Ethylene Inhibits Root Elongation during Alkaline …...Ethylene Inhibits Root Elongation during Alkaline Stress through AUXIN1 and Associated Changes in Auxin Accumulation1 Juan Li,

Ethylene Inhibits Root Elongation during AlkalineStress through AUXIN1 and Associated Changes inAuxin Accumulation1

Juan Li, Heng-Hao Xu, Wen-Cheng Liu, Xiao-Wei Zhang, and Ying-Tang Lu*

State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China (J.L.,W.-C.L., X.-W.Z., Y.-T.L.); and Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening andCo-Innovation Center for Jiangsu Marine Bio-Industry Technology, Huaihai Institute of Technology,Lianyungang 222005, China (H.-H.X.)

ORCID ID: 0000-0002-8875-9091 (Y.-T.L.).

Soil alkalinity causes major reductions in yield and quality of crops worldwide. The plant root is the first organ sensing soilalkalinity, which results in shorter primary roots. However, the mechanism underlying alkaline stress-mediated inhibition ofroot elongation remains to be further elucidated. Here, we report that alkaline conditions inhibit primary root elongation ofArabidopsis (Arabidopsis thaliana) seedlings by reducing cell division potential in the meristem zones and that ethylene signalingaffects this process. The ethylene perception antagonist silver (Ag+) alleviated the inhibition of root elongation by alkalinestress. Moreover, the ethylene signaling mutants ethylene response1-3 (etr1-3), ethylene insensitive2 (ein2), and ein3-1 showedless reduction in root length under alkaline conditions, indicating a reduced sensitivity to alkalinity. Ethylene biosynthesisalso was found to play a role in alkaline stress-mediated root inhibition; the ethylene overproducer1-1 mutant, whichoverproduces ethylene because of increased stability of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE5,was hypersensitive to alkaline stress. In addition, the ethylene biosynthesis inhibitor cobalt (Co2+) suppressed alkaline stress-mediated inhibition of root elongation. We further found that alkaline stress caused an increase in auxin levels by promotingexpression of auxin biosynthesis-related genes, but the increase in auxin levels was reduced in the roots of the etr1-3 andein3-1 mutants and in Ag+/Co2+-treated wild-type plants. Additional genetic and physiological data showed that AUXIN1(AUX1) was involved in alkaline stress-mediated inhibition of root elongation. Taken together, our results reveal that ethylenemodulates alkaline stress-mediated inhibition of root growth by increasing auxin accumulation by stimulating the expressionof AUX1 and auxin biosynthesis-related genes.

Soil alkalinity limits agricultural productivity world-wide by affecting 434 million ha of land in more than100 countries (Jin et al., 2006). Because only a few plantscan grow well in alkalinized soil, soil alkalinity limitsthe availability of arable land and results in low agri-cultural productivity. Accordingly, studies on the mech-anisms underlying plant responses and adaptation toalkaline stress are important for both basic plant bio-logy knowledge and agricultural applications.

Alkaline stress can lead to reduced seed germination,decreased shoot growth, and smaller leaves as well asinhibited root growth and elongation (Cha-Um et al.,

2009; Patil et al., 2012). In addition to this inhibitionof plant growth, alkaline stress influences numer-ous metabolic and physiological processes, such asphotosynthesis, microfilament depolymerization, cel-lular ionic homeostasis, metabolic balance of reactiveoxygen species, selectivity of the membrane system,and osmotic absorption of water (Shi and Sheng, 2005;Zhang and Mu, 2009; Zhou et al., 2010; Liu and Guo,2011).

The plant root is the first organ that senses soil al-kalinity, which inhibits primary root growth (Fuglsanget al., 2007; Yang et al., 2010; Xu et al., 2012). Severalrecent reports show that the plasma membrane H+-ATPsynthase (H+-ATPase) plays an important role in theadaptation of roots to alkaline stress by mediating pro-ton secretion (Fuglsang et al., 2007; Yang et al., 2010; Xuet al., 2013). For example, the protein kinase PROTEINKINASE SOS2-LIKE5 (PKS5) inhibits the plasma mem-brane H+-ATPase by preventing its interaction with14-3-3 proteins, resulting in higher alkaline tolerance ofArabidopsis (Arabidopsis thaliana) pks5 plants (Fuglsanget al., 2007). Conversely, the chaperone J3 activatesthe plasma membrane H+-ATPase by repressing PKS5kinase activity; thus, the j3 mutant is hypersensitive toalkaline stress (Yang et al., 2010).

1 This work was supported by the National Natural Science Foun-dation of China (grant no. 31470378).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ying-Tang Lu ([email protected]).

J.L. and H.-H.X. designed the experiments and analyzed the data;J.L. conceived the project and wrote the article with contributions ofall the authors; W.-C.L. and X.-W.Z. provided technical assistance toJ.L.; Y.-T.L. supervised and complemented the writing.

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Auxin, a well-established regulator of root growth,is subject to modulation at the levels of biosynthesis,distribution, and polar transport (Dharmasiri et al.,2005; Tanaka et al., 2006; Petrásek and Friml, 2009;Wang et al., 2009; Zhao, 2010; Li et al., 2011). Polarauxin transport (PAT) mediated by influx carriers ofthe AUXIN1 (AUX1)/LIKE AUXIN1 (LAX) family andefflux carriers of the PINFORMED (PIN) family(Vieten et al., 2007; K�re�cek et al., 2009; Péret et al.,2012) is involved in plant responses to environmentalstresses, such as phosphate starvation, ammoniumtoxicity, salt stress, and excess metals (Al and Cu;Pérez-Torres et al., 2008; Wang et al., 2009; Li et al.,2011; Yuan et al., 2013). PAT also strongly influencesprimary root growth (Blilou et al., 2005; Grieneisenet al., 2007). A recent report indicates that PIN2 is in-volved in the PKS5-mediated signaling cascade andrequired for plant adaptation to alkaline stress bymodulating proton secretion in root tips to maintainprimary root elongation (Xu et al., 2012).

In addition to auxin, ethylene is often associated withstress responses, such as heat, drought, ozone, phos-phate starvation, and aluminum excess (Sun et al., 2007,2010; Lei et al., 2011; Khan et al., 2013; Habben et al.,2014; Ludwików et al., 2014). The ethylene signalingpathway begins with ethylene binding to receptorproteins. The ethylene receptors exist as a multimemberfamily that is composed of ETHYLENE RESPONSE1(ETR1), ETHYLENE RESPONSE SENSOR1 (ERS1),ETR2, ERS2, and ETHYLENE INSENSITIVE4 (EIN4)in Arabidopsis (Grefen et al., 2008; Plett et al., 2009;Kim et al., 2011; Liu and Wen, 2012; McDaniel andBinder, 2012). Upon ethylene binding, the receptortransmits the signal to the CONSTITUTIVE TRIPLERESPONSE1 (CTR1) protein kinase, resulting in theinhibition of the ability of CTR1 to phosphorylateEIN2 and causing the translocation into the nucleusof the C-terminal end of EIN2, which then leads tothe stabilization of EIN3 and the activation of theEIN3/ETHYLENE-INSENSITIVE3-LIKE (EIL)-dependenttranscriptional cascade (Mayerhofer et al., 2012; Wenet al., 2012; Dharmasiri et al., 2013; Merchante et al.,2013).

