Plant Physiology Preview. Published on March 27, 2015, as DOI:10.1104/pp.15.00030

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Running head: Salt reduces meristem size via NO and auxin

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Author for correspondence: Ying-Tang Lu

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State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University,

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Wuhan 430072, China

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Phone number: +86 27 68752619

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Email: [email protected]

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Research Area: Signaling and Response

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Copyright 2015 by the American Society of Plant Biologists

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Salt stress reduces root meristem size by nitric oxide-mediated modulation of

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auxin accumulation and signaling in Arabidopsis

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Wen Liu, Rong-Jun Li, Tong-Tong Han, Wei Cai, Zheng-Wei Fu and Ying-Tang Lu*

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State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University,

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Wuhan, China

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One-sentence summary: Nitric oxide functions downstream of salt stress to

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modulate auxin response for salt-mediated inhibition of root meristem development.

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Footnotes:

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This work was supported by Major State Basic Research Program (2013CB126901)

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to YTL.

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*Corresponding author; e-mail [email protected]

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Abstract

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The development of the plant root system is highly plastic, which allows the plant to

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adapt to various environmental stresses. Salt stress inhibits root elongation by

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reducing the size of the root meristem. However, the mechanism underlying this

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process remains unclear. In this study, we explored whether and how auxin and nitric

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oxide (NO) are involved in salt-mediated inhibition of root meristem growth in

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Arabidopsis (Arabidopsis thaliana) using physiological, pharmacological, and genetic

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approaches. We found that salt stress significantly reduced root meristem size by

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down-regulating the expression of PINs, thereby reducing auxin levels. In addition,

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salt stress promoted AXR3/IAA17 stabilization, which repressed auxin signaling

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during this process. Furthermore, salt stress stimulated NO accumulation, whereas

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blocking NO production with the inhibitor Nω-nitro-L-Arg-methylester (L-NAME)

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compromised the salt-mediated reduction of root meristem size, PIN down-regulation

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and stabilization of AXR3/IAA17, indicating that NO is involved in salt-mediated

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inhibition of root meristem growth. Taken together, these findings suggest that salt

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stress inhibits root meristem growth by repressing PIN expression (thereby reducing

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auxin levels) and stabilizing IAA17 (thereby repressing auxin signaling) via

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increasing NO levels.

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Introduction

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Due to agricultural practices and climate change, soil salinity has become a

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serious factor limiting the productivity and quality of agricultural crops (Zhu, 2007).

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Worldwide, high salinity in the soil damages approximately 20% of total irrigated

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lands and takes 1.5 million hectares out of production each year (Munns and Tester,

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2008). In general, high salinity affects plant growth and development by reducing

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plant water potential, altering nutrient uptake and increasing the accumulation of toxic

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ions (Hasegawa et al., 2000; Munns, 2002; Zhang and Shi, 2013). Together, these

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effects severely reduce plant growth and survival.

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Since the root is the first organ to sense high salinity, salt stress plays a direct,

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important role in modulating root system architecture (Wang et al., 2009). For

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instance, salt stress negatively regulates root hair formation and gravitropism (Sun et

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al., 2008; Wang et al., 2008). The role of salt in lateral root formation depends on the

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NaCl concentration. While high NaCl levels inhibit lateral root formation, lower NaCl

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levels stimulate lateral root formation in an auxin-dependent manner (Zolla et al.,

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2010; Ji et al., 2013). The root meristem plays an essential role in sustaining root

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growth (Perilli et al., 2012). Salt stress inhibits primary root elongation by

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suppressing root meristem activity (West et al., 2004). However, how this inhibition

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occurs remains largely unclear.

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Plant hormones are important intermediary signaling compounds that function

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downstream of environmental stimuli. Among plant hormones, indole-3-acetic acid

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(IAA) is thought to play a fundamental role in root system architecture by regulating

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cell division, expansion, and differentiation. In Arabidopsis (Arabidopsis thaliana)

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root tips, a distal auxin maximum is formed and maintained by polar auxin transport

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(PAT), which determines the orientation and extent of cell division in the root

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meristem as well as root pattern formation (Sabatini et al., 1999). PIN-FORMED

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(PIN) proteins, which are components of the auxin efflux machinery, regulate primary

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root elongation and root meristem size (Blilou et al., 2005; Dello Ioio et al., 2008;

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Yuan et al., 2013; Yuan et al., 2013). The auxin signal transduction pathway is

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activated by direct binding of auxin to its receptor protein, TIR1/AFB, promoting the 5 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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degradation of Aux/IAA proteins, releasing auxin response factors (ARFs) and

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activating the expression of auxin-responsive genes (Gray et al., 2001; Dharmasiri et

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al., 2005a; Kepinski and Leyser, 2005). Aux/IAA proteins are short-lived,

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nuclear-localized proteins that play key roles in auxin signal activation and root

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growth modulation (Rouse et al., 1998). Other hormones and stresses often regulate

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auxin signaling by affecting Aux/IAA protein stability (Lim and Kunkel, 2004;

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Nemhauser et al., 2004; Wang et al., 2007; Kushwah and Laxmi, 2013).

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Nitric oxide (NO) is a signaling molecule with diverse biological functions in

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plants (He et al., 2004; Fernandez-Marcos et al., 2011; Shi et al., 2012), including

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important roles in the regulation of root growth and development. NO functions

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downstream of auxin during the adventitious rooting process in cucumber (Cucumis

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sativus; Pagnussat et al., 2002). Exogenous auxin-induced NO biosynthesis is

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associated with nitrate reductase activity during lateral root formation, and NO is

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necessary for auxin-induced lateral root and root hair development (Pagnussat et al.,

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2002; Lombardo et al., 2006). Pharmacological and genetic analyses in Arabidopsis

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indicate that NO suppresses primary root growth and root meristem activity

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(Fernandez-Marcos et al., 2011). Additionally, both exogenous application of the NO

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donor sodium nitroprusside (SNP) and over-accumulation of NO in the mutant

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chlorophyll a/b binding protein underexpressed 1/NO overproducer 1 (cue1/nox1)

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result in reduced PIN1 expression and auxin accumulation in root tips. The auxin

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receptors protein TIR1 is S-nitrosylated by NO, suggesting that this protein is a direct

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target of NO in the regulation of root development (Terrile et al., 2012).

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Because NO is a free radical, NO levels are dynamically regulated by endogenous

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and environmental cues. Many phytohormones, including ABA, auxin, cytokinin, SA,

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JA, and ethylene, induce NO biosynthesis (Zottini et al., 2007; Kolbert et al., 2008;

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Tun et al., 2008; Garcia et al., 2011). In addition, many abiotic and biotic stresses or

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stimuli, such as cold, heat, salt, drought, heavy metals, and pathogens/elicitors, also

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stimulate NO biosynthesis (Zhao et al., 2009; Mandal et al., 2012). Salt stress

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stimulates NO and ONOO- accumulation in roots (Corpas et al., 2009), but the

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contribution of NO to root meristem growth under salinity stress has yet to be 6 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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examined in detail.

