root meristem inhibition (Fig. 3, C–F). These results imply that salt stress inhibits root meristem development by stabilizing IAA17. This notion was further reinforced by analysis of axr3/iaa17, a loss-of-function mutant (Overvoorde et al., 2005). Whereas both root meristem size and cell number in axr3/iaa17 were similar to those of the wild type under normal conditions (Fig. 3, C and D), longer root meristems and higher root meristem cell numbers were observed in axr3/iaa17 compared with wild-type plants under salt stress (Fig. 3, C and D), indicating that IAA17 is required for salt-mediated inhibition of root meristem growth.

Salt Stress Affects Root Meristem Size through Overaccumulation of NO

Salt stress stimulates NO accumulation in roots (Corpas et al., 2009), and NO was recently shown to modulate root meristem development (Fernández-Marcos et al., 2011). First, we confirmed that NaCl-induced NO accumulation in the roots was significantly reduced in plants treated with Nω-nitro-l-arginine-methylester (l-NAME), an inhibitor of animal NO synthase that is also effective in plant systems, or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a widely used NO scavenger (Fig. 4, A and B; Flores et al., 2008; Besson-Bard et al., 2009; Zhao et al., 2009). Then, we transferred 5-d-old seedlings germinated on normal one-half-strength MS plates to new plates containing 100 mm NaCl with or without 1 mm l-NAME or 250 μm cPTIO and measured root meristem length and cell number after two additional days of growth. Combined application of either NaCl and l-NAME or NaCl and cPTIO reduced the inhibitory effect of salt treatment on root meristem length and cell number compared with salt treatment alone (Fig. 4, C and D), suggesting that NO is involved in salt-mediated inhibition of root meristem development. In addition, when the mitotic cyclin B1 (CYCB1) GUS reporter line was treated with either NaCl and l-NAME or NaCl and cPTIO, the roots had more GUS-stained cells compared with the roots of plants treated with NaCl alone (Fig. 4, E and F), revealing that salt stress represses meristem cell division through overaccumulation of NO.

Figure 4.

Figure 4.

Open in a new tab

Salt stress reduces root meristem size through NO overaccumulation. A, NO contents in the roots of wild-type seedlings treated without or with 100 mm NaCl, 100 mm NaCl plus 1 mm l-NAME, 100 mm NaCl plus 250 μm cPTIO, 1 mm l-NAME, or 250 μm cPTIO for 4 h as revealed by the NO-specific fluorescent probe DAF-2 DA. B, Quantification of the fluorescence intensities of plants treated as in A. The fluorescence intensity in untreated roots was set to 1. At least 15 seedlings were imaged per treatment for each of three replicates. C and D, Root meristem length (C) and root meristem cell number (D) of wild-type seedlings treated without or with 100 mm NaCl, 100 mm NaCl plus 1 mm l-NAME, 100 mm NaCl plus 250 μm cPTIO, 1 mm l-NAME, or 250 μm cPTIO for 2 d. E, GUS staining images of CYCB1;1::GUS seedlings treated without or with 100 mm NaCl, 100 mm NaCl plus 1 mm l-NAME, 100 mm NaCl plus 250 μm cPTIO, 1 mm l-NAME, or 250 μm cPTIO for 24 h. F, Number of GUS-stained cells in root tips of CYCB1;1::GUS seedlings. Error bars represent sd, and different letters indicate significantly different values (P < 0.05 by Tukey’s test). Bars = 50 µm.

Salt Stress Reduces Auxin Levels Possibly through NO Overaccumulation, Leading to Repressed Root Meristem Development

The above results demonstrate that salt stress inhibits root meristem development by reducing auxin levels in roots. We next explored whether salt stress modulates auxin accumulation though salt-induced NO accumulation. Thus, we first monitored possible changes in auxin signaling in the DR5::GFP marker line under salt stress in the presence or absence of l-NAME. Analysis of GFP fluorescence revealed that additional application of l-NAME reversed the attenuated DR5 activity in salt-treated roots (Fig. 5, A and B), suggesting that NO contributes to the role of salt stress in reducing auxin accumulation. This notion was further confirmed through direct measurement of endogenous IAA levels via GC-mass spectrometry. The results show that the reduced IAA levels in salt-treated roots were partially rescued by additional application of l-NAME (Fig. 5C).

Figure 5.

Figure 5.

Open in a new tab

Salt stress reduces auxin levels through overaccumulation of NO. A, GFP expression in the roots of DR5::GFP seedlings treated without or with 100 mm NaCl, 100 mm NaCl plus 1 mm l-NAME, or 1 mm l-NAME for 24 h. B, Quantification of DR5::GFP fluorescence intensities in plants treated as in A. The fluorescence intensity of untreated roots was set to 1. At least 15 seedlings were imaged per treatment for each of three replicates. C, IAA contents in the roots of wild-type seedlings treated without or with 100 mm NaCl, 100 mm NaCl plus 1 mm l-NAME, or 1 mm l-NAME for 24 h. Error bars represent sd, and different letters indicate significantly different values (P < 0.05 by Tukey’s test). FW, Fresh weight. Bars = 50 µm.