Ethylene is produced fromMet through S-adenosyl-L-Metand 1-aminocyclopropane-1-carboxylic acid (ACC), whichare catalyzed by 1-aminocyclopropane-1-carboxylic acidsynthase (ACS) and 1-aminocyclopropane-1-carboxylicacid oxidase (ACO), respectively (Kende, 1993). ACScontrols the rate-limiting step in ethylene biosynthesis(Chae and Kieber, 2005). ACS and ACO are encodedby multigene families and regulated by many bioticand abiotic factors (Wang et al., 2002). When subjectedto exogenously supplied ACC, Arabidopsis seed-lings exhibit reduced root elongation (R�uzicka et al.,2007).

The ethylene and auxin pathways show crosstalk at multiple levels (Muday et al., 2012; Robleset al., 2013). For example, auxin increases ACStranscription, thus stimulating ethylene biosyn-thesis (Tsuchisaka and Theologis, 2004), and ethylene

up-regulates the expression of the auxin biosynthesis-related genes WEAK ETHYLENE INSENSITIVE2 (WEI2)/ANTHRANILATE SYNTHASE ALPHA SUBUNIT1 (ASA1),WEI7/ANTHRANILATE SYNTHASE BETA SUBUNIT1(ASB1), andWEI8/TRYPTOPHAN AMINOTRANSFERASEOF ARABIDOPSIS1 (TAA1) in roots, leading to in-creased auxin production and inhibition of root growth(Stepanova et al., 2005, 2008). In addition, ethylene pro-motes basipetal auxin transport to affect root elongation(R�uzicka et al., 2007).

Here, we report that alkaline stress inhibits primaryroot elongation of Arabidopsis seedlings by affectingthe meristem zone through reduced meristematic celldivision potential. Our data indicate for the first time,to our knowledge, that ethylene modulates this alka-line stress-mediated inhibition of root growth by in-creasing auxin accumulation by stimulating expressionof auxin biosynthesis-related genes and AUX1.

RESULTS

Alkaline Stress Inhibits Root Elongation by AffectingMeristem, Elongation, and Differentiation Zones

Previous reports indicated that root elongation isinhibited in media of pH 8.0 to pH 8.4 (Fuglsanget al., 2007; Xu et al., 2012). To further explore howalkaline stress modulates primary root growth, we grewArabidopsis seedlings on one-half-strength Murashigeand Skoog medium (MS) at the standard pH of 5.8 andthen, subjected 5-d-old seedlings to one-half-strengthMS at pH 8.0 or pH 9.0 as alkaline stress or pH 5.8 ascontrol for 12 h. Then, the treated seedlings weretransferred to one-half-strength MS (pH 5.8) for con-tinued growth for 2 d, and the lengths of newly grownprimary roots were measured. Our results showed thatalkaline stress inhibited primary root elongation (Fig.1A), consistent with previous reports (Fuglsang et al.,2007; Xu et al., 2012). Our data also indicated that theinhibition was positively associated with alkalinity(Fig. 1B). The primary root length was inhibited by47.1% in seedlings exposed to pH 8.0 and up to 62.8%at pH 9.0 (Fig. 1B). To examine this inhibition in detail,the lengths of both root meristem and elongation zoneswere assayed in the alkaline stress-treated roots. Bothroot meristem and elongation zones were reduced inresponse to pH 8.0 or pH 9.0 compared with pH 5.8(Fig. 1, C–E). Alkaline stress also decreased the celllength of the differentiation zone (Fig. 1F).

A decrease in root meristematic size can be causedby a reduction in stem cell niche activity, the loss ofdivision potential of meristematic cells in the proximalmeristem, or the acceleration of the elongation anddifferentiation of meristematic cells in the transitionzone (Dello Ioio et al., 2007; Baluška et al., 2010). Thus,we first analyzed whether the reduction in meristemsize was caused by possible changes in stem cellniche activity using both quiescent center (QC)25::GUSand QC46::GUS (Sabatini et al., 2003). GUS stainingshowed that both reporter constructs were expressed

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in the roots of these two lines treated with pH 8.0 orpH 9.0 (Fig. 2A), indicating that stem cell niche activityis not responsible for the reduced meristematic zone ofplant roots treated with alkaline stress.Then, we assayed meristematic cell division poten-

tial with CYCLIN B1;1 (CYCB1;1)::GUS, an excellentmarker to monitor cell cycle progression (Colón-Carmona et al., 1999). The percentage of GUS-stainedcells in the root meristem of this reporter line wassignificantly reduced in alkaline stress-treated rootscompared with that in control roots (Fig. 2B), sug-gesting that alkaline stress reduces the competence ofmeristematic cells to divide. This was further sup-ported by flow cytometry analysis of the nuclear DNAcontent in the cells of alkaline stress-treated or un-treated root tips. Indeed, alkaline stress-treated rootshad fewer cells in G2/M phases than untreated roots(Fig. 2, C and D).Furthermore, we examined the effect of alkaline

stress on the expression of a set of cell cycle-relatedgenes, including A-TYPE CYCLIN-DEPENDENT KI-NASE1 (CDKA;1), CYCLIN A2;1 (CYCA2;1), CYCB1;1,CYCB3;1, CYCLIN D1;1 (CYCD1;1), CYCD2;1, CYCD3;1,CYCD4;1, CYCD4;2, and CYCD6;1, as well as the cellcycle-related transcription factor genes E2Fa and E2Fb,all of which promote cell cycle progression and stimulatecell division (De Veylder et al., 2002; Masubelele et al.,2005; Kono et al., 2006; Sozzani et al., 2006; Cruz-Ramírezet al., 2012). Quantitative real-time (qRT)-PCR analysis

indicated that alkaline stress down-regulated the expres-sion of all of the assayed genes, except for CDKA;1 andE2Fa (Fig. 2E). By contrast, E2Fc andRETINOBLASTOMA-RELATED (RBR), which repress cell division (del Pozoet al., 2002; Wildwater et al., 2005), were significantlyup-regulated under alkaline stress (Fig. 2E).

Taken together, our data indicated that alkaline stressinhibits primary root growth by affecting the lengthsof the meristem and elongation zones and cell lengthsin the differentiation zone. Alkaline stress-induced re-duction of meristem size is caused by inhibition of celldivision in the meristem.