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In this study, we found that salt stress significantly down-regulated the expression

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of PINs and promoted AXR3/IAA17 stabilization. Furthermore, salt stress stimulated

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NO accumulation, and pharmacological inhibition of NO biosynthesis compromised

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the salt-mediated reduction in root meristem size. Our results support a model in

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which salt stress reduces root meristem size by increasing NO accumulation, which

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represses PIN expression and stabilizes IAA17, thereby reducing auxin levels and

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repressing auxin signaling.

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Results

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Salt-mediated inhibition of root meristem development is due to reduced auxin

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accumulation in roots

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To begin to elucidate how salt stress reduces root meristem size, we transferred

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5-day-old seedlings germinated on half-strength MS plates to new plates

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supplemented with or without 100 mM NaCl and measured primary root growth 2

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days after transfer. We chose to move the seedlings after germination because salt

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stress inhibits seed germination in Arabidopsis (Park et al., 2011). We found that

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primary root elongation was inhibited and root meristem size was reduced in 100 mM

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NaCl-treated seedlings (Supplemental Fig. S1), similar to the results of West et al.

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(2004). Based on these results, 100 mM NaCl was used in subsequent experiments.

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Auxin plays an essential role in root meristem maintenance (Swarup et al., 2002;

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Overvoorde et al., 2010). The defective root meristem patterning observed under salt

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stress raised the question of whether auxin content or auxin signaling is affected by

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salt stress. Hence, we looked for changes in auxin signaling in salt-treated roots using

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the auxin-responsive DR5::GFP marker line, which reports auxin accumulation and

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distribution (Friml et al., 2003). For this purpose, 5-day-old seedlings were treated

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with or without 100 mM NaCl for 24 h and the expression of DR5::GFP was

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monitored. The fluorescence intensity in salt-treated DR5::GFP roots was

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significantly lower than that in untreated roots (Fig. 1, A and B). We directly

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measured endogenous IAA in roots using GC-MS and found that IAA levels were

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significantly lower in salt-treated roots than in the untreated control (Fig. 1C),

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suggesting that decreased auxin accumulation may be responsible for the reduced root

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meristem size under salt stress. We tested this notion by experiments employing

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exogenous application of auxin. We transferred 5-day-old seedlings germinated on

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half-strength MS plates to new plates containing 100 mM NaCl supplemented with

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various concentrations of IAA and measured the root meristem length and cell number

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2 days after transfer. Whereas the root meristem length and cell number were

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significantly reduced upon 100 mM NaCl treatment, application of IAA led to a

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longer root meristem and increased root meristem cell number in roots subjected to 8 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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100 mM NaCl treatment (Fig. 1, D and E), indicating that exogenous auxin partially

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rescues the salt-related inhibition of root meristem size.

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Auxin accumulation in root tips is modulated by PAT though auxin carriers 9 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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(Blilou et al., 2005; Overvoorde et al., 2010). The changes in auxin levels in roots

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subjected to salt stress may be due to changes in PAT. Thus, we treated wild-type

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plants with NaCl in the presence or absence of naphthylphthalamic acid (NPA), an

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auxin transport inhibitor, and examined both root meristem length and cell number.

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Whereas treatment with NaCl alone decreased root meristem size, root meristem

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length and cell number were not further reduced by the presence of NPA (Fig. 1, F

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and G), suggesting that PAT is required for the modulation of root meristem size by

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salt stress.

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PIN1, PIN3, and PIN7 are involved in regulating root meristem development

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under salt stress

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An auxin gradient and maximum in the root apex is established by PAT via auxin

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carriers, such as PIN1, PIN3, and PIN7 (Blilou et al., 2005; Dello Ioio et al., 2008;

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Hong et al., 2014). The reduced auxin accumulation observed in roots subjected to

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salt stress may have been due to the suppression of auxin carriers. Indeed, we found

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that the mRNA levels of PIN1, PIN3, and PIN7 were significantly reduced in roots

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subjected to 6 h of salt stress (Fig. 2A). This conclusion was further confirmed by

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analyzing PIN1::PIN1-GFP, PIN3::PIN3-GFP, and PIN7::PIN7-GFP lines. As

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visualized by PIN-GFP fluorescence, the protein levels of PIN1, PIN3, and PIN7 were

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reduced in salt-treated roots (Fig. 2, B and C), although their distribution patterns

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were not altered under salt stress.

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We then explored the role of PINs in salt-mediated inhibition of root meristem

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growth using pin mutants. While both pin3 and pin7 had similar phenotypes to those

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of wild-type seedlings, pin1 exhibited less of a reduction in root meristem length and

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cell number under salt stress compared with wild-type seedlings (Fig. 2, D and E),

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implying that PIN1 plays a role in the response to salt stress. In addition, the

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pin1pin3pin7 triple mutant was even more tolerant to salt stress than pin1 in terms of

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root meristem length and cell number (Fig. 2, D and E). Taken together, these results

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suggest that these PIN genes function additively in salt-mediated root meristem

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inhibition and that PIN1 plays a major role whereas PIN3 and/or PIN7 function in a 10 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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lesser capacity in this process.

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Salt stress stabilizes IAA17, leading to salt-mediated inhibition of root meristem 11 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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growth

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Next, we wondered whether auxin signaling was affected by salt stress. Aux/IAA

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proteins are generally thought to be transcriptional repressors of auxin-responsive

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reporter genes expression (Rouse et al., 1998; Overvoorde et al., 2005; Wang et al.,

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2013), and Aux/IAA protein levels are maintained by the E3 ubiquitin ligase complex

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SCFTIR1 (Tan et al., 2007; Maraschin Fdos et al., 2009). Accordingly, we monitored

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the effect of salt stress on the stability of IAA17 in the reporter line

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HS::AXR3NT-GUS, harboring a construct encoding the amino terminus of the

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Aux/IAA protein AXR3/IAA17 and the GUS reporter under the control of a

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heat-shock inducible promoter (Gray et al., 2001). In this system, GUS activity is

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reduced upon recognition and degradation of AXR3NT-GUS by the SCFTIR1 complex

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(Gray et al., 2001). We incubated HS::AXR3NT-GUS seedlings at 37°C for 2 h to

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enable accumulation of AXR3NT-GUS, and we assayed GUS activity in the roots of

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seedlings transferred to medium containing 100 mM NaCl and incubated at 23°C for

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45 min. GUS activity was significantly higher in salt-treated roots than in untreated

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roots (Fig. 3, A and B). These results suggest that salt stress stabilizes IAA17, an

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important component of auxin signaling.