PIN1, PIN3, and PIN7 Are Involved in NO-Mediated Inhibition of Root Meristem Development upon Salt Stress

The above results indicate that PIN1, PIN3, and PIN7 function in regulating root meristem development under salt stress (Fig. 2) and that salt-treated roots accumulate more NO than the control (Fig. 4, A and B). Thus, we examined whether salt-induced NO overaccumulation modulates the expression of PIN1, PIN3, and PIN7. The mRNA accumulation of PIN1, PIN3, and PIN7 was decreased in the roots of plants treated with 20 μm SNP (Fig. 6A), a NO donor previously used to study the role of NO in regulating root growth and development (Fernández-Marcos et al., 2011; Bai et al., 2012). In addition, PIN-GFP fluorescence, reflecting the protein levels of PIN1, PIN3, and PIN7, was significantly reduced in SNP-treated roots of PIN1::PIN1-GFP, PIN3::PIN3-GFP, and PIN7::PIN7-GFP lines (Fig. 6, B and C). These effects of NO on the expression of three PIN genes were also verified using another NO donor, S-nitrosoglutathione (GSNO; Supplemental Fig. S2, A–C). These results, together with the finding that salt stress stimulates NO accumulation in the roots, which represses root meristem size, suggest that salt stress may down-regulate the expression of PIN genes by increasing NO accumulation. The reduced mRNA levels of PIN1, PIN3, and PIN7 in salt-treated roots were partially rescued in the roots treated with NaCl and l-NAME together (Fig. 6A). This result was further confirmed by analyzing GFP fluorescence in PIN1::PIN1-GFP, PIN3::PIN3-GFP, and PIN7::PIN7-GFP lines. The protein levels of PIN1, PIN3, and PIN7 were reduced to a lesser extent in roots treated with NaCl and l-NAME together compared with those in salt-treated roots (Fig. 6, B and C).

Figure 6.

Figure 6.

Open in a new tab

NO is required for repressing PIN expression and reducing root meristem size upon salt stress. A, qRT-PCR analysis of PIN1, PIN3, and PIN7 expression in the roots of wild-type seedlings treated without or with 100 mm NaCl, 20 μm SNP, 100 mm NaCl plus 1 mm l-NAME, or 1 mm l-NAME for 6 h. The expression level of the indicated gene in untreated roots was set to 1. B, Expression of PIN1-GFP, PIN3-GFP, and PIN7-GFP in the roots of PIN1::PIN1-GFP, PIN3::PIN3-GFP, and PIN7::PIN7-GFP seedlings treated without or with 100 mm NaCl, 20 μm SNP, 100 mm NaCl plus 1 mm l-NAME, or 1 mm l-NAME for 12 h. C, Quantification of the fluorescence intensities in plants treated as in B. The fluorescence intensity of the indicated line in untreated roots was set to 1. At least 15 seedlings were imaged per line for each of three replicates. D and E, Relative root meristem length (D) and root meristem cell number (E) of the wild type and pin mutants treated with 20 μm SNP compared with untreated plants. Error bars represent sd, and different letters indicate significantly different values (P < 0.05 by Tukey’s test). Col-0, Ecotype Columbia. Bars = 50 µm.

Next, we assayed NO-mediated inhibition of both root meristem length and cell number in the pin1, pin3, and pin7 mutants compared with that in wild-type plants upon SNP treatment. All three single mutants exhibited similar inhibition to that of wild-type plants, whereas the triple mutant pin1pin3pin7 was less sensitive to SNP treatment compared with wild-type plants in both root meristem size and cell number (Fig. 6, D and E). This result, combined with the above findings that pin1pin3pin7 is less sensitive to salt stress than the wild type in terms of root meristem inhibition and that treatment with either l-NAME or cPTIO rescues salt-mediated inhibition of root meristem development, suggests that PIN1, PIN3, and PIN7 are involved in NO-mediated inhibition of root meristem development in seedlings under salt stress.

IAA17 Functions in NO-Regulated Root Meristem Development upon Salt Stress

Because IAA17 is required for salt stress-mediated inhibition of root meristem growth, we next used the HS::AXR3NT-GUS line to examine whether salt-induced overaccumulation of NO modulates the stability of IAA17. GUS activity was significantly higher in roots treated with NO donor (SNP or GSNO) than in untreated roots (Fig. 7, A and B; Supplemental Fig. S2, D and E), which is similar to the observation that salt treatment promotes IAA17 stabilization (Fig. 3, A and B). This salt-induced increase in GUS activity was attenuated by l-NAME treatment (Fig. 7, A and B), suggesting that NO contributes to the effect of salt stress on the stability of IAA17. Next, we verified the role of IAA17 in NO-mediated inhibition of root meristem size by examining both root meristem length and cell number in iaa17 plants subjected to SNP treatment. While the axr3/iaa17 mutant exhibited less reduction in both root meristem length and cell number compared with wild-type seedlings upon SNP treatment, the inhibition was not exacerbated in the axr3-3 mutant by SNP treatment (Fig. 7, C–F). By contrast, the tir1afb2afb3 mutant was more sensitive than wild-type plants to SNP treatment (Fig. 7, C–F). These results imply that NO inhibits root meristem growth by stabilizing IAA17. In addition, we crossed HS::AXR3NT-GUS plants with the mutant nox1, which has higher endogenous NO levels and reduced root meristem development compared with the wild type (Fernández-Marcos et al., 2011), and we assayed AXR3NT-GUS activity in the resulting nox1 HS::AXR3NT-GUS line. The results show that the stability of IAA17 was greater in nox1 HS::AXR3NT-GUS plants than in HS::AXR3NT-GUS plants (Fig. 7, G and H). These results, combined with the observation that salt inhibited root meristem growth by increasing NO levels (Fig. 4, A–D), suggest that IAA17 functions in NO-regulated root meristem development upon salt stress.

Figure 7.

Figure 7.

Open in a new tab

NO is necessary for stabilization of IAA17 by salt stress. A, GUS staining images of HS::AXR3NT-GUS. Seedlings were heat shocked at 37°C for 2 h and treated without or with 100 mm NaCl, 20 μm SNP, 100 mm NaCl plus 1 mm l-NAME, or 1 mm l-NAME for 45 min at 23°C, followed by GUS staining. B, Relative GUS activity of HS::AXR3NT-GUS as treated in A. The GUS activity in untreated plants was set to 1. C and D, Root meristem length (C) and root meristem cell number (D) of wild-type, axr3-3, tir1afb2afb3, and axr3/iaa17 seedlings treated without or with 20 μm SNP for 2 d. E and F, Relative root meristem length (E) and root meristem cell number (F) of each genotype treated with 20 μm SNP compared with untreated plants. G, GUS staining images of HS::AXR3NT-GUS in the wild type (WT) and nox1 mutant background. Seedlings were heat shocked at 37°C for 2 h and transferred to 23°C. GUS staining was performed at 0, 30, or 120 min after transfer. H, Relative GUS activity of HS::AXR3NT-GUS as treated in G. The GUS activity in wild-type plants at 0 min after heat shock was set to 1. Error bars represent sd, and different letters indicate significantly different values (P < 0.05 by Tukey’s test). Col-0, Ecotype Columbia. Bars = 50 µm.