Ethylene Is Involved in Alkaline Stress-MediatedInhibition of Primary Root Elongation

Ethylene is often associated with stress responses(Potters et al., 2009) and plays a role in the inhibitionof primary root growth by affecting cell elongation(Swarup et al., 2007). Because our above data showedthat alkaline stress substantially inhibited root elonga-tion, we analyzed the possible involvement of ethylenesignaling in alkaline stress-mediated root inhibitionusing Ag+, an antagonist of ethylene perception. Al-though Ag+ at 10 mM slightly inhibited root elongation,alkaline stress-induced inhibition of root elongation wasmarkedly recovered in wild-type plants exposed to pH8.0 and Ag+ together (Fig. 3A), suggesting that ethylene

Figure 1. Alkaline stress inhibits primary root growth by reducing the lengths of meristem and elongation zones and the celllengths of the differentiation zone. A and B, Five-day-old wild-type seedlings were exposed to pH 5.8, pH 8.0, or pH 9.0 for12 h and then transferred (straight line) to one-half-strength MS of pH 5.8 for another 2 d. Bar = 1 cm (A). The lengths of newlygrown roots were measured after the alkaline stress-treated plants were transferred to one-half-strength MS of pH 5.8 for another2 d (B). C, Longitudinal views of Arabidopsis root meristems treated with pH 5.8, pH 8.0, or pH 9.0 for 12 h. Arrows indicateQCs. Bars = 100 mm. *, Proximal boundary of the root meristem. D to F, Root meristem zone lengths (D), root elongation zonelengths (E), and differentiation zone cell lengths (F) of 5-d-old Arabidopsis seedlings treated with pH 5.8, pH 8.0, or pH 9.0 for12 h. Data shown are means 6 SEM. Different letters indicate significant differences between treatments (P , 0.05 by one-wayANOVA with Tukey’s multiple comparison test).

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Alkalinity Inhibits Root Growth by ET and Auxin



perception participates in the inhibition of primary rootelongation by alkaline stress. Alkaline stress also in-duced the expression of ETR1, EIN2, and EIN3, pivotalcomponents of ethylene signaling (Fig. 3B). When theethylene reporter line EIN3-Binding Site (EBS)::GUS, inwhich the GUS reporter gene is driven by a syntheticEIN3-responsive promoter (Stepanova et al., 2007), wasanalyzed, a marked increase in the activity of EBS::GUSin the root apices was observed in the seedlings exposedto alkaline stress or ACC compared with that in un-treated roots (Fig. 3, C and D). These data suggest thatthe ethylene signaling pathway is involved in alkalinestress-mediated inhibition of root growth. To examinethis idea further, we used a genetic approach using theethylene-insensitive mutants etr1-3, ein2-1, and ein3-1,which were used extensively in deciphering the physi-ological functions of ethylene (Alonso et al., 1999;Binder et al., 2006; Stepanova et al., 2007). When grownat pH 8.0 and pH 9.0, the etr1-3, ein2-1, and ein3-1 mu-tants showed less-pronounced inhibition of root growthcompared with wild-type plants (Fig. 3, E and F). Col-lectively, these results indicate that ethylene signalingparticipates in alkaline stress-mediated inhibition ofroot elongation.

The enhanced ethylene signaling in alkaline stress-induced inhibition of root elongation could be causedby changes in ethylene biosynthesis. Thus, we first

assayed the primary root elongation of the seedlingstreated with ACC. We found that, although the rootlength was reduced by 35.2%, 45.9%, and 67.1% afterACC treatment for 6, 12, and 24 h, respectively, asimilar but more severe inhibition of root elongationby alkaline stress was observed (39.3%, 51.4%, and84.8% after alkaline stress treatment for 6, 12, and 24 h,respectively; Fig. 4A). Next, an antagonist of ethylenebiosynthesis (Co2+) was used to further test whetherethylene biosynthesis was involved in alkaline stress-mediated inhibition of root elongation. Although Co2+

at 10 mM had no effect on root elongation comparedwith untreated seedlings, Co2+ substantially amelio-rated the alkaline stress-mediated inhibition of rootelongation (Fig. 4B), suggesting a role of ethylene bio-synthesis in alkaline stress-mediated root elongation.Furthermore, we analyzed the expression of four genes(ACS2, ACS5, ACS6, and ACS8) involved in ethylenebiosynthesis in roots (Kende, 1993) by qRT-PCR. Asexpected, the expression of these genes was dramati-cally induced under alkaline stress treatment (Fig. 4C).Finally, we directly measured ethylene levels in theroots using gas chromatography (GC) and found thatthe ethylene level was significantly higher in alkalinestress-treated roots than that in the untreated con-trol (Fig. 4D), suggesting that increased ethylene couldbe responsible for reduced primary root length under

Figure 2. Alkaline stress reduces cell division potential. A and B, Images of GUS staining of 5-d-old QC25::GUS andQC46::GUS (A) and CYCB1;1::GUS (B) seedlings exposed to pH 5.8, 8.0, or 9.0 for 12 h. Bars = 100 mm. C and D, Flowcytometric analysis of the nuclear DNA content in the cells of root tips treated with pH 5.8 (C) or pH 8.0 (D) for 12 h and stainedwith PI. E, The relative expression of cell cycle-related genes in wild-type plants treated by pH 5.8 or pH 8.0 assayed by qRT-PCR. The expression level of the indicated gene in pH 5.8-treated roots is set to one. Data shown are means 6 SEM. Asterisksindicate significant differences with respect to each control (Student’s t test): *, P , 0.05; **, P , 0.01; and ***, P , 0.001.


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alkaline stress. To further verify this, we examined anethylene overproducer1-1 (eto1-1) mutant, in which ACS5stability is increased, leading to overproduction of eth-ylene (Chae et al., 2003). When exposed to pH 8.0 andpH 9.0, primary root elongation was inhibited by 52.9%and 77.8%, respectively, in eto1-1 seedlings but only46.6% and 63.2%, respectively, in wild-type plantscompared with the controls, indicating that the mutantis more sensitive to alkaline stress (Fig. 4, E and F).Consistent with this, ethylene accumulation in eto1-1roots was higher than that in wild-type roots (Fig. 4G).

Alkaline Stress Represses Root Elongation throughEthylene-Induced Auxin Accumulation

Ethylene stimulates auxin accumulation in root mer-istem and elongation zones (R�u�zi�cka et al., 2007),and alkaline stress increases auxin distribution in roottips (Xu et al., 2012). Auxin, as a critical phytohormonefor root growth, may also be involved in ethylene-mediated root inhibition of the seedlings exposed to

alkaline stress. Accordingly, we visualized auxin sig-naling using a domain II (DII)-VENUS line, whichexpresses the VENUS fast-maturing form of yellowfluorescent protein fused with the Aux/indole-3-aceticacid (IAA) auxin interaction domain. DII-VENUS israpidly degraded in response to auxin (Brunoud et al.,2012). Our results showed that the DII-VENUS signaldecreased in the roots subjected to alkaline stress(pH 8.0 for 6 and 12 h or pH 9.0 for 12 h) comparedwith the untreated control (Fig. 5, A and B), suggestingan increase in auxin abundance under alkaline stress.This was further supported by measurements of IAAcontent using GC-mass spectrometry. The IAA concen-trations in the roots treated with either pH 8.0 for 6 and12 h or pH 9.0 for 12 h were 135.846 13.54 and 194.21615.04 ng g21 of fresh weight or 236.96 6 15.02 ng g21 offresh weight, respectively, whereas IAA concentrationof untreated roots was 107.81 6 9.03 ng g21 of freshweight (Fig. 5C).