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To further investigate whether IAA17 is involved in salt-induced inhibition of

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meristem development, we analyzed the root meristem length and cell number of

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iaa17 plants upon salt stress. The axr3-3 mutant, in which IAA17 protein is stabilized

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due to a single point-mutation (V89G) in domain II (Rouse et al., 1998), had much

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shorter root meristems with fewer cells compared with wild-type seedlings (Fig. 3, C

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and D). However, this repression of root meristem growth was not affected by salt

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stress (Fig. 3, C and D), perhaps because salt treatment could not further stabilize the

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mutant AXR3/IAA17 in axr3-3 to further repress root meristem size. We also

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examined the responsiveness of the tir1afb2afb3 mutant to NaCl treatment, as this

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mutant also has enhanced AXR3/IAA17 stability (Dharmasiri et al., 2005b). The

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mutant was more sensitive to NaCl treatment compared with the wild type in terms of

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root meristem inhibition (Fig. 3, C-F). These results imply that salt stress inhibits root

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meristem development by stabilizing IAA17. This notion was further reinforced by 12 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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analysis of axr3/iaa17, a loss-of-function mutant (Overvoorde et al., 2005). Whereas

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both root meristem size and cell number in axr3/iaa17 were similar to those of the

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wild type under normal conditions (Fig. 3, C and D), longer root meristems and 13 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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higher root meristem cell numbers were observed in axr3/iaa17 compared with

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wild-type plants under salt stress (Fig. 3, C and D), indicating that IAA17 is required

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for salt-mediated inhibition of root meristem growth.

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Salt stress affects root meristem size through over-accumulation of NO

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Salt stress stimulates NO accumulation in roots (Corpas et al., 2009), and NO was

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recently shown to modulate root meristem development (Fernandez-Marcos et al.,

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2011). First, we confirmed that NaCl-induced NO accumulation in the roots was

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significantly reduced in plants treated with L-NAME, an inhibitor of animal NO

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synthase that is also effective in plant systems, or cPTIO, a widely used NO scavenger

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(Fig. 4, A and B; Flores et al., 2008; Besson-Bard et al., 2009; Zhao et al., 2009).

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Then, we transferred 5-day-old seedlings germinated on normal half-strength MS

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plates to new plates containing 100 mM NaCl with or without 1 mM L-NAME or 250

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μM cPTIO and measured root meristem length and cell number after 2 additional days

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of growth. Combined application of either NaCl and L-NAME or NaCl and cPTIO

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reduced the inhibitory effect of salt treatment on root meristem length and cell

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number compared with salt treatment alone (Fig. 4, C and D), suggesting that NO is

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involved in salt-mediated inhibition of root meristem development. In addition, when

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the CYCB1;1::GUS line was treated with either NaCl and L-NAME or NaCl and

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cPTIO, the roots had more GUS-stained cells compared with the roots of plants

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treated with NaCl alone (Fig. 4, E and F), revealing that salt stress represses meristem

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cell division through over-accumulation of NO.

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Salt stress reduces auxin levels possibly through NO over-accumulation, leading

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to repressed root meristem development

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The above results demonstrate that salt stress inhibits root meristem development by

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reducing auxin levels in roots. We next explored whether salt stress modulates auxin

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accumulation though salt-induced NO accumulation. Thus, we first monitored

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possible changes in auxin signaling in the DR5::GFP marker line under salt stress in

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the presence or absence of L-NAME. Analysis of GFP fluorescence revealed that 14 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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additional application of L-NAME reversed the attenuated DR5 activity in salt-treated

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roots (Fig. 5, A and B), suggesting that NO contributes to the role of salt stress in

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reducing auxin accumulation. This notion was further confirmed through direct 15 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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measurement of endogenous IAA levels via GC-MS. The results show that the

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reduced IAA levels in salt-treated roots were partially rescued by additional

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application of L-NAME (Fig. 5C). 16 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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PIN1, PIN3, and PIN7 are involved in NO-mediated inhibition of root meristem

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development upon salt stress

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The above results indicate that PIN1, PIN3, and PIN7 function in regulating root

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meristem development under salt stress (Fig. 2) and that salt-treated roots accumulate

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more NO than the control (Fig. 4, A and B). Thus, we examined whether salt-induced

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NO over-accumulation modulates the expression of PIN1, PIN3, and PIN7. The

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mRNA accumulation of PIN1, PIN3, and PIN7 was decreased in the roots of plants

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treated with 20 μM sodium nitroprusside (SNP; Fig. 6A), a NO donor previously used

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to study the role of NO in regulating root growth and development

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(Fernandez-Marcos et al., 2011; Bai et al., 2012). In addition, PIN-GFP fluorescence,

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reflecting the protein levels of PIN1, PIN3, and PIN7, was significantly reduced in

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SNP-treated roots of PIN1::PIN1-GFP, PIN3::PIN3-GFP, and PIN7::PIN7-GFP

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lines (Fig. 6, B and C). These effects of NO on the expression of three PIN genes

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were also verified using another NO donor, GSNO (Supplemental Fig. S2, A-C).

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These results, together with the finding that salt stress stimulates NO accumulation in

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the roots, which represses root meristem size, suggest that salt stress may

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down-regulate the expression of PINs by increasing NO accumulation. Indeed, the

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reduced mRNA levels of PIN1, PIN3, and PIN7 in salt-treated roots were partially

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rescued in the roots treated with NaCl and L-NAME together (Fig. 6A). This result

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was further confirmed by analyzing GFP fluorescence in PIN1::PIN1-GFP,

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PIN3::PIN3-GFP, and PIN7::PIN7-GFP lines. The protein levels of PIN1, PIN3, and

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PIN7 were reduced to a lesser extent in roots treated with NaCl and L-NAME

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together compared with those in salt-treated roots (Fig. 6, B and C).

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Next, we assayed NO-mediated inhibition of both root meristem length and cell

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number in the pin1, pin3, and pin7 mutants compared with that in wild-type plants

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upon SNP treatment. All three single mutants exhibited similar inhibition to that of

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wild-type plants, whereas the triple mutant pin1pin3pin7 was less sensitive to SNP

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treatment compared with wild-type plants in both root meristem size and cell number

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(Fig. 6, D and E). This result, combined with the above findings that pin1pin3pin7 is 17 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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less sensitive to salt stress than wild type in terms of root meristem inhibition and that

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treatment with either L-NAME or cPTIO rescues salt-mediated inhibition of root

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meristem development, suggest that PIN1, PIN3, and PIN7 are involved in 18 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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NO-mediated inhibition of root meristem development in seedlings under salt stress.