Vieten et al. (2005) reported that exogenous auxin application induces the expression of PIN1, PIN3, and PIN7, but the induction of these genes by auxin is repressed in solitary root1, an IAA14 gain-of-function mutant, indicating that IAA14 plays a negative role in auxin-induced expression of PIN1, PIN3, and PIN7 (Vieten et al., 2005). Accordingly, we examined whether AXR3/IAA17 is involved in NO-repressed expression of PIN1, PIN3, and PIN7. In contrast to the reduced expression of these genes in wild-type plants treated with the NO donor SNP (which enhances AXR3/IAA17 stability), axr3-3, harboring stabilized AXR3/IAA17, exhibited wild-type levels of expression of these genes (Supplemental Fig. S3). Furthermore, SNP efficiently suppressed the expression of these three PIN genes in axr3/iaa17, as was also observed in wild-type plants (Supplemental Fig. S3). These results suggest that ARX3/IAA17 is not essential for NO-repressed expression of PIN1, PIN3, and PIN7.

DISCUSSION

Salt stress, like many other abiotic stresses, has a dramatic effect on root system architecture. By altering its growth pattern, the plant root system is able to reach larger domains of the soil environment or to escape from potential harmful areas; this strategy allows plants to survive biotic and abiotic stresses. The root system architecture of higher plants is primarily established postembryonically through maintaining the root meristem, generating lateral roots, forming root hairs, and determining the direction of growth in root tips by gravitropism (Osmont et al., 2007; Galvan-Ampudia and Testerink, 2011). Previous studies have demonstrated the role of salt stress in lateral root development, root hair formation, and root gravitropism (Sun et al., 2008; Wang et al., 2009; Zolla et al., 2010; Zhao et al., 2011). Salt stress was previously reported to inhibit primary root elongation by reducing root meristem size (West et al., 2004). However, how salt stress regulates root meristem development has remained largely unknown. In this study, we found that salt stress significantly reduces root meristem size by reducing auxin accumulation and suppressing auxin signaling via increasing NO levels.

Auxin plays critical roles during root growth and development. Proper auxin signaling relies on the interplay between its biosynthesis, transport, and signaling. Auxin is biosynthesized in young leaves and the shoot apex (Ljung et al., 2001), and shoot-derived auxin is required for root growth (Friml et al., 2003; Wisniewska et al., 2006). Decreasing auxin resources or disturbing shoot-to-root PAT often results in reduced auxin accumulation in roots and the formation of a shorter root meristem. For example, plants either with ectopic expression of the bacterial gene IAA-Lys synthetase in the shoot apex or with parts of their shoots excised display shorter roots and less expanded root meristems (Wisniewska et al., 2006; Sassi et al., 2012; Hong et al., 2014). Mutation of PIN genes or application of the PAT inhibitor NPA also reduces root elongation and root meristem size (Blilou et al., 2005). In this study, we found that salt stress reduced auxin levels in the roots, and NPA did not increase salt-mediated inhibition of root meristem development, suggesting that salt stress modulates root meristem size, at least in part, by affecting PAT. Our data further indicate that PIN genes are involved in this process. In addition, auxin can be biosynthesized in roots, and root-generated auxin also contributes to root development (Overvoorde et al., 2010). To date, several IAA biosynthesis pathways have been documented, including one Trp-independent and four Trp-dependent pathways (Zhao, 2010). However, our quantitative reverse transcription (qRT)-PCR results show that the expression of all auxin biosynthesis genes examined was similar in salt-treated and untreated roots (Supplemental Fig. S4), suggesting that local auxin biosynthesis in roots may not be affected by salt stress.

We noted that although the sensitivity of pin3, pin7, and wild-type plants to salt stress was similar in terms of root meristem length and cell number, pin1pin3pin7 was less sensitive to salt stress than wild-type plants and even pin1. These results suggest that in addition to PIN1, PIN3 and/or PIN7 also function in salt-mediated root meristem inhibition. Similar results were also obtained in a previous study of the role of PIN3 and PIN7 in pulse-induced phototropism. Haga and Sakai (2012) demonstrate that the curvature response in pulse-induced phototropism is reduced significantly in pin3, but not in pin7, and impairment of the phototropic curvature of the pin3pin7 double mutant is greater than that observed in pin3, indicating that PIN3 and PIN7 function additively. Similar observations were also reported for other gene families such as the TGACG motif binding factor (TGA) gene family. While tga4-1 mutant and wild-type plants exhibit similar susceptibility to pathogen infection, tga1-1 plants exhibit significantly higher pathogen growth than the wild type, and the tga1-1tga4-1 double mutant has even greater susceptibility than tga1-1 (Kesarwani et al., 2007). These findings suggest that TGA1 and TGA4 play partially redundant roles in plant basal resistance to pathogen infection, with TGA1 having a greater effect than TGA4 (Kesarwani et al., 2007). Similarly, Hutchison et al. (2006) determined that Arabidopsis histidine phosphotransfer protein1 (AHP1), AHP2, and AHP3 play overlapping roles in affecting root elongation in response to cytokinin treatment, as ahp2ahp3 is slightly less sensitive (and ahp1ahp2ahp3 is substantially less sensitive) than the wild type in terms of 6-benzyladenine-mediated root elongation, while the sensitivity of all three single mutants to cytokinin treatment is similar to that of the wild type.