Next, we examined the expression of genes en-coding auxin biosynthesis-related proteins of theindole-3-acetaldoxime, tryptamine, and indole-3-pyruvic

Figure 3. Ethylene signaling is involved in alkaline stress-induced inhibition of primary root elongation. A, The effect of AgNO3

on alkaline stress-mediated inhibition of primary root elongation. Five-day-old wild-type seedlings were incubated in one-half-strength MS of pH 5.8 or pH 8.0 with additional application of 10 mM AgNO3 for 12 h, and the lengths of newly grown rootswere measured after the treated plants were transferred to one-half-strength MS of pH 5.8 for another 2 d. Values are relative toplants treated with pH 5.8 as a control. B, qRT-PCR analysis of ETR1, EIN2, and EIN3 expression in the roots of 5-d-old plantstreated with pH 8.0 for 6 or 12 h. Data are relative to those of seedlings subjected to pH 5.8 as the control. C, The effect ofalkaline stress and ACC on the expression of the ethylene reporter EBS::GUS. Five-day-old seedlings were treated with pH 8.0for 6 and 12 h or 10 mM ACC for 12 h. Bars = 100 mm. D, Relative GUS activity of EBS::GUS treated with pH 8.0 for 6 and 12 hor ACC for 12 h. The GUS activity in pH 5.8-treated EBS::GUS is set to 100%. E and F, The seedlings of the wild type (WT), etr1-3, ein2-1, and ein3-1 were exposed to pH 5.8, pH 8.0, or pH 9.0 for 12 h, and the lengths of newly grown roots were measuredafter the treated plants were transferred to one-half-strength MS of pH 5.8 for another 2 d (E). Data are relative to control valuesobtained from wild-type, etr1-3, ein2-1, and ein3-1 seedlings, respectively, treated with one-half-strength MS of pH 5.8 (F).Error bars represent SEM of three independent experiments. Asterisks indicate significant differences based on Student’s t test(B and D) or two-way ANOVA with Bonferroni posttests compared with related control (A) or the wild type at each pH con-dition (E and F): *, P , 0.05; and ***, P , 0.001.





acid pathways (Cheng et al., 2007; Ikeda et al., 2009; Zhaoand Hasenstein, 2009; Brumos et al., 2014; Gao et al., 2014).Upon treatment at pH 8.0 for 6 and 12 h, the transcriptlevels of the Trp biosynthetic genes (i.e. ASA1, ASB1,PHOSPHORIBOSYLANTHRANILATE TRANSFERASE1[PAT1], PHOSPHORIBOSYLANTHRANILATE ISOMERASE1[PAI1], and INDOLE-3-GLYCEROL PHOSPHATESYNTHASE1 [IGPS1]) increased (Fig. 5D), and those ofgenes believed to be involved in Trp-dependent IAAbiosynthesis (i.e. CYTOCHROME P450, FAMILY 79,SUBFAMILY B, POLYPEPTIDE2 [CYP79B2], YUCCA1,YUCCA6, TAA1, TRYPTOPHAN AMINOTRANSFERASERELATED1 [TAR1], TAR2, andNITRILASE3 [NIT3]) alsoincreased (Fig. 5E). The expression of SUPERROOT1(SUR1) and SUR2, which are involved in indoleglucosinolate biosynthesis, was also up-regulated bypH 8.0 for 12 h but not pH 8.0 for 6 h (Fig. 5E). Thesedata suggest that the transcriptional promotion of theauxin biosynthetic genes could be responsible for the

alkaline stress-induced increase in auxin levels. Ad-ditionally, we also assayed the expression of genesfor auxin conjugation, because IAA can be releasedby hydrolytic cleavage of IAA conjugates, contrib-uting to local auxin concentration (Ludwig-Müller,2011; Korasick et al., 2013). One of the IAA-amino acidconjugate hydrolases, IAA-LEUCINE RESISTANT-LIKE2 (ILL2), which releases free IAA by cleavingIAA-amino conjugates, was up-regulated by alka-line stress, whereas genes encoding other hydrolases,such as ILL3 and IAA-ALANINE RESISTANT3, re-mained unchanged (Fig. 5F). Not surprisingly, the ex-pression of IAA-amino synthase genes GH3.2, GH3.3,GH3.4, and GH3.6 as members of primary auxin-responsive GH3 family (Hagen and Guilfoyle, 1985;Conner et al., 1990) was also up-regulated under al-kaline stress (Fig. 5F).

Furthermore, we verified the role of auxin biosyn-thesis in alkaline stress-induced root inhibition with

Figure 4. Ethylene biosynthesis participates in alkaline stress-induced inhibition of primary root elongation. A, The effect ofACC and alkaline stress on primary root elongation. Five-day-old wild-type seedlings were treated with 10 mM ACC of pH 8.0for 6, 12, or 24 h and then transferred to one-half-strength MS of pH 5.8 for continued growth for 2 d. The lengths of newlygrown roots were measured and statistically analyzed. Data are presented as relative root elongation compared with controlvalues. B, The effect of CoCl2 on alkaline stress-mediated inhibition of primary root elongation. Five-day-old wild-type seedlingswere incubated in one-half-strength MS of pH 5.8 or pH 8.0 with additional application of 10 mM CoCl2 for 12 h, and thelengths of newly grown roots were measured after the treated plants were transferred to one-half-strength MS of pH 5.8 foranother 2 d. Values are relative to plants treated with pH 5.8 as the control. C, qRT-PCR analysis of ACS2, ACS5, ACS6, andACS8 expression in the roots of 5-d-old wild-type plants treated with pH 8.0 for 6 or 12 h. Data are relative to those of seedlingssubjected to pH 5.8 as the control. D, Ethylene evolution from roots of 5-d-old wild-type plants upon exposure to pH 8.0 for 6 or12 h or 10 mM ACC for 12 h. E and F, The seedlings of the wild type (WT) and eto1-1 were exposed to pH 5.8, 8.0, or 9.0 for12 h, and the lengths of newly grown roots were measured after the treated plants were transferred to one-half-strength MS ofpH 5.8 for another 2 d (E). Data are relative to control values obtained from wild-type and eto1-1 seedlings, respectively, treatedwith one-half-strength MS of pH 5.8 (F). G, Ethylene evolution from the roots of 5-d-old wild-type and eto1-1 plants uponexposure to pH 8.0 for 12 h. Error bars represent SEM of three independent experiments. Different letters indicate significantdifferences between treatments (P , 0.05 by one-way ANOVA with Tukey’s multiple comparison test; A). Asterisks indicatesignificant differences based on Student’s t test (C and D) or two-way ANOVA with Bonferroni posttests compared with relatedcontrol (B) or the wild type at each pH condition (E–G): FW, fresh weight; *, P , 0.05; **, P , 0.01; and ***, P , 0.001.


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wei2-2 and wei8-1mutants, in which auxin biosynthesisis impaired (Stepanova et al., 2005, 2008; Yang et al.,2014), because their expression is enhanced by alkalinestress as shown above. When exposed to pH 8.0 or pH9.0, the inhibitions of root elongations of both wei2-2and wei8-1 were less pronounced than those of wild-type plants (Fig. 5, G and H), suggesting involvementof both WEI2 (ASA1) and WEI8 (TAA1) in alkalinestress-induced inhibition of root growth. Taken to-gether, these results reveal that alkaline stress repressesroot elongation by increasing auxin accumulation

through increased expression of auxin biosynthesisgenes.