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IAA17 functions in NO-regulated root meristem development upon salt stress

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Since IAA17 is required for salt stress-mediated inhibition of root meristem growth,

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we next used the HS::AXR3NT-GUS line to examine whether salt-induced

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over-accumulation of NO modulates the stability of IAA17. GUS activity was

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significantly higher in roots treated with NO donor (SNP or GSNO) than in untreated

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roots (Fig. 7, A and B; Supplemental Fig. S2, D and E), which is similar to the

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observation that salt treatment promotes IAA17 stabilization (Fig. 3, A and B). This

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salt-induced increase in GUS activity was attenuated by L-NAME treatment (Fig. 7, A

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and B), suggesting that NO contributes to the effect of salt stress on the stability of

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IAA17. Next, we verified the role of IAA17 in NO-mediated inhibition of root

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meristem size by examining both root meristem length and cell number in iaa17

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plants subjected to SNP treatment. While the axr3/iaa17 mutant exhibited less

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reduction in both root meristem length and cell number compared with wild-type

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seedlings upon SNP treatment, the inhibition was not exacerbated in the axr3-3

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mutant by SNP treatment (Fig. 7, C-F). By contrast, the tir1afb2afb3 mutant was more

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sensitive than wild-type plants to SNP treatment (Fig. 7, C-F). These results imply

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that NO inhibits root meristem growth by stabilizing IAA17. In addition, we crossed

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HS::AXR3NT-GUS plants with the mutant nox1, which has higher endogenous NO

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levels and reduced root meristem development compared with wild type

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(Fernandez-Marcos et al., 2011), and we assayed AXR3NT-GUS activity in the

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resulting nox1 HS::AXR3NT-GUS line. The results show that the stability of IAA17

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was greater in nox1 HS::AXR3NT-GUS plants than in HS::AXR3NT-GUS plants (Fig.

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7, G and H). These results, combined with the observation that salt inhibited root

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meristem growth by increasing NO levels (Fig. 4, A-D), suggest that IAA17 functions

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in NO-regulated root meristem development upon salt stress.

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Vieten et al. (2005) reported that exogenous auxin application induces the

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expression of PIN1, PIN3, and PIN7, but the induction of these genes by auxin is

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repressed in slr1, an IAA14 gain-of-function mutant, indicating that IAA14 plays a 19 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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negative role in auxin-induced expression of PIN1, PIN3, and PIN7 (Vieten et al.,

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2005). Accordingly, we examined whether AXR3/IAA17 is involved in NO-repressed

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expression of PIN1, PIN3, and PIN7. In contrast to the reduced expression of these 20 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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genes in wild-type plants treated with the NO-donor SNP (which enhances

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AXR3/IAA17 stability), axr3-3, harboring stabilized AXR3/IAA17, exhibited

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wild-type levels of expression of these genes (Supplemental Fig. S3). Furthermore,

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SNP efficiently suppressed the expression of these three PIN genes in axr3/iaa17, as

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was also observed in wild-type plants (Supplemental Fig. S3). These results suggest

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that ARX3/IAA17 is not essential for NO-repressed expression of PIN1, PIN3, and

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PIN7.

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Discussion

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Salt stress, like many other abiotic stresses, has a dramatic effect on root system

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architecture. By altering its growth pattern, the plant root system is able to reach

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larger domains of the soil environment or to escape from potential harmful areas; this

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strategy allows plants to survive biotic and abiotic stresses. The root system

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architecture of higher plants is primarily established post-embryonically through

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maintaining the root meristem, generating lateral roots, forming root hairs and

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determining the direction of growth in root tips by gravitropism (Osmont et al., 2007;

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Galvan-Ampudia and Testerink, 2011). Previous studies have demonstrated the role of

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salt stress in lateral root development, root hair formation and root gravitropism (Sun

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et al., 2008; Wang et al., 2009; Zhao et al., 2010; Zolla et al., 2010). Salt stress was

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previously reported to inhibit primary root elongation by reducing root meristem size

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(West et al., 2004). However, how salt stress regulates root meristem development has

414

remained largely unknown. In this study, we found that salt stress significantly

415

reduces root meristem size by reducing auxin accumulation and suppressing auxin

416

signaling via increasing NO levels.

417

Auxin plays critical roles during root growth and development. Proper auxin

418

signaling relies on the interplay between its biosynthesis, transport and signaling.

419

Auxin is biosynthesized in young leaves and the shoot apex (Ljung et al., 2001), and

420

shoot-derived auxin is required for root growth (Friml et al., 2003; Wisniewska et al.,

421

2006). Decreasing auxin resources or disturbing shoot-to-root PAT often results in

422

reduced auxin accumulation in roots and the formation of a shorter root meristem. For

423

example, plants either with ectopic expression of the bacterial gene iaaL in the shoot

424

apex or with parts of their shoots excised display shorter roots and less expanded root

425

meristems (Wisniewska et al., 2006; Sassi et al., 2012; Hong et al., 2014). Mutation of

426

PIN genes or application of the PAT inhibitor NPA also reduces root elongation and

427

root meristem size (Blilou et al., 2005). In the current study, we found that salt stress

428

reduced auxin levels in the roots, and NPA did not increase salt-mediated inhibition of

429

root meristem development, suggesting that salt stress modulates root meristem size,

430

at least in part, by affecting PAT. Indeed, our data further indicate the PINs are 22 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

431

involved in this process. In addition, auxin can be biosynthesized in roots, and

432

root-generated auxin also contributes to root development (Overvoorde et al., 2010).

433

To date, several IAA biosynthesis pathways have been documented, including one

434

tryptophan (Trp)-independent and four Trp-dependent pathways (Zhao, 2010).

435

However, our qRT-PCR results show that the expression of all auxin biosynthesis

436

genes examined was similar in salt-treated and untreated roots (Supplemental Fig. S4),

437

suggesting that local auxin biosynthesis in roots may not be affected by salt stress.

438

We noted that although the sensitivity of pin3, pin7, and wild-type plants to salt

439

stress was similar in terms of root meristem length and cell number, pin1pin3pin7 was

440

less sensitive to salt stress than wild-type plants and even pin1. These results suggest

441

that in addition to PIN1, PIN3 and/or PIN7 also function in salt-mediated root

442

meristem inhibition. Similar results were also obtained in a previous study of the role

443

of PIN3 and PIN7 in pulse-induced phototropism. Haga et al. (2012) demonstrate that

444

the curvature response in pulse-induced phototropism is reduced significantly in pin3,

445

but not in pin7, and impairment of the phototropic curvature of the pin3pin7 double

446

mutant is greater than that observed in pin3, indicating that PIN3 and PIN7 function

447

additively. Similar observations were also reported for other gene families such as the

448

TGA gene family. While tga4-1 mutant and wild-type plants exhibit similar

449

susceptibility to pathogen infection, tga1-1 plants exhibit significantly higher

450

pathogen growth than wild type, and the tga1-1tga4-1 double mutant has even greater

451

susceptibility than tga1-1 (Kesarwani et al., 2007). These findings suggest that TGA1

452

and TGA4 play partially redundant roles in plant basal resistance to pathogen infection,

453

with TGA1 having a greater effect than TGA4 (Kesarwani et al., 2007). Similarly,

454

Hutchison et al. (2006) determined that AHP1, AHP2, and AHP3 play overlapping

455

roles in affecting root elongation in response to cytokinin treatment, as ahp2ahp3 is

456

slightly less sensitive (and ahp1ahp2ahp3 is substantially less sensitive) than the wild

457

type in terms of 6-BA-mediated root elongation, while the sensitivity of all three

458

single mutants to cytokinin treatment is similar to that of the wild type.