In this study, we found that the single mutant pin1 was less sensitive to salt stress (but not to NO application) than the wild type in terms of root meristem length and cell number, although the pin1pin3pin7 mutant was less sensitive to both salt and NO treatment. This difference remains to be further investigated. A recent report indicates that other phytohormones, such as brassinosteroid, GA, abscisic acid, and jasmonic acid, also function in the plant response to salt stress (Geng et al., 2013).

Galvan-Ampudia et al. (2013) reported that PIN2 plays a role in mediating a salt avoidance mechanism. Thus, we further explored the possible involvement of PIN2 in salt-mediated root meristem inhibition. We found that although the expression of PIN2 was reduced by treatment with either salt or NO, the pin2 mutant displayed a wild-type phenotype upon salt and NO treatment in terms of root meristem inhibition (Supplemental Fig. S5).

Auxin signaling components, including Aux/IAA and ARF proteins, contribute to root development (Overvoorde et al., 2010; Yan et al., 2013). Gain-of-function IAA3, IAA12, and IAA17 mutants and loss-of-function ARF5 mutants display defective root development (Hamann et al., 2002; Dello Ioio et al., 2008). In this study, salt stress increased the stability of AXR3/IAA17, which functions in salt-mediated inhibition of root meristem development. The expression of a number of auxin response genes and cell wall-related genes, which is reduced in the gain-of-function mutant axr3-1 (Overvoorde et al., 2005), was also significantly reduced by salt treatment (Supplemental Fig. S6), further supporting the notion that salt stress increases the stability of IAA17, thereby repressing auxin signaling. We also observed increased responsiveness of tir1afb2afb3 to salt stress compared with the wild type in terms of root meristem development, whereas NaCl treatment did not exacerbate the severe root meristem phenotypes associated with the axr3-3 mutant. It could be that the stabilizing effect of IAA17/AXR3 in the triple mutant was less pronounced than that in axr3-3 because of functional redundancy within the TIR1/AFB family.

NO, as an endogenous signaling molecule, plays a key role during plant adaptation to various environmental stresses (Qiao and Fan, 2008; Zhao et al., 2009; Shi et al., 2012). A previous study involving increasing exogenous or endogenous NO levels revealed that NO regulates root meristem size through affecting PIN1-mediated auxin accumulation in roots (Fernández-Marcos et al., 2011). Here, we demonstrated that salt stress reduced auxin accumulation by increasing NO levels through repressing PIN expression in salt-treated roots. NO also affected auxin signaling in the salt-mediated reduction of root meristem size, because NO is essential for salt-promoted stabilization of IAA17 in roots upon salt stress treatment. Interestingly, through investigating a HS::AXR3NT-GUS line, Terrile et al. (2012) showed that NO promotes the degradation of IAA17 in the leaves of 6-d-old seedlings. Here, we found that exogenous application of NO stabilizes IAA17 in roots, which was also observed in the nox1 mutant. The difference in these effects on IAA17 may be due to the different tissues examined, as auxin responses differ in different tissues. For example, auxin accumulates on the illuminated sides of plant roots, promoting negative phototropism in the root, whereas auxin accumulation is detected on the shaded side of the plant hypocotyl, promoting positive phototropism in the shoot (Ding et al., 2011; Zhang et al., 2013).

Like NO, reactive oxygen species (ROS) play an important role in plant growth and environmental responses (Verslues et al., 2007; Miller et al., 2010; Tsukagoshi et al., 2010). Tsukagoshi et al. (2010) demonstrated that transcriptional regulation of ROS by UPBEAT1 regulates the balance between cellular proliferation and differentiation in the roots. Moreover, crosstalk between ROS and auxin regulatory networks is also involved in modulating plant stress responses (Tognetti et al., 2012; Gao et al., 2014). Whether ROS also function in salt-induced root meristem inhibition requires further investigation.

In conclusion, our results indicate that salt stress reduces root meristem length and cell numbers, thereby generating short primary roots, by increasing NO levels. The elevated NO accumulation further down-regulates the expression of PIN genes, leading to reduced auxin levels, and thus stabilizes IAA17 for repressed auxin signaling.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used in this study. The transgenic and mutant lines used in this study include the following: DR5::GFP (Friml et al., 2003), PIN1::PIN1-GFP (Benková et al., 2003), PIN2::PIN2-GFP (Blilou et al., 2005), PIN3::PIN3-GFP (Blilou et al., 2005), PIN7::PIN7-GFP (Blilou et al., 2005), CYCB1;1::GUS (Colón-Carmona et al., 1999), pin1pin3pin7 (Blilou et al., 2005), tir1afb2afb3 (Dharmasiri et al., 2005b), and cue1/nox1 (He et al., 2004). Lines HS::AXR3NT-GUS (CS9571), pin1 (SALK_047613), pin2 (CS8058), pin3 (CS9364), pin7 (CS9367), axr3/iaa17 (SALK_065697), and axr3-3 (CS57505) were obtained from the Arabidopsis Biological Resource Center. The transgenic and mutant lines were confirmed by PCR. Arabidopsis seeds were surface sterilized for 5 min with 5% (w/v) bleach, washed three times with sterile water, incubated for 3 d at 4°C in the dark, and plated onto agar medium containing one-half-strength MS medium (Sigma-Aldrich), pH 5.8, supplemented with 0.8% (w/v) agar and 1% (w/v) Suc. Seedlings were grown in a growth chamber maintained at 23°C, 80 µmol photons m–2 s–1 light under a 16-h-light/8-h-dark cycle.

Measurement of Root Meristem Size

Seeds were germinated on one-half-strength MS medium as described above and grown in a vertical position. Five-day-old seedlings were transferred onto plates supplemented with various components and grown for an additional 2 d. Digital images of seedlings were captured for subsequent measurement of the lengths of newly grown roots, and the roots were then excised, mounted immediately on glass slides with clearing solution (50 g of chloral hydrate, 15 mL of water, and 10 mL of glycerol), examined under an Olympus BX60 differential interference contrast microscope, and photographed using a CCD Olympus dp72 camera. The root meristem zone was defined according to published methods (Dello Ioio et al., 2007). Measurements of newly grown root length and root meristem length were carried out as previously described (Yuan et al., 2013). At least 30 seedlings were analyzed per treatment and genotype.