To test whether ethylene is involved in alkalinestress-induced auxin accumulation and signaling for theinhibition of root elongation, we treated DII-VENUSwith ACC and assayed the changes of the DII-VENUSsignal. The fluorescence intensity decreased to 68.5%and 54.1% in the presence of ACC for 6 and 12 h, re-spectively (Fig. 6, A and B), indicating that ethyleneincreased auxin signaling as reported by R�uzicka et al.(2007). Then, we investigated the effect of Ag+ and Co2+

Figure 5. Alkaline stress modulates auxin accumulation in the roots. A, The effect of alkaline stress on auxin distribution andaccumulation in the root meristems was monitored using an auxin signaling sensor (DII-VENUS). The fluorescence signals of 5-d-old DII-VENUS seedlings treated with pH 8.0 for 6 and 12 h or pH 9.0 for 12 h are shown. Bars = 100 mm. B, Quantification of theDII-VENUS fluorescence intensities in the root meristem zones of the DII-VENUS seedlings in A. Values are relative to those ofseedlings subjected to pH 5.8. C, IAA content in the root of 5-d-old wild-type seedlings treated with pH 8.0 for 6 and 12 h or pH9.0 for 12 h. FW, Fresh weight. D and E, qRT-PCR analysis of auxin biosynthesis-related gene expression in the roots of 5-d-oldwild-type seedlings treated with pH 8.0 for 6 or 12 h. Transcript levels of the Trp biosynthetic genes (D) and genes involved in Trp-dependent IAA biosynthesis (E). Values are relative to those of the seedlings subjected to pH 5.8 as the control. F, qRT-PCR analysisof auxin conjugation-related gene expression in the roots of 5-d-old wild-type seedlings treated with pH 8.0 for 6 or 12 h. Valuesare relative to those of the seedlings subjected to pH 5.8 as the control. G and H, The seedlings of the wild type (WT), wei2-2, andwei8-1 were exposed to pH 5.8, 8.0, or 9.0 for 12 h, and the lengths of newly grown roots were measured after the treated plantswere transferred to one-half-strength MS of pH 5.8 for another 2 d (G). Data are relative to control values obtained from wild-type,wei2-2, and wei8-1 seedlings, respectively, treated with one-half-strength MS of pH 5.8 (H). Error bars represent SEM. Asterisksindicate significant differences based on Student’s t test (B–F) or two-way ANOVA with Bonferroni posttests compared with thewild type at each pH condition (G and H): *, P , 0.05; **, P , 0.01; and ***, P , 0.001.





on DII-VENUS expression in alkaline stress-treatedroots and found that DII-VENUS fluorescence inten-sity increased by 21.2% or 22.1% in the roots subjectedto alkaline stress in the presence of either Ag+ or Co2+,respectively, compared with that in the roots treatedwith alkaline stress alone (Fig. 6, C and D), implyingthe involvement of ethylene biosynthesis and signalingin alkaline stress-increased auxin signaling. This viewwas further verified using an auxin-insensitive mutantauxin resistant1-3 (axr1-3), which carries a mutation in

AXR1, encoding a subunit of a heterodimeric RELATEDTO UBIQUITIN1-activating enzyme, resulting in a fail-ure to degrade Aux/IAA proteins (Lincoln et al., 1990;Cernac et al., 1997; Pozo et al., 1998). Our results showedthat, although the eto1-1 mutant with higher ethylenelevel was more sensitive to alkaline stress (Fig. 6, E andF), eto1-1 did not enhance the sensitivity to alkaline stressdisplayed by axr1-3 in terms of relative root elongation ofeto1-1, axr1-3, and eto1-1 axr1-3 under alkaline stress (Fig.6, E and F). Ethylene-promoted auxin signaling could be

Figure 6. Alkaline stress-increased auxin level is regulated by ethylene. A, The effect of ACC on the expression of DII-VENUS.Five-day-old DII-VENUS seedlings were treated with 10 mM ACC for 6 or 12 h. Bars = 100 mm. B, Quantification of theDII-VENUS fluorescence intensities in the meristem zones of the DII-VENUS roots in A. Values are relative to those of the seedlingssubjected to pH 5.8 as the control. C, The effect of AgNO3 or CoCl2 on alkaline stress-decreased expression of DII-VENUS.Five-day-old DII-VENUS seedlings were incubated in one-half-strength MS of pH 5.8 or 8.0 with additional application of 10mM AgNO3 or 10 mM CoCl2 for 12 h. Bars = 100 mm. D, Quantification of the DII-VENUS fluorescence intensities in themeristem zones of DII-VENUS roots in C. Values are relative to those of the seedlings subjected to pH 5.8. E and F, The seedlingsof the wild type (WT), eto1-1, axr1-3, aux1-7, eto1-1 axr1-3, and eto1-1 aux1-7 were exposed to pH 5.8, 8.0, or 9.0 for 12 h,and the lengths of newly grown roots were measured after the treated plants were transferred to one-half-strength MS of pH 5.8for another 2 d (E). Data are relative to control values obtained from wild-type, eto1-1, axr1-3, aux1-7, eto1-1 axr1-3, and eto1-1 aux1-7 seedlings, respectively, treated with one-half-strength MS of pH 5.8 (F). G, IAA content in the root of 5-d-old wild-type,etr1-3, and ein3-1 seedlings treated with pH 5.8 or 8.0 for 12 h. Data shown are mean 6 SEM. Asterisks indicate significantdifferences based on Student’s t test (B) or two-way ANOVAwith Bonferroni posttests compared with related control (D) or thewild type at each pH condition (E–G): FW, fresh weight; *, P , 0.05; **, P , 0.01; and ***, P , 0.001.


Li et al.



because of the change of auxin accumulation in alkalinestress-mediated root inhibition. Accordingly, we directlymeasured auxin contents of the roots of wild-type, etr1-3,and ein3-1 plants exposed to alkaline stress. Our resultsrevealed that auxin contents increased only by 32.1% and41.6% in etr1-3 and ein3-1, respectively, compared with80.1% in the roots of wild-type plants (Fig. 6G). Takentogether with our above data for ethylene involvement inalkaline stress-induced inhibition of root elongation(Figs. 3, A, E, and F and 4, B, E, and F), these resultsindicate that ethylene plays its role in alkaline stress-mediated root inhibition by increasing auxin levels.