459

In the current study, we found that the single mutant pin1 was less sensitive to

460

salt stress (but not to NO application) than the wild type in terms of root meristem 23 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

461

length and cell number, although the pin1pin3pin7 mutant was less sensitive to both

462

salt and NO treatment. This difference remains to be further investigated. A recent

463

report indicates that other phytohormones, such as brassinosteroid (BR), gibberellic

464

acid (GA), abscisic acid (ABA), and jasmonic acid (JA), also function in the plant

465

response to salt stress (Geng et al., 2013).

466

Galvan-Ampudia et al. (2013) reported that PIN2 plays a role in mediating a salt

467

avoidance mechanism. Thus, we further explored the possible involvement of PIN2 in

468

salt-mediated root meristem inhibition. We found that although the expression of

469

PIN2 was reduced by treatment with either salt or NO, the pin2 mutant displayed a

470

wild-type phenotype upon salt and NO treatment in terms of root meristem inhibition

471

(Supplemental Fig. S5).

472

Auxin signaling components, including Aux/IAA and ARF proteins, contribute to

473

root development (Overvoorde et al., 2010; Yan et al., 2013). Gain-of-function IAA3,

474

IAA12, and IAA17 mutants and loss-of-function ARF5 mutants display defective root

475

development (Hamann et al., 2002; Dello Ioio et al., 2008). In the current study, salt

476

stress increased the stability of AXR3/IAA17, which functions in salt-mediated

477

inhibition of root meristem development. The expression of a number of auxin

478

response genes and cell wall-related genes, which is reduced in the gain-of-function

479

mutant axr3-1 (Overvoorde et al., 2005), was also significantly reduced by salt

480

treatment (Supplemental Fig. S6), further supporting the notion that salt stress

481

increases the stability of IAA17, thereby repressing auxin signaling. We also observed

482

increased responsiveness of tir1afb2afb3 to salt stress compared with the wild type in

483

terms of root meristem development, whereas NaCl treatment did not exacerbate the

484

severe root meristem phenotypes associated with the axr3-3 mutant. It could be that

485

the stabilizing effect of IAA17/AXR3 in the triple mutant was less pronounced than

486

that in axr3-3 because of functional redundancy within the TIR1/AFB family.

487

NO, as an endogenous signaling molecule, plays a key role during plant

488

adaptation to various environmental stresses (Qiao and Fan, 2008; Zhao et al., 2009;

489

Shi et al., 2012). A previous study involving increasing exogenous or endogenous NO

490

levels revealed that NO regulates root meristem size through affecting PIN1-mediated 24 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

491

auxin accumulation in roots (Fernandez-Marcos et al., 2011). Here, we demonstrated

492

that salt stress reduced auxin accumulation by increasing NO levels through

493

repressing PIN expression in salt-treated roots. NO also affected auxin signaling in

494

the salt-mediated reduction of root meristem size, since NO is essential for

495

salt-promoted stabilization of IAA17 in roots upon salt stress treatment. Interestingly,

496

through investigating an HS::AXR3NT-GUS line, Terrile et al. (2012) showed that NO

497

promotes the degradation of IAA17 in the leaves of six-day-old seedlings. Here, we

498

found that exogenous application of NO stabilizes IAA17 in roots, which was also

499

observed in the nox1 mutant. The difference in these effects on IAA17 may be due to

500

the different tissues examined, as auxin responses differ in different tissues. For

501

example, auxin accumulates on the illuminated sides of plant roots, promoting

502

negative phototropism in the root, whereas auxin accumulation is detected on the

503

shaded side of the plant hypocotyl, promoting positive phototropism in the shoot

504

(Ding et al., 2011; Zhang et al., 2013).

505

Like NO, reactive oxygen species (ROS) play an important role in plant growth

506

and environmental responses (Verslues et al., 2007; Miller et al., 2010; Tsukagoshi et

507

al., 2010). Tsukagoshi et al. (2010) demonstrated that transcriptional regulation of

508

ROS by UPB1 regulates the balance between cellular proliferation and differentiation

509

in the roots. Moreover, crosstalk between ROS and auxin regulatory networks is also

510

involved in modulating plant stress responses (Tognetti et al., 2012; Gao et al., 2014).

511

Whether ROS also function in salt-induced root meristem inhibition requires further

512

investigation.

513

In conclusion, our results indicate that salt stress reduces root meristem length

514

and cell numbers, thereby generating short primary roots, by increasing NO levels.

515

The elevated NO accumulation further down-regulates the expression of PINs,

516

leading to reduced auxin levels, and thus stabilizes IAA17 for repressed auxin

517

signaling.

518 519

Materials and Methods

520

Plant materials and growth conditions 25 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

521

Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used in this study. The

522

transgenic and mutant lines used in this study include the following: DR5::GFP

523

(Friml et al., 2003); PIN1::PIN1-GFP (Benkova et al., 2003); PIN2::PIN2-GFP

524

(Blilou et al., 2005); PIN3::PIN3-GFP (Blilou et al., 2005); PIN7::PIN7-GFP (Blilou

525

et al., 2005); CYCB1;1::GUS (Colon-Carmona et al., 1999); pin1pin3pin7 (Blilou et

526

al., 2005); tir1afb2afb3 (Dharmasiri et al., 2005b); and cue1/nox1 (He et al., 2004).

527

Lines HS::AXR3NT-GUS (CS9571), pin1 (SALK_047613), pin2 (CS8058), pin3

528

(CS9364), pin7 (CS9367), axr3/iaa17 (SALK_065697), and axr3-3 (CS57505) were

529

obtained from the Arabidopsis Biological Resource Center. The transgenic and mutant

530

lines were confirmed by PCR. Arabidopsis seeds were surface sterilized for 5 min

531

with 5% bleach, washed three times with sterile water, incubated for 3 days at 4°C in

532

the dark and plated onto agar medium containing half-strength Murashige and Skoog

533

(MS) medium (Sigma-Aldrich), pH 5.8, supplemented with 0.8% agar and 1%

534

sucrose. Seedlings were grown in a growth chamber maintained at 23°C, 80 µmol

535

photons m-2 s-1 light under a 16/8 h light/dark cycle.