Measurement of GUS Activity

GUS histochemical staining was performed as previously described (Hu et al., 2010). Seedlings harboring the GUS reporter gene were incubated at 37°C in staining solution (100 mm sodium phosphate buffer, pH 7.5, 10.0 mm EDTA, 0.5 mm potassium ferricyanide, 0.5 mm potassium ferrocyanide, 1 mm 5-bromo-chloro-3-indolyl-β-d-glucuronide, and 0.1% [v/v] Triton X-100). The duration of GUS staining was chosen based on the transgenic marker line: 6 h for CYCB1;1::GUS and 12 h for HS::AXR3NT-GUS. A quantitative GUS activity assay was performed according to previously described methods (Gao et al., 2014).

Determination of NO Contents

The endogenous NO levels in root meristems were visualized using the specific NO fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA; He et al., 2004; Moreau et al., 2008; Shi et al., 2012). For DAF-2 DA imaging, seedlings were incubated in 10 μm DAF-2 DA in 20 mm HEPES-NaOH, pH 7.5, for 1 h and rinsed three times with HEPES-NaOH buffer prior to visualization under an Olympus BX60 differential interference contrast microscope equipped with a CCD Olympus dp72 camera with excitation set at 488 nm and emission set at 515 nm. At least 15 seedlings were analyzed per treatment. Quantitative measurement of fluorescence intensity was performed using Photoshop CS5 (Adobe).

Confocal Microscopy

Confocal images were captured using an Olympus FluoView 1000 confocal laser-scanning microscope according to the manufacturer's instructions as previously described (Yuan et al., 2013). Briefly, 5-d-old seedlings were transferred to plates containing different compounds and treated for the indicated time. The root tips of GFP lines were then mounted onto microscope slides for observation. The emission wavelength for GFP detection was 500 to 540 nm. For each treatment and genotype, photographs of at least 15 seedlings were taken and analyzed. Quantitative measurement of GFP signal intensity was performed using Photoshop CS5.

RNA Extraction and qRT-PCR

As previously described (Gao et al., 2014), total RNA from roots was isolated using PureLink Plant RNA Reagent (Invitrogen) according to the manufacturer’s instructions. To remove contaminating DNA, all RNA samples were digested with RQ1 RNase-free DNase I (Promega). Reverse transcription was then carried out using ReverTra Ace (TOYOBO). Quantitative PCR was performed on a Bio-Rad CFX96 apparatus with SYBR Green I dye (Invitrogen). PCR was carried out in 96-well plates as following: 3-min incubation at 95°C for complete denaturation, followed by 40 cycles of 95°C for 15 s and 60°C for 45 s. The Protein Phosphatase 2A Subunit A3 (AT1G13320) and Eukaryotic Translation Initiation Factor 4A1 (AT3G13920) were chosen as the best reference genes for our conditions based on analysis with geNorm software (Czechowski et al., 2005). All experiments were performed with three independent biological replicates and three technical repetitions. The primer sequences used to amplify auxin-related genes are listed in Gao et al. (2014), and the other primer sequences can be found in Supplemental Table S1.

Quantification of IAA Levels by GC-Selected Ion Monitoring-Mass Spectrometry

Endogenous IAA levels were quantified according to a previously described protocol (Gao et al., 2014). For each sample, root tips of at least 100 mg of fresh weight were collected and immediately frozen in liquid nitrogen. After extraction, the endogenous IAA was purified, methylated in a stream of diazomethane gas, and resuspended in 100 μL of ethyl acetate. The endogenous IAA content was analyzed by GC-selected ion monitoring-mass spectrometry. A Shimadzu GCMS-QP2010 Plus system equipped with an HP-5MS column (30 m long, 0.25-mm i.d., 0.25-μm film; Agilent) was used to determine IAA levels.

Statistical Analysis

All experiments were performed with at least three repetitions. The significance of differences was determined by ANOVA or Student’s t test, as indicated in the figure legends.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers At1G70940 (PIN3), At1G23080 (PIN7), At1G04250 (AXR3/IAA17), AT3G62980 (TIR1), AT3G26810 (AFB2), and AT1G12820 (AFB3).

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_168_1_343__index.html (964B, html)

Glossary

NO

nitric oxide

PAT

polar auxin transport

SNP

sodium nitroprusside

MS

Murashige and Skoog

GC

gas chromatography

NPA

naphthylphthalamic acid

l-NAME

Nω-nitro-l-arginine-methylester

qRT

quantitative reverse transcription

ROS

reactive oxygen species

Footnotes

1

This work was supported by the Major State Basic Research Program (grant no. 2013CB126901 to Y.-T.L.).

[OPEN]

Articles can be viewed without a subscription.