AUX1 Is Required in Alkaline Stress-Induced Inhibition ofPrimary Root Elongation

The polar transport of auxin is critical for auxin dis-tribution and accumulation in roots and mediated byinflux carriers of the AUX/LAX family and efflux car-riers of the PIN family (Blilou et al., 2005; Overvoordeet al., 2010). The auxin efflux carrier PIN2 is involved inalkaline stress-induced inhibition of root elongation (Xuet al., 2012). The auxin influx carrier AUX1 also plays acrucial role in root elongation (R�u�zi�cka et al., 2007;Stepanova et al., 2007). We explored the possible role ofAUX1 in alkaline stress-induced inhibition of primaryroot elongation. We found that the expression of AUX1was up-regulated in the roots exposed to alkaline stresswith either pH 8.0 for 6 and 12 h or pH 9.0 for 12 h(Fig. 7A). In agreement with the qRT-PCR results, theAUX1-Yellow Fluorescent Protein (YFP) signal wasenhanced to 157.5%, 200.6%, or 254.8% in the roots ofAUX1::AUX1-YFP seedlings treated with pH 8.0 for 6and 12 h or pH 9.0 for 12 h compared with that ofuntreated control (Fig. 7, B and C), showing that AUX1expression is induced by alkaline stress. Then, the aux1-7mutant was used to assay the role of AUX1 in alkalinestress-mediated root inhibition. For this purpose, both

wild-type and aux1-7 seedlings were exposed to alka-line stress, and the root lengths were statistically ana-lyzed as above. We found that, when the seedlings weresubjected to pH 8.0 and pH 9.0, root elongation in wild-type plants was reduced to 53.1% and 36.8%, respec-tively, whereas root elongation of aux1-7 seedlings wasreduced only to 85.6% and 67.4%, indicating that rootelongation in aux1-7was relatively insensitive to alkalinestress (Fig. 6, E and F). Taken together, our data revealthat AUX1 participates in alkaline stress-mediated in-hibition of primary root elongation.

We next examined whether ethylene was involved inalkaline stress-inducedAUX1 expression. Alkaline stress-enhanced AUX1 expression was dramatically repressedin the roots of wild-type plants by additional applicationof either Ag+ or Co2+ (Fig. 8A). This repression of AUX1expression was further verified by its protein level,which was indicated by reduced AUX1-YFP fluorescenceintensity in the AUX1::AUX1-YFP roots subjected to Ag+

or Co2+ and alkaline stress compared with the AUX1::AUX1-YFP plants treated with alkaline stress alone (Fig.8, B and C). As expected, the alkaline stress-promotedAUX1 expression was also inhibited in etr1-3, ein2-1, andein3-1 (Fig. 8D). Furthermore, eto1-1 did not affect thesensitivity of aux1-7 to alkaline stress in terms of rootelongation of eto1-1, aux1-7, and eto1-1 aux1-7 subjectedto pH 8.0 or pH 9.0 (Fig. 6, E and F). Collectively, ourdata suggest that alkaline stress-promoted AUX1 ex-pression is mediated by ethylene in alkaline stress-mediated inhibition of primary root elongation.

DISCUSSION

Alkaline pH serves as an important factor in plantgrowth and environmental responses by modulatingnumerous metabolic and physiological processes, in-cluding photosynthesis, microfilament depolymeriza-tion, ionic homeostasis, reactive oxygen species balance,

Figure 7. AUX1 plays a role in alkaline stress-mediated inhibition of the primary root elongation. A, qRT-PCR analysis of AUX1expression in the roots of 5-d-old wild-type seedlings treated with pH 8.0 for 6 and 12 h or pH 9.0 for 12 h. Values are relativeto those of the seedlings subjected to pH 5.8 as the control. B, The effect of alkaline stress on AUX1-YFP expression in AUX1::AUX1-YFP plants. The AUX1-YFP fluorescence is shown in the roots of 5-d-old seedlings treated with pH 8.0 for 6 and 12 h orpH 9.0 for 12 h. Bars = 100 mm. C, Quantification of AUX1-YFP fluorescence intensities in the root meristem zones of theAUX1::AUX1-YFP seedlings in B. Values are relative to those of the seedlings subjected to pH 5.8. Error bars represent SEM ofthree independent experiments. ***, Significant differences based on Student’s t test (P , 0.001).





and selectivity of the membrane system, and controllingplant growth, such as root hair formation and primaryroot elongation (Shi and Sheng, 2005; Monshausenet al., 2007; Zhang and Mu, 2009; Zhou et al., 2010; Liuand Guo, 2011). When subjected to alkaline stress,plants can respond by modifying auxin distribution,plasma membrane H+-ATPase activity, and microfila-ment stabilization, resulting in shorter roots (Fuglsanget al., 2007; Yang et al., 2010; Zhou et al., 2010; Liu and

Guo, 2011; Xu et al., 2012, 2013). Alkaline stress inhibitsprimary root elongation (Fuglsang et al., 2007; Xu et al.,2012). Here, additional detailed examination indicatedthat the shorter root length was caused by reducedmeristematic cell division. This phenomenon is alsoobserved in plant responses to other stresses, such asexcess copper, phosphate starvation, and toxic levels ofchromium (Sánchez-Calderón et al., 2005; Castro et al.,2007; Yuan et al., 2013). However, the root meristematic

Figure 8. Alkaline stress-increased AUX1 expression is regulated by ethylene. A, qRT-PCR analysis of AUX1 expression in the rootstreated with pH 5.8 or pH 8.0 with additional application of 10 mM AgNO3 or 10 mM CoCl2. Values are relative to those of seedlingssubjected to pH 5.8 as the control. B, The effect of AgNO3 or CoCl2 on alkaline stress-increased expression of AUX1-YFP. TheAUX1-YFP fluorescence is shown in the roots of 5-d-old AUX1::AUX1-YFP seedlings incubated in one-half-strength MS of pH 5.8or 8.0 with additional application of 10 mM AgNO3 or 10 mM CoCl2 for 12 h. Bars = 100 mm. C, Quantification of AUX1-YFPfluorescence intensities in the meristem zones of AUX1::AXU1-YFP roots in B. Values are relative to those of the seedlings sub-jected to pH 5.8 as the control. D, qRT-PCR analysis of AUX1 expression in the root of 5-d-old wild-type (WT), etr1-3, ein2-1, andein3-1 seedlings treated with pH 5.8 or pH 8.0 for 12 h. Data are relative to control values obtained from wild-type, etr1-3, ein2-1,and ein3-1 seedlings, respectively, treated with one-half-strength MS of pH 5.8. Mean and SEM were calculated from three inde-pendent experiments. Asterisks indicate significant differences based on two-way ANOVAwith Bonferroni posttests compared withrelated control (A and C) or the wild type at each pH condition (D): *, P , 0.05; **, P , 0.01; and ***, P , 0.001.


Li et al.



cell division potential is not affected during Glc-mediated inhibition of root meristem growth (Yuanet al., 2014). The reduced meristematic cell divisioncould be caused by the change in the expression levelof cell cycle-related genes. Indeed, alkaline stress re-pressed CYCA2;1, CYCB1;1, CYCB3;1, CYCD1;1,CYCD2;1, CYCD3;1, CYCD4;1, CYCD4;2, CYCD6;1, andE2Fb, which are positive regulators of cell cycle pro-gression, and enhanced the expression of E2Fc andRBR, which are negative regulators of cell proliferation.Considering that ethylene negatively regulates cell di-vision in both hypocotyls and leaf primordia (Dan et al.,2003; Skirycz et al., 2011; Vandenbussche et al., 2012),this is consistent with our observation that alkalinestress increases ethylene level in the roots. In addition,the increased auxin accumulation in plant response toalkaline stress may also involve this process. R�u�zi�ckaet al. (2009) showed that 2,4-dichlorophenoxyacetic acidreduces the root meristem size and the mitotic activityin the root meristem. It is also reported that aluminumstress can result in increased accumulation of bothethylene and auxin in the roots (Sun et al., 2010) andreduce primary root growth by inhibiting cell cycleprogression (Herrera and Bucio, 2013), suggesting thataluminum-promoted accumulation of these two phy-tohormones acts in the inhibition of root cell division.Ethylene is associated with environmental stresses