536 537

Measurement of root meristem size

538

Seeds were germinated on half-strength MS medium as described above and grown in

539

a vertical position. Five-day-old seedlings were transferred onto plates supplemented

540

with various components and grown for an additional 2 days. Digital images of

541

seedlings were captured for subsequent measurement of the lengths of newly grown

542

roots, and the roots were then excised, mounted immediately on glass slides with

543

clearing solution (50 g chloral hydrate, 15 mL water, and 10 mL glycerol), examined

544

under an Olympus BX60 differential interference contrast (DIC) microscope and

545

photographed using a Charge-Coupled Device (CCD) Olympus dp72 camera. The

546

root meristem zone was defined according to published methods (Dello Ioio et al.,

547

2007). Measurements of newly grown root length and root meristem length were

548

carried out as previously described (Yuan et al., 2013). At least 30 seedlings were

549

analyzed per treatment and genotype.

550 26 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

551

Measurement of GUS activity

552

GUS histochemical staining was performed as previously described (Hu et al., 2010).

553

Seedlings harboring the GUS reporter gene were incubated at 37°C in staining

554

solution (100 mM sodium phosphate buffer pH 7.5, 10.0 mM EDTA, 0.5 mM

555

potassium

556

5-bromo-chloro-3-indolyl-b-D-glucuronide, and 0.1% Triton X-100). The duration of

557

GUS staining was chosen based on the transgenic marker line: 6 h for CYCB1;1::GUS

558

and 12 h for HS::AXR3NT-GUS. A quantitative GUS activity assay was performed

559

according to previously described methods (Gao et al., 2013).

ferricyanide,

0.5

mM

potassium

ferrocyanide,

1

mM

560 561

Determination of NO contents

562

The endogenous NO levels in root meristems were visualized using the specific NO

563

fluorescent probe DAF-2 DA (He et al., 2004; Moreau et al., 2008; Shi et al., 2012).

564

For DAF-2 DA imaging, seedlings were incubated in 10 μM DAF-2 DA in 20 mM

565

HEPES-NaOH (pH 7.5) for 1 h and rinsed three times with HEPES-NaOH buffer

566

prior to visualization under an Olympus BX60 DIC microscope equipped with a CCD

567

Olympus dp72 camera with excitation set at 488 nm and emission set at 515 nm. At

568

least 15 seedlings were analyzed per treatment. Quantitative measurement of

569

fluorescence intensity was performed using Photoshop CS5 (Adobe, San Jose, CA,

570

USA).

571 572

Confocal microscopy

573

Confocal images were captured using an Olympus FluoView 1000 confocal laser

574

scanning microscope according to the manufacturer's instructions as previously

575

described (Yuan, TT et al., 2013). Briefly, 5-day-old seedlings were transferred to

576

plates containing different compounds and treated for the indicated time. The root tips

577

of GFP lines were then mounted onto microscope slides for observation. The emission

578

wavelength for GFP detection was 500 to 540 nm. For each treatment and genotype,

579

photographs of at least 15 seedlings were taken and analyzed. Quantitative

580

measurement of GFP signal intensity was performed using Photoshop CS5 (Adobe, 27 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

581

San Jose, CA, USA).

582 583

RNA extraction and quantitative RT-PCR

584

As previously described (Gao et al., 2013), total RNA from roots was isolated using

585

PureLink™ Plant RNA Reagent (Invitrogen) according to the manufacturer’s

586

instructions. To remove contaminating DNA, all RNA samples were digested with

587

RQ1 RNase-free DNase I (Promega). Reverse transcription was then carried out using

588

ReverTra Ace® (TOYOBO). Quantitative PCR was performed on a Bio-Rad CFX96

589

apparatus with SYBR Green I dye (Invitrogen). PCR was carried out in 96-well plates

590

as following: 3 min incubation at 95°C for complete denaturation, followed by 40

591

cycles of 95°C for 15 s and 60°C for 45 s. The PP2A subunit gene PDF2

592

(AT1G13320) and EIF4A (AT3G13920) were chosen as the best reference genes for

593

our conditions based on analysis with geNorm software (Czechowski et al., 2005). All

594

experiments were performed with three independent biological replicates and three

595

technical repetitions. The primer sequences used to amplify auxin-related genes are

596

listed in Gao et al. (2013), and the other primer sequences can be found in

597

Supplemental Table S1.

598 599

Quantification of IAA levels by GC-SIM-MS

600

Endogenous IAA levels were quantified according to a previously described protocol

601

(Gao et al., 2013). For each sample, root tips of at least 100 mg fresh weight were

602

collected and immediately frozen in liquid nitrogen. After extraction, the endogenous

603

IAA was purified, methylated in a stream of diazomethane gas and resuspended in

604

100 μL ethyl acetate. The endogenous IAA content was analyzed by gas

605

chromatography-selected ion monitoring mass spectrometry (GC-SIM-MS). A

606

Shimadzu GCMS-QP2010 Plus system (Shimadzu, Kyoto, Japan) equipped with an

607

HP-5MS column (30 m long, 0.25 mm i.d., 0.25 μm Film; Agilent, Palo Alto, CA,

608

USA) was used to determine IAA levels.

609 610

Statistical analysis 28 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

611

All experiments were performed with at least three repetitions. The significance of

612

differences was determined by ANOVA or Student’s t-test, as indicated in the figure

613

legends.

614 615

Acknowledgements

616

This work was supported by Major State Basic Research Program (2013CB126901)

617

to YTL.

618 619

Figure Legends

620

Fig. 1 Salt stress reduces auxin accumulation in roots, inhibiting root meristem

621

growth. (A) GFP fluorescence in the roots of DR5::GFP seedlings treated without or

622

with 100 mM NaCl for 24 h. Bars=50 µm. (B) Quantification of DR5::GFP

623

fluorescence intensities in plants treated as in (A). The fluorescence intensity of

624

untreated roots was set to 1. At least 15 seedlings were imaged per treatment for each

625

of three replicates. (C) IAA contents in the roots of wild-type seedlings treated

626

without or with 100 mM NaCl for 24 h. (D and E) Root meristem length (D) and root

627

meristem cell number (E) of wild-type seedlings treated without or with 100 mM

628

NaCl plus 0 nM IAA, 0.1 nM, or 0.5 nM IAA for 2 d. (F and G) Root meristem length

629

(F) and root meristem cell number (G) of wild-type seedlings treated without or with

630

100 mM NaCl in the presence or absence of 5 μM NPA for 2 d. Error bars represent

631

SD. Asterisks (***) indicate significant differences with respect to the corresponding

632

control (Student’s t-test, P < 0.001), and different letters indicate significantly

633

different values (P < 0.05 by Tukey’s test)

634 635

Fig. 2 Salt stress represses the expression of PIN genes, resulting in short root

636

meristems. (A) Quantitative RT-PCR analysis of PIN1, PIN3, and PIN7 expression in

637

the roots of wild-type seedlings treated without or with 100 mM NaCl for 6 h. The

638

expression levels of the indicated genes in untreated roots were set to 1. (B)

639

Expression of PIN1-GFP, PIN3-GFP, and PIN7-GFP in the roots of PIN1::PIN1-GFP,

640

PIN3::PIN3-GFP, and PIN7::PIN7-GFP seedlings treated without or with 100 mM 29 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

641

NaCl for 12 h. Bars=50 µm. (C) Quantification of the fluorescence intensities in

642

plants treated as in (B). The fluorescence intensity of the indicated line in untreated

643

roots was set to 1. At least 15 seedlings were imaged per line for each of three

644

replicates. (D and E) Relative root meristem length (D) and root meristem cell number

645

(E) of each genotype treated with 100 mM NaCl compared with untreated plants.