References

  1. Bai S, Li M, Yao T, Wang H, Zhang Y, Xiao L, Wang J, Zhang Z, Hu Y, Liu W, et al. (2012) Nitric oxide restrain root growth by DNA damage induced cell cycle arrest in Arabidopsis thaliana. Nitric Oxide 26: 54–60 [DOI] [PubMed] [Google Scholar]
  2. Benková E, Michniewicz M, Sauer M, Teichmann T, Seifertová D, Jürgens G, Friml J (2003) Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115: 591–602 [DOI] [PubMed] [Google Scholar]
  3. Besson-Bard A, Gravot A, Richaud P, Auroy P, Duc C, Gaymard F, Taconnat L, Renou JP, Pugin A, Wendehenne D (2009) Nitric oxide contributes to cadmium toxicity in Arabidopsis by promoting cadmium accumulation in roots and by up-regulating genes related to iron uptake. Plant Physiol 149: 1302–1315 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J, Heidstra R, Aida M, Palme K, Scheres B (2005) The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433: 39–44 [DOI] [PubMed] [Google Scholar]
  5. Colón-Carmona A, You R, Haimovitch-Gal T, Doerner P (1999) Technical advance: spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein. Plant J 20: 503–508 [DOI] [PubMed] [Google Scholar]
  6. Corpas FJ, Hayashi M, Mano S, Nishimura M, Barroso JB (2009) Peroxisomes are required for in vivo nitric oxide accumulation in the cytosol following salinity stress of Arabidopsis plants. Plant Physiol 151: 2083–2094 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139: 5–17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dello Ioio R, Linhares FS, Scacchi E, Casamitjana-Martinez E, Heidstra R, Costantino P, Sabatini S (2007) Cytokinins determine Arabidopsis root-meristem size by controlling cell differentiation. Curr Biol 17: 678–682 [DOI] [PubMed] [Google Scholar]
  9. Dello Ioio R, Nakamura K, Moubayidin L, Perilli S, Taniguchi M, Morita MT, Aoyama T, Costantino P, Sabatini S (2008) A genetic framework for the control of cell division and differentiation in the root meristem. Science 322: 1380–1384 [DOI] [PubMed] [Google Scholar]
  10. Dharmasiri N, Dharmasiri S, Estelle M (2005a) The F-box protein TIR1 is an auxin receptor. Nature 435: 441–445 [DOI] [PubMed] [Google Scholar]
  11. Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M, Hobbie L, Ehrismann JS, Jürgens G, Estelle M (2005b) Plant development is regulated by a family of auxin receptor F box proteins. Dev Cell 9: 109–119 [DOI] [PubMed] [Google Scholar]
  12. Ding Z, Galván-Ampudia CS, Demarsy E, Łangowski Ł, Kleine-Vehn J, Fan Y, Morita MT, Tasaka M, Fankhauser C, Offringa R, et al. (2011) Light-mediated polarization of the PIN3 auxin transporter for the phototropic response in Arabidopsis. Nat Cell Biol 13: 447–452 [DOI] [PubMed] [Google Scholar]
  13. Fernández-Marcos M, Sanz L, Lewis DR, Muday GK, Lorenzo O (2011) Nitric oxide causes root apical meristem defects and growth inhibition while reducing PIN-FORMED 1 (PIN1)-dependent acropetal auxin transport. Proc Natl Acad Sci USA 108: 18506–18511 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Flores T, Todd CD, Tovar-Mendez A, Dhanoa PK, Correa-Aragunde N, Hoyos ME, Brownfield DM, Mullen RT, Lamattina L, Polacco JC (2008) Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development. Plant Physiol 147: 1936–1946 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jürgens G (2003) Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426: 147–153 [DOI] [PubMed] [Google Scholar]
  16. Galvan-Ampudia CS, Julkowska MM, Darwish E, Gandullo J, Korver RA, Brunoud G, Haring MA, Munnik T, Vernoux T, Testerink C (2013) Halotropism is a response of plant roots to avoid a saline environment. Curr Biol 23: 2044–2050 [DOI] [PubMed] [Google Scholar]
  17. Galvan-Ampudia CS, Testerink C (2011) Salt stress signals shape the plant root. Curr Opin Plant Biol 14: 296–302 [DOI] [PubMed] [Google Scholar]
  18. Gao X, Yuan HM, Hu YQ, Li J, Lu YT (2014) Mutation of Arabidopsis CATALASE2 results in hyponastic leaves by changes of auxin levels. Plant Cell Environ 37: 175–188 [DOI] [PubMed] [Google Scholar]
  19. García MJ, Suárez V, Romera FJ, Alcántara E, Pérez-Vicente R (2011) A new model involving ethylene, nitric oxide and Fe to explain the regulation of Fe-acquisition genes in Strategy I plants. Plant Physiol Biochem 49: 537–544 [DOI] [PubMed] [Google Scholar]
  20. Geng Y, Wu R, Wee CW, Xie F, Wei X, Chan PM, Tham C, Duan L, Dinneny JR (2013) A spatio-temporal understanding of growth regulation during the salt stress response in Arabidopsis. Plant Cell 25: 2132–2154 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M (2001) Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Nature 414: 271–276 [DOI] [PubMed] [Google Scholar]
  22. Haga K, Sakai T (2012) PIN auxin efflux carriers are necessary for pulse-induced but not continuous light-induced phototropism in Arabidopsis. Plant Physiol 160: 763–776 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hamann T, Benkova E, Bäurle I, Kientz M, Jürgens G (2002) The Arabidopsis BODENLOS gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo patterning. Genes Dev 16: 1610–1615 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51: 463–499 [DOI] [PubMed] [Google Scholar]
  25. He Y, Tang RH, Hao Y, Stevens RD, Cook CW, Ahn SM, Jing L, Yang Z, Chen L, Guo F, et al. (2004) Nitric oxide represses the Arabidopsis floral transition. Science 305: 1968–1971 [DOI] [PubMed] [Google Scholar]
  26. Hong LW, Yan DW, Liu WC, Chen HG, Lu YT (2014) TIME FOR COFFEE controls root meristem size by changes in auxin accumulation in Arabidopsis. J Exp Bot 65: 275–286 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hu YQ, Liu S, Yuan HM, Li J, Yan DW, Zhang JF, Lu YT (2010) Functional comparison of catalase genes in the elimination of photorespiratory H2O2 using promoter- and 3′-untranslated region exchange experiments in the Arabidopsis cat2 photorespiratory mutant. Plant Cell Environ 33: 1656–1670 [DOI] [PubMed] [Google Scholar]