(Potters et al., 2009), such as heat, drought, ozone,phosphate starvation, aluminum excess, and low bo-ron supply (Sun et al., 2007, 2010; Lei et al., 2011;Martín-Rejano et al., 2011; Khan et al., 2013; Habbenet al., 2014; Ludwików et al., 2014). Excess aluminumand low boron can elicit rapid production of ethylene,leading to marked inhibition of root elongation (Sunet al., 2007, 2010; Martín-Rejano et al., 2011). However,whether ethylene is involved in plant response to al-kaline stress had not been examined. Our results here,using physiological, pharmacological, and geneticapproaches, provided solid evidence for the involve-ment of ethylene in alkaline stress-mediated inhibitionof root elongation. Although root elongation in theetr1-3, ein2-1, and ein3-1 mutants was less sensitive toalkaline stress, the eto1-1 mutant, which overproducesethylene, was hypersensitive to alkaline stress. Fur-thermore, an ethylene perception antagonist (Ag+) andan ethylene biosynthesis inhibitor (Co2+) alleviatedalkaline stress-mediated inhibition of root elongation.Staal et al. (2011) reported that exogenous ACC reducescell elongation of the elongation zone of Arabidopsisroots by changes in apoplastic alkalinization by mod-ulating plasma membrane H+-ATPase. Thus, althoughboth ethylene biosynthesis and signaling are pro-moted in the roots sensing alkaline stress, ethylenecan also affect apoplastic pH of the roots. We alsonoted more severe inhibition of primary root elon-gation in alkaline stress-treated seedlings comparedwith that in ACC-treated plants. Taken together withthe data that ethylene levels in ACC-treated roots weresignificantly higher than those in alkaline stress-treatedroots, these data suggest that another factor(s) other

than ethylene is also involved in alkaline stress-mediated inhibition of primary root elongation. Thisview is consistent with our data that Ag+ and Co2+

only partially alleviate root elongation inhibition underalkaline stress.

In addition to ethylene, auxin functions in responses tovarious environmental stimuli by modifying root de-velopment (Overvoorde et al., 2010). Exposure to excessCu, Cd, Al, or As(III) or high Glc or salt stresses disturbsnormal auxin accumulation and distribution, reducingroot growth (Wang et al., 2009; Sun et al., 2010; Hu et al.,2013; Krishnamurthy and Rathinasabapathi, 2013; Yuanet al., 2013, 2014). Auxin efflux or influx carriers are in-volved in these changes in auxin accumulation anddistribution (Yan et al., 2013; Yuan et al., 2013, 2014;Zhang et al., 2013, 2014; Hong et al., 2014; Liu et al., 2015;Zhu et al., 2015). Moreover, PIN2 participates in alkalinestress-mediated root inhibition (Xu et al., 2012). Our re-sults suggested that AUX1 mediates the alkaline stress-induced accumulation of auxin for the inhibition of rootelongation. Furthermore, auxin biosynthesis was alsoconcluded to be involved in this process, because theexpression of auxin biosynthesis-related genes was ele-vated and the inhibitions of root elongations of bothwei2-2 and wei8-1 were less pronounced than those ofwild-type plants when subjected to alkaline stress.

Ethylene and auxin act synergistically to control rootelongation (Rahman et al., 2001; Chilley et al., 2006;Swarup et al., 2007). The possibility that ethylene andauxin function together in plant responses to alkalinestress was explored in our study, which verified thecross talk between ethylene and auxin. The increase inauxin level was reduced in the roots of the mutantsetr1-3 and ein3-1 or Ag+/Co2+-treated wild-type plants,and eto1-1 did not strengthen the sensitivity to alkalinestress displayed by axr1-3 and aux1-7. However, auxinmay not be involved in alkaline-induced ethylene ac-cumulation, because alkaline stress can efficiently in-crease ethylene level to about 3-fold in the roots of thewild type (287.91%), axr1-3 (303.11%), and aux1-7(281.87%; Supplemental Fig. S1, A and B). Also, theEBS::GUS expression in the roots of both axr1-3 EBS::GUS and aux1-7 EBS::GUS was significantly promotedby alkaline stress, although to a slightly lower degreethan in EBS::GUS (Supplemental Fig. S1, C and D).

Taken together, our results suggest that ethylenemodulates alkaline stress-mediated inhibition of rootgrowth by increasing auxin accumulation by stimu-lating expression of AUX1 and auxin biosynthesis-related genes.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 was used. The pub-lished transgenic and mutant lines used in this study areQC25::GUS,QC46::GUS(Sabatini et al., 2003), CYCB1;1::GUS (Colón-Carmona et al., 1999), DII-VENUS(Brunoud et al., 2012), AUX1::AUX1-YFP (Swarup et al., 2005), and eto1-1 (Chaeet al., 2003). The EBS::GUS reporter line originally generated by Anna Stepanovain the laboratory of Joe Ecker (Stepanova et al., 2005) was provided by




http://www.plantphysiol.org/cgi/content/full/pp.15.00523/DC1



Jose Alonso (Department of Genetics, North Carolina State University); eto1-1 aux1-7and eto1-1 axr1-3 were provided by Bonnie Bartel (Department of Biochemistryand Cell Biology, Rice University) and Lucia C. Strader (Department of Biology,Washington University, St. Louis; Strader et al., 2010; Thole et al., 2014), aux1-7EBS::GUS and axr1-3 EBS::GUSwere provided by Anna Stepanova (Departmentof Genetics, North Carolina State University, Raleigh; Stepanova et al., 2007),etr1-3 was provided by W.H. Zhang (Institute of Botany, Chinese Academy ofSciences; Sun et al., 2010), and wei8-1 was provided by Z.J. Ding (School of LifeSciences, Shandong University; Yang et al., 2014). The lines ein2-1 (CS3071),ein3-1 (CS8052), wei2-2 (Salk_017444), aux1-7 (CS3047), and axr1-3 (CS3075) wereobtained from the Arabidopsis Biological Resource Centre.

Arabidopsis seeds were surface sterilized with 5% (w/v) bleach for 5 min,washed three times with sterile water, placed at 4°C for 3 d, and then plantedon medium containing one-half-strength MS (Murashige and Skoog, 1962),1% (m/v) Suc, 1% (m/v) agar, and 5 mM HEPES; the medium pH was ad-justed with 1 M KOH. Seedlings were grown at 23°C and 100 mmol m22 s21 ofillumination under 16-h-light/8-h-dark conditions.