646

Error bars represent SD. Asterisks (***) indicate significant differences with respect

647

to the corresponding control (Student’s t-test, P < 0.001), and different letters indicate

648

significantly different values (P < 0.05 by Tukey’s test).

649 650

Fig. 3 Salt stress stabilizes IAA17, which functions in salt-mediated inhibition of root

651

meristem growth. (A) GUS staining images of HS::AXR3NT-GUS. Seedlings were

652

heat-shocked at 37°C for 2 h and treated without or with 100 mM NaCl for 45 min at

653

23°C, followed by GUS staining. Bars=50 µm. (B) Relative GUS activity of

654

HS::AXR3NT-GUS as treated in (A). The GUS activity in untreated plants was set to 1.

655

(C and D) Root meristem length (C) and root meristem cell number (D) of wild-type,

656

axr3-3, tir1afb2afb3, and axr3/iaa17 seedlings treated without or with 100 mM NaCl

657

for 2 d. (E and F) Relative root meristem length (E) and root meristem cell number (F)

658

of each genotype treated with 100 mM NaCl compared with untreated plants. Error

659

bars represent SD. Asterisks (***) indicate significant differences with respect to the

660

corresponding control (Student’s t-test, P < 0.001), and different letters indicate

661

significantly different values (P < 0.05 by Tukey’s test).

662 663

Fig. 4 Salt stress reduces root meristem size through NO over-accumulation. (A) NO

664

contents in the roots of wild-type seedlings treated without or with 100 mM NaCl,

665

100 mM NaCl+1 mM L-NAME, 100 mM NaCl+250 μM cPTIO, 1 mM L-NAME, or

666

250 μM cPTIO for 4 h, as revealed by the NO-specific fluorescent probe DAF-2 DA.

667

Bars=50 µm. (B) Quantification of the fluorescence intensities of plants treated as in

668

(A). The fluorescence intensity in untreated roots was set to 1. At least 15 seedlings

669

were imaged per treatment for each of three replicates. (C and D) Root meristem

670

length (C) and root meristem cell number (D) of wild-type seedlings treated without 30 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

671

or with 100 mM NaCl, 100 mM NaCl+1 mM L-NAME, 100 mM NaCl+250 μM

672

cPTIO, 1 mM L-NAME, or 250 μM cPTIO for 2 d. (E) GUS staining images of

673

CYCB1;1::GUS seedlings treated without or with 100 mM NaCl, 100 mM NaCl+1

674

mM L-NAME, 100 mM NaCl+250 μM cPTIO, 1 mM L-NAME, or 250 μM cPTIO

675

for 24 h. Bars=50 µm. (F) Number of GUS-stained cells in root tips of

676

CYCB1;1::GUS seedlings. Error bars represent SD, and different letters indicate

677

significantly different values (P < 0.05 by Tukey’s test).

678 679

Fig. 5 Salt stress reduces auxin levels through over-accumulation of NO. (A) GFP

680

expression in the roots of DR5::GFP seedlings treated without or with 100 mM NaCl,

681

100 mM NaCl+1 mM L-NAME, or 1 mM L-NAME for 24 h. Bars=50 µm. (B)

682

Quantification of DR5::GFP fluorescence intensities in plants treated as in (A). The

683

fluorescence intensity of untreated roots was set to 1. At least 15 seedlings were

684

imaged per treatment for each of three replicates. (C) IAA contents in the roots of

685

wild-type seedlings treated without or with 100 mM NaCl, 100 mM NaCl+1 mM

686

L-NAME, or 1 mM L-NAME for 24 h. Error bars represent SD, and different letters

687

indicate significantly different values (P < 0.05 by Tukey’s test).

688 689

Fig. 6 NO is required for repressing PIN expression and reducing root meristem size

690

upon salt stress. (A) Quantitative RT-PCR analysis of PIN1, PIN3, and PIN7

691

expression in the roots of wild-type seedlings treated without or with 100 mM NaCl,

692

20 μM SNP, 100 mM NaCl+1 mM L-NAME, or 1 mM L-NAME for 6 h. The

693

expression level of the indicated gene in untreated roots was set to 1. (B) Expression

694

of PIN1-GFP, PIN3-GFP, and PIN7-GFP in the roots of PIN1::PIN1-GFP,

695

PIN3::PIN3-GFP, and PIN7::PIN7-GFP seedlings treated without or with 100 mM

696

NaCl, 20 μM SNP, 100 mM NaCl+1 mM L-NAME, or 1 mM L-NAME for 12 h.

697

Bars=50 µm. (C) Quantification of the fluorescence intensities in plants treated as in

698

(B). The fluorescence intensity of the indicated line in untreated roots was set to 1. At

699

least 15 seedlings were imaged per line for each of three replicates. (D and E)

700

Relative root meristem length (D) and root meristem cell number (E) of wild-type and 31 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

701

pin mutants treated with 20 μM SNP compared with untreated plants. Error bars

702

represent SD, and different letters indicate significantly different values (P < 0.05 by

703

Tukey’s test).

704 705

Fig. 7 NO is necessary for stabilization of IAA17 by salt stress. (A) GUS staining

706

images of HS::AXR3NT-GUS. Seedlings were heat-shocked at 37°C for 2 h and

707

treated without or with 100 mM NaCl, 20 μM SNP, 100 mM NaCl+1 mM L-NAME,

708

or 1 mM L-NAME for 45 min at 23°C, followed by GUS staining. Bars=50 µm. (B)

709

Relative GUS activity of HS::AXR3NT-GUS as treated in (A). The GUS activity in

710

untreated plants was set to 1. (C and D) Root meristem length (C) and root meristem

711

cell number (D) of wild-type, axr3-3, tir1afb2afb3, and axr3/iaa17 seedlings treated

712

without or with 20 μM SNP for 2 d. (E and F) Relative root meristem length (E) and

713

root meristem cell number (F) of each genotype treated with 20 μM SNP compared

714

with untreated plants. (G) GUS staining images of HS::AXR3NT-GUS in the WT and

715

nox1 mutant background. Seedlings were heat-shocked at 37°C for 2 h and transferred

716

to 23°C. GUS staining was performed at 0 min, 30 min, or 120 min after transfer.