  28. Hutchison CE, Li J, Argueso C, Gonzalez M, Lee E, Lewis MW, Maxwell BB, Perdue TD, Schaller GE, Alonso JM, et al. (2006) The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling. Plant Cell 18: 3073–3087 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ji H, Pardo JM, Batelli G, Van Oosten MJ, Bressan RA, Li X (2013) The Salt Overly Sensitive (SOS) pathway: established and emerging roles. Mol Plant 6: 275–286 [DOI] [PubMed] [Google Scholar]
  30. Kepinski S, Leyser O (2005) The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435: 446–451 [DOI] [PubMed] [Google Scholar]
  31. Kesarwani M, Yoo J, Dong X (2007) Genetic interactions of TGA transcription factors in the regulation of pathogenesis-related genes and disease resistance in Arabidopsis. Plant Physiol 144: 336–346 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kolbert Z, Bartha B, Erdei L (2008) Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia. J Plant Physiol 165: 967–975 [DOI] [PubMed] [Google Scholar]
  33. Kushwah S, Laxmi A (2014) The interaction between glucose and cytokinin signal transduction pathway in Arabidopsis thaliana. Plant Cell Environ 37: 235–253 [DOI] [PubMed] [Google Scholar]
  34. Lim MT, Kunkel BN (2004) The Pseudomonas syringae type III effector AvrRpt2 promotes virulence independently of RIN4, a predicted virulence target in Arabidopsis thaliana. Plant J 40: 790–798 [DOI] [PubMed] [Google Scholar]
  35. Ljung K, Bhalerao RP, Sandberg G (2001) Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J 28: 465–474 [DOI] [PubMed] [Google Scholar]
  36. Lombardo MC, Graziano M, Polacco JC, Lamattina L (2006) Nitric oxide functions as a positive regulator of root hair development. Plant Signal Behav 1: 28–33 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mandal MK, Chandra-Shekara AC, Jeong RD, Yu K, Zhu S, Chanda B, Navarre D, Kachroo A, Kachroo P (2012) Oleic acid-dependent modulation of NITRIC OXIDE ASSOCIATED1 protein levels regulates nitric oxide-mediated defense signaling in Arabidopsis. Plant Cell 24: 1654–1674 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Maraschin FS, Memelink J, Offringa R (2009) Auxin-induced, SCFTIR1-mediated poly-ubiquitination marks AUX/IAA proteins for degradation. Plant J 59: 100–109 [DOI] [PubMed] [Google Scholar]
  39. Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 33: 453–467 [DOI] [PubMed] [Google Scholar]
  40. Moreau M, Lee GI, Wang Y, Crane BR, Klessig DF (2008) AtNOS/AtNOA1 is a functional Arabidopsis thaliana cGTPase and not a nitric-oxide synthase. J Biol Chem 283: 32957–32967 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Munns R. (2002) Comparative physiology of salt and water stress. Plant Cell Environ 25: 239–250 [DOI] [PubMed] [Google Scholar]
  42. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59: 651–681 [DOI] [PubMed] [Google Scholar]
  43. Nemhauser JL, Mockler TC, Chory J (2004) Interdependency of brassinosteroid and auxin signaling in Arabidopsis. PLoS Biol 2: E258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Osmont KS, Sibout R, Hardtke CS (2007) Hidden branches: developments in root system architecture. Annu Rev Plant Biol 58: 93–113 [DOI] [PubMed] [Google Scholar]
  45. Overvoorde P, Fukaki H, Beeckman T (2010) Auxin control of root development. Cold Spring Harb Perspect Biol 2: a001537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Overvoorde PJ, Okushima Y, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Liu A, Onodera C, Quach H, et al. (2005) Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana. Plant Cell 17: 3282–3300 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pagnussat GC, Simontacchi M, Puntarulo S, Lamattina L (2002) Nitric oxide is required for root organogenesis. Plant Physiol 129: 954–956 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Park J, Kim YS, Kim SG, Jung JH, Woo JC, Park CM (2011) Integration of auxin and salt signals by the NAC transcription factor NTM2 during seed germination in Arabidopsis. Plant Physiol 156: 537–549 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Perilli S, Di Mambro R, Sabatini S (2012) Growth and development of the root apical meristem. Curr Opin Plant Biol 15: 17–23 [DOI] [PubMed] [Google Scholar]
  50. Qiao W, Fan LM (2008) Nitric oxide signaling in plant responses to abiotic stresses. J Integr Plant Biol 50: 1238–1246 [DOI] [PubMed] [Google Scholar]
  51. Rouse D, Mackay P, Stirnberg P, Estelle M, Leyser O (1998) Changes in auxin response from mutations in an AUX/IAA gene. Science 279: 1371–1373 [DOI] [PubMed] [Google Scholar]
  52. Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold N, Weisbeek P, et al. (1999) An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 99: 463–472 [DOI] [PubMed] [Google Scholar]
  53. Sassi M, Lu Y, Zhang Y, Wang J, Dhonukshe P, Blilou I, Dai M, Li J, Gong X, Jaillais Y, et al. (2012) COP1 mediates the coordination of root and shoot growth by light through modulation of PIN1- and PIN2-dependent auxin transport in Arabidopsis. Development 139: 3402–3412 [DOI] [PubMed] [Google Scholar]
  54. Shi HT, Li RJ, Cai W, Liu W, Wang CL, Lu YT (2012) Increasing nitric oxide content in Arabidopsis thaliana by expressing rat neuronal nitric oxide synthase resulted in enhanced stress tolerance. Plant Cell Physiol 53: 344–357 [DOI] [PubMed] [Google Scholar]