Root Length Measurement

To study the inhibitory effect of alkaline stress on root elongation, 5-d-oldArabidopsis seedlings were exposed to one-half-strength MS of pH 5.8, 8.0, or9.0 for 12 h and then transferred to one-half-strength MS of pH 5.8 for another2 d. The lengths of newly grown roots were measured and statistically analyzed.For the effect of ACC on root elongation, seedlings of the wild type were ex-posed to 10 mM ACC for 6, 12, and 24 h and then grown in one-half-strength MSof pH 5.8 for an additional 2 d. To assay the effect of AgNO3 and CoCl2 on rootelongation in the presence of alkaline stress, seedlings were incubated in one-half-strength MS of pH 5.8 or pH 8.0 with additional application of either 10 mM

AgNO3 or 10 mM CoCl2 for 12 h and then transferred to one-half-strength MS ofpH 5.8 for another 2 d. Seedlings of the wild type, etr1-3, ein2-1, ein3-1, eto1-1,wei2-2, wei8-1, axr1-3, aux1-7, eto1-1 axr1-3, and eto1-1 aux1-7 were exposed topH 5.8, 8.0, or 9.0, and root elongation was measured 2 d after the seedlingswere transferred and grown in one-half-strength MS of pH 5.8. The results areexpressed as the mean 6 SEM (n . 30 roots). Each measurement was repeatedwith at least three biological replicates.

GUS Staining

GUS staining was carried out according to the methods described previously(Hu et al., 2010). Briefly, seedlings were incubated at 37°C in staining solution(100 mM sodium phosphate buffer, pH 7.5, containing 10.0 mM EDTA, pH 8.0,0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6], 0.1% (v/v) Triton X-100, and 1.0 mM

5-bromo-chloro-3-indolyl-b-D-glucuronide). The GUS staining time was depen-dent on the transgenic marker lines: 6 h for QC25::GUS, 12 h for QC46::GUS andCYCB1;1::GUS, and 4 h for EBS::GUS. The 4-methyl ketone of umbrella gluco-side acid assays were carried out according to the method described previously(Yuan et al., 2014). The experiments were repeated three times.

Flow Cytometry

Nuclear isolation was performed according to the previously describedmethod (Zhao et al., 2014). Briefly, the samples were chopped with nucleusisolation buffer (10 mM MgSO4, 50 mM KCl, 5 mM HEPES, 1 mg mL21 dithio-threitol [Sigma], and 0.2% [v/v] Triton X-100) and filtered through a 33-mmnylon mesh. The nuclei were fixed in 4% (m/v) paraformaldehyde for 30 min,precipitated (200g for 10 min at 4°C), and resuspended in the isolation buffer.Propidium iodide (PI) was added to the resuspended samples at 50 mg mL21.The nuclear DNA content was analyzed using flow cytometry (BeckManCoulter) with a 488-nm solid-state laser (100 mW) for excitation, and emissiondata were collected after a 590-nm long-pass filter.

Microscopic Analysis

For phenotypic analysis of root or GUS staining microexamination, seed-lings were cleared and mounted with clearing solution (8 g of chloral hydrate,2 mL of water, and 1 mL of glycerol) on glass slides. The slides were examinedunder differential interference contrast optics (Olympus BX63; Olympus) andphotographed using a CCD camera (Olympus DP72; Olympus).

Confocal microscopy was performed using an Olympus FluoView 1000Confocal Laser-ScanningMicroscope according to themanufacturer’s instructions.The YFP lines were mounted onto microscope slides with 20 mg mL21 PI. At least

24 seedlings were analyzed per treatment. The signal intensity was measuredusing Photoshop CS5 (Adobe), and error bars were obtained based on mea-surements of more than 20 seedlings per treatment. The intensity ratio wasobtained by comparing DII-VENUS or AUX1::AUX1-YFP fluorescence intensitywith that of seedlings subjected to one-half-strength MS of pH 5.8.

RNA Extraction and qRT-PCR Analysis

The roots were collected for total RNA isolation using TRIzol Reagent(Invitrogen) according to the manufacturer’s instructions. After treatment withRQ1 RNase-free DNase I (Promega), first strand complementary DNA syn-thesis was carried out using Superscrip II Reverse Transcriptase (Invitrogen)or M-MLV Reverse Transcriptase (NOVA; LCP Biomed Co.) according to themanufacturer’s instructions.

The qRT-PCR analysis was performed on a Bio-Rad CFX96 apparatus (Bio-Rad) using the dye SYBR Green I (Invitrogen). PCR was carried out in 96-wellplates according the following protocol: 3 min at 95°C followed by 40 cycles ofdenaturation for 15 s at 95°C, annealing for 15 s at 58°C, and extension for 20 sat 72°C. Primers were designed using Beacon Designer 7.0 (Premier BiosoftInternational). ACTIN2 (AT3G18780) and EUKARYOTIC TRANSLATIONINITIATION FACTOR 4A (EIF4A; AT3G13920) were used as internal controlsusing GeNorm (Czechowski et al., 2005). All experiments were performedwith three independent biological replicates and three technical repetitions.The genes analyzed and the corresponding specific primers are listed inSupplemental Table S1 (Sun et al., 2010; Yan et al., 2013; Gao et al., 2014; Honget al., 2014).

The related abundance of transcripts was calculated according to the Bio-Rad CFX Manager (Version1.5.534) software of BIO-RAD CFX96 using thecomparative DDCT method with ACTIN2 and ELF4A as internal standards.The related expression of specific genes was calculated by the formulas DCT =CT-specific gene 2 CT reference gene and DDCT = DCT alkaline-treatedsample 2 DCT untreated sample. The related level was calculated as 22DDCT. All calculations were automatically carried on Bio-Rad CFX Manager(version 1.5.534) of BIO-RAD CFX96.

Quantification of IAA by GC-Mass Spectrometry

IAA was quantified as described previously (Gao et al., 2014). Briefly,150 mg (fresh weight) of whole root for each treatment was immediatelyfrozen in liquid nitrogen, extracted, and purified for endogenous IAA. Thepurified samples were methylated by a stream of diazomethane gas,resuspended in 100 mL of ethyl acetate, and analyzed by GC-mass spec-trometry. A Shimadzu GCMS-QP2010 Plus equipped with an HP-5MSColumn (30-m long, 0.25-mm i.d., 0.25-lm Film; Agilent) was used to de-termine the level of IAA.

Quantification of Ethylene by GC

The ethylene level was measured according to the previously describedmethod (Sun et al., 2010; Du et al., 2014). Whole-root samples (200 mg) wereexcised and put into 8-mL gas-tight vials containing 2 mL of agar medium(1% [m/v] agar). A 1-mL sample of the headspace from the vials was injectedinto a gas chromatograph equipped with a flame ionization detector columnpacked with activated aluminum at 100°C. Ethylene was detected by anionization detector and recorded by an integrator. The sample injected tem-perature was 80°C, whereas the column was 150°C.

Statistical Analysis

All experiments in this study were performed with at least three repetitions.The significance of differences was determined by one-way ANOVA withTukey’s multiple comparison test, two-way ANOVA with Bonferroni post-tests, or Student’s t test using GraphPad Prism version 5.0 (GraphPadSoftware; www.graphpad.com) as indicated in the figures.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Alkaline stress promoted ethylene evolution.

Supplemental Table S1. List of primers used in this study.


Li et al.



http://www.graphpad.com




ACKNOWLEDGMENTS

We thank Jose Alonso, Wenhao Zhang, Anna Stepanova, Bonnie Bartel,Lucia C. Strader, and Zhaojun Ding for sharing published materials andLizhong Xiong for help with ethylene content measurement.

Received April 9, 2015; accepted June 23, 2015; published June 24, 2015.

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