717

Bars=50 µm. (H) Relative GUS activity of HS::AXR3NT-GUS as treated in (G). The

718

GUS activity in WT plants at 0 min after heat shock was set to 1. Error bars represent

719

SD, and different letters indicate significantly different values (P < 0.05 by Tukey’s

720

test).

32 Downloaded from www.plantphysiol.org on April 17, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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1

Supplemental Data

2 3

Supplemental Figure S1 Salt stress reduces primary root elongation and root

4

meristem size. Five-d-old wild-type seedlings were treated without or with 100 mM

5

NaCl for 2 d. (A-C) Newly grown root length (A), root meristem length (B), and root

6

meristem cell number (C) were measured. Error bars represent SD, and asterisks

7

indicate significant differences with respect to the corresponding control by Student’s

8

t-test at ***, P < 0.001.

9 10 11 12 13 14 15 16 17 1

18

19 20

Supplemental Figure S2 The effects of GSNO on PIN expression and AXR3 protein

21

stability. (A) Quantitative RT-PCR analysis of PIN1, PIN3, and PIN7 expression in

22

the roots of 5-d-old wild-type plants treated without or with 200 μM GSNO for 6 h.

23

The expression level of the indicated gene in untreated roots was set to 1. (B)

24

Expression of PIN1-GFP, PIN3-GFP, and PIN7-GFP in the roots of 5-d-old

25

PIN1::PIN1-GFP, PIN3::PIN3-GFP, and PIN7::PIN7-GFP seedlings treated without

26

or with 200 μM GSNO for 12 h. Bars=50 µm. (C) Quantification of the fluorescence

27

intensities in plants treated as in (B). The fluorescent intensity of the indicated line in

28

untreated roots was set to 1. At least 15 seedlings were imaged per line for each of

29

three replicates. (D) GUS staining images of HS::AXR3NT-GUS. Seedlings were

30

heat-shocked at 37°C for 2 h and treated without or with 100 μM GSNO for 45 min at

31

23°C, followed by GUS staining. Bars=50 µm. (E) Relative GUS activity of

32

HS::AXR3NT-GUS as treated in (D). The GUS activity in untreated plants was set to 1.

33

Error bars represent SD, and asterisks indicate significant differences with respect to

34

the corresponding control by Student’s t-test at **, P < 0.01 and ***, P < 0.001. 2

35

36 37

Supplemental Figure S3 AXR3/IAA17 is not essential for NO-repressed expression

38

of PIN1, PIN3, and PIN7. Quantitative RT-PCR analysis of PIN1, PIN3, and PIN7

39

expression in the roots of untreated wild-type plants, SNP-treated wild-type plants,

40

untreated axr3-3 plants, untreated axr3/iaa17 plants, or SNP-treated axr3/iaa17 plants.

41

Error bars represent SD, and different letters indicate significantly different values (P

42

< 0.05 by Tukey’s test).

43 44 45 46 47 48 49 50 51 52 53 54 55 56 3

57

58 59

Supplemental Figure S4 Expression of auxin biosynthesis-related genes in roots

60

under salt stress. Quantitative RT-PCR analysis of the expression of auxin

61

biosynthesis genes in the roots of 5-d-old wild-type plants treated without or with 100

62

mM NaCl for 6 h. Error bars represent SD.

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 4

78

79 80

Supplemental Figure S5 The effects of PIN2 on salt-mediated root meristem

81

inhibition. (A) Quantitative RT-PCR analysis of PIN2 expression in the roots of

82

5-d-old wild-type plants treated without or with 100 mM NaCl or 20 μM SNP for 6 h.

83

The expression level of the indicated gene in untreated roots was set to 1. (B)

84

Expression of PIN2-GFP in the roots of 5-d-old PIN2::PIN2-GFP seedlings treated

85

without or with 100 mM NaCl or 20 μM SNP for 12 h. Bars=50 µm. (C)

86

Quantification of the fluorescence intensities in plants as treated in (B). The

87

fluorescent intensity of the indicated line in untreated roots was set to 1. At least 15

88

seedlings were imaged per line for each of three replicates. (D and F) Root meristem

89

length (D) and root meristem cell number (F) of 5-d-old wild-type seedlings treated

90

without or with 100 mM NaCl or 20 μM SNP for 2 d. (E and G) Relative root

91

meristem length (E) and root meristem cell number (G) of each genotype treated with

92

100 mM NaCl or 20 μM SNP compared with untreated plants. Error bars represent

93

SD, and different letters indicate significantly different values (P < 0.05 by Tukey’s 5

94

test).

95 96

Supplemental Figure S6 Expression of auxin response and cell wall-related genes in

97

roots under salt stress. Quantitative RT-PCR analysis of the expression of auxin

98

response and cell wall-related genes in the roots of 5-d-old wild-type plants treated

99

without or with 100 mM NaCl for 6 h. Error bars represent SD, and asterisks indicate

100

significant differences with respect to the corresponding control by Student’s t-test at

101

***, P < 0.001.

102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 6

122

Supplemental Table S1 List of the Primers Used in this study.

123

Primer name PDF2 F PDF2 R eIF4A F eIF4A R PIN1 F PIN1 R PIN2 F PIN2 R PIN3 F PIN3 R PIN7 F PIN7 R SAUR15 F SAUR15 R SAUR23 F SAUR23 R IAA2 F IAA2 R IAA5 F IAA5 R IAA6 F IAA6 R GH3-2 F GH3-2 R XTH8 F XTH8 R XTH10 F XTH10 R

124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151

Sequence (5’ to 3’) TAACGTGGCCAAAATGATGC GTTCTCCACAACCGCTTGGT TCATAGATCTGGTCCTTGAAACC GGCAGTCTCTTCGTGCTGAC GGAGACTTAAGTAGGAGCTCAGCA CCAAAAGAGGAAACACGAATG TTACTCCGTTCAATCGTCAC ACGCCTTTAGAAGACTGAAG TCTTTGATTAGGTTCGGGTAACTC GCTCATGTGAAACTGGAACAAG CCAAGATTAGTGGAACGCAAC GAAAAGGGTTTTTGGATCCTC ATGGCTTTTTTGAGGAGTTTCTTGGG TCATTGTATCTGAGATGTGACTGTG ATGGCTTTGGTGAGAAGTCTATTGGT TCAATGGAGCCGAGAAGTCACATTGA TTGTAAGAGACTCAGAATCATGAAGG CAGCTTCTCTGGATCATAAGGAA TCTGCAAATTCTGTTCGGATGCT CTCTTGCACGATCCAAGGAACATT AATCTCTTCGGCTGTCTTGGCATA TGGAGACCAAAACCAGTTGCAT CTTAGACCGACGTCAGCTTTTATACAG GGTAACCCACCTGACGTCTTTG ACCCGACCAAGGATTATCACACC TTTCCAGTCAGTCTTCTCCAGACC CCTAACCGGGATGAGATTGACTTTGA CAACCTTATCGGAATCTGATCCACCA

152 153

7