  55. Sun F, Zhang W, Hu H, Li B, Wang Y, Zhao Y, Li K, Liu M, Li X (2008) Salt modulates gravity signaling pathway to regulate growth direction of primary roots in Arabidopsis. Plant Physiol 146: 178–188 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Swarup R, Parry G, Graham N, Allen T, Bennett M (2002) Auxin cross-talk: integration of signalling pathways to control plant development. Plant Mol Biol 49: 411–426 [DOI] [PubMed] [Google Scholar]
  57. Tan X, Calderon-Villalobos LI, Sharon M, Zheng C, Robinson CV, Estelle M, Zheng N (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446: 640–645 [DOI] [PubMed] [Google Scholar]
  58. Terrile MC, París R, Calderón-Villalobos LI, Iglesias MJ, Lamattina L, Estelle M, Casalongué CA (2012) Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor. Plant J 70: 492–500 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tognetti VB, Mühlenbock P, Van Breusegem F (2012) Stress homeostasis: the redox and auxin perspective. Plant Cell Environ 35: 321–333 [DOI] [PubMed] [Google Scholar]
  60. Tsukagoshi H, Busch W, Benfey PN (2010) Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root. Cell 143: 606–616 [DOI] [PubMed] [Google Scholar]
  61. Tun NN, Livaja M, Kieber JJ, Scherer GF (2008) Zeatin-induced nitric oxide (NO) biosynthesis in Arabidopsis thaliana mutants of NO biosynthesis and of two-component signaling genes. New Phytol 178: 515–531 [DOI] [PubMed] [Google Scholar]
  62. Verslues PE, Batelli G, Grillo S, Agius F, Kim YS, Zhu J, Agarwal M, Katiyar-Agarwal S, Zhu JK (2007) Interaction of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals a point of connection between salt stress and H2O2 signaling in Arabidopsis thaliana. Mol Cell Biol 27: 7771–7780 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Vieten A, Vanneste S, Wisniewska J, Benková E, Benjamins R, Beeckman T, Luschnig C, Friml J (2005) Functional redundancy of PIN proteins is accompanied by auxin-dependent cross-regulation of PIN expression. Development 132: 4521–4531 [DOI] [PubMed] [Google Scholar]
  64. Wang D, Pajerowska-Mukhtar K, Culler AH, Dong X (2007) Salicylic acid inhibits pathogen growth in plants through repression of the auxin signaling pathway. Curr Biol 17: 1784–1790 [DOI] [PubMed] [Google Scholar]
  65. Wang J, Yan DW, Yuan TT, Gao X, Lu YT (2013) A gain-of-function mutation in IAA8 alters Arabidopsis floral organ development by change of jasmonic acid level. Plant Mol Biol 82: 71–83 [DOI] [PubMed] [Google Scholar]
  66. Wang Y, Li K, Li X (2009) Auxin redistribution modulates plastic development of root system architecture under salt stress in Arabidopsis thaliana. J Plant Physiol 166: 1637–1645 [DOI] [PubMed] [Google Scholar]
  67. Wang Y, Zhang W, Li K, Sun F, Han C, Wang Y, Li X (2008) Salt-induced plasticity of root hair development is caused by ion disequilibrium in Arabidopsis thaliana. J Plant Res 121: 87–96 [DOI] [PubMed] [Google Scholar]
  68. West G, Inzé D, Beemster GT (2004) Cell cycle modulation in the response of the primary root of Arabidopsis to salt stress. Plant Physiol 135: 1050–1058 [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wisniewska J, Xu J, Seifertová D, Brewer PB, Ruzicka K, Blilou I, Rouquié D, Benková E, Scheres B, Friml J (2006) Polar PIN localization directs auxin flow in plants. Science 312: 883. [DOI] [PubMed] [Google Scholar]
  70. Yan DW, Wang J, Yuan TT, Hong LW, Gao X, Lu YT (2013) Perturbation of auxin homeostasis by overexpression of wild-type IAA15 results in impaired stem cell differentiation and gravitropism in roots. PLoS ONE 8: e58103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Yuan HM, Xu HH, Liu WC, Lu YT (2013) Copper regulates primary root elongation through PIN1-mediated auxin redistribution. Plant Cell Physiol 54: 766–778 [DOI] [PubMed] [Google Scholar]
  72. Yuan TT, Xu HH, Zhang KX, Guo TT, Lu YT (2014) Glucose inhibits root meristem growth via ABA INSENSITIVE 5, which represses PIN1 accumulation and auxin activity in Arabidopsis. Plant Cell Environ 37: 1338–1350 [DOI] [PubMed] [Google Scholar]
  73. Zhang JL, Shi H (2013) Physiological and molecular mechanisms of plant salt tolerance. Photosynth Res 115: 1–22 [DOI] [PubMed] [Google Scholar]
  74. Zhang KX, Xu HH, Yuan TT, Zhang L, Lu YT (2013) Blue-light-induced PIN3 polarization for root negative phototropic response in Arabidopsis. Plant J 76: 308–321 [DOI] [PubMed] [Google Scholar]
  75. Zhao MG, Chen L, Zhang LL, Zhang WH (2009) Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis. Plant Physiol 151: 755–767 [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Zhao Y. (2010) Auxin biosynthesis and its role in plant development. Annu Rev Plant Biol 61: 49–64 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Zhao Y, Wang T, Zhang W, Li X (2011) SOS3 mediates lateral root development under low salt stress through regulation of auxin redistribution and maxima in Arabidopsis. New Phytol 189: 1122–1134 [DOI] [PubMed] [Google Scholar]
  78. Zhu JK. (2007) Plant salt stress. In Encyclopedia of Life Sciences. Wiley, Chichester, UK http://dx.doi.org/10.1002/9780470015902.a0001300.pub2 (April 21, 2015) [Google Scholar]
  79. Zolla G, Heimer YM, Barak S (2010) Mild salinity stimulates a stress-induced morphogenic response in Arabidopsis thaliana roots. J Exp Bot 61: 211–224 [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Zottini M, Costa A, De Michele R, Ruzzene M, Carimi F, Lo Schiavo F (2007) Salicylic acid activates nitric oxide synthesis in Arabidopsis. J Exp Bot 58: 1397–1405 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data
supp_168_1_343__index.html (964B, html)
supp_pp.15.00030_00030R1_Supplemental_Material.pdf (349.4KB, pdf)

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES