Ethylene Inhibits Root Elongation during Alkaline Stress through AUXIN1 and Associated Changes in Auxin Accumulation

Ethylene promotes auxin transporter expression and auxin biosynthesis to modulate root elongation during alkaline stress.

Abstract

Soil alkalinity causes major reductions in yield and quality of crops worldwide. The plant root is the first organ sensing soil alkalinity, which results in shorter primary roots. However, the mechanism underlying alkaline stress-mediated inhibition of root elongation remains to be further elucidated. Here, we report that alkaline conditions inhibit primary root elongation of Arabidopsis (Arabidopsis thaliana) seedlings by reducing cell division potential in the meristem zones and that ethylene signaling affects this process. The ethylene perception antagonist silver (Ag⁺) alleviated the inhibition of root elongation by alkaline stress. Moreover, the ethylene signaling mutants ethylene response1-3 (etr1-3), ethylene insensitive2 (ein2), and ein3-1 showed less reduction in root length under alkaline conditions, indicating a reduced sensitivity to alkalinity. Ethylene biosynthesis also was found to play a role in alkaline stress-mediated root inhibition; the ethylene overproducer1-1 mutant, which overproduces 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 promoting expression of auxin biosynthesis-related genes, but the increase in auxin levels was reduced in the roots of the etr1-3 and ein3-1 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 ethylene modulates alkaline stress-mediated inhibition of root growth by increasing auxin accumulation by stimulating the expression of AUX1 and auxin biosynthesis-related genes.

Soil alkalinity limits agricultural productivity worldwide by affecting 434 million ha of land in more than 100 countries (Jin et al., 2006). Because only a few plants can grow well in alkalinized soil, soil alkalinity limits the availability of arable land and results in low agricultural productivity. Accordingly, studies on the mechanisms underlying plant responses and adaptation to alkaline stress are important for both basic plant biology knowledge and agricultural applications.

Alkaline stress can lead to reduced seed germination, decreased shoot growth, and smaller leaves as well as inhibited root growth and elongation (Cha-Um et al., 2009; Patil et al., 2012). In addition to this inhibition of plant growth, alkaline stress influences numerous metabolic and physiological processes, such as photosynthesis, microfilament depolymerization, cellular ionic homeostasis, metabolic balance of reactive oxygen 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 alkalinity, which inhibits primary root growth (Fuglsang et al., 2007; Yang et al., 2010; Xu et al., 2012). Several recent reports show that the plasma membrane H⁺-ATP synthase (H⁺-ATPase) plays an important role in the adaptation of roots to alkaline stress by mediating proton secretion (Fuglsang et al., 2007; Yang et al., 2010; Xu et al., 2013). For example, the protein kinase PROTEIN KINASE SOS2-LIKE5 (PKS5) inhibits the plasma membrane H⁺-ATPase by preventing its interaction with 14-3-3 proteins, resulting in higher alkaline tolerance of Arabidopsis (Arabidopsis thaliana) pks5 plants (Fuglsang et al., 2007). Conversely, the chaperone J3 activates the plasma membrane H⁺-ATPase by repressing PKS5 kinase activity; thus, the j3 mutant is hypersensitive to alkaline stress (Yang et al., 2010).

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). Polar auxin transport (PAT) mediated by influx carriers of the AUXIN1 (AUX1)/LIKE AUXIN1 (LAX) family and efflux carriers of the PINFORMED (PIN) family (Vieten et al., 2007; Křeček et al., 2009; Péret et al., 2012) is involved in plant responses to environmental stresses, such as phosphate starvation, ammonium toxicity, 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 influences primary root growth (Blilou et al., 2005; Grieneisen et al., 2007). A recent report indicates that PIN2 is involved in the PKS5-mediated signaling cascade and required for plant adaptation to alkaline stress by modulating proton secretion in root tips to maintain primary root elongation (Xu et al., 2012).

In addition to auxin, ethylene is often associated with stress responses, such as heat, drought, ozone, phosphate 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 signaling pathway begins with ethylene binding to receptor proteins. The ethylene receptors exist as a multimember family 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 and Binder, 2012). Upon ethylene binding, the receptor transmits the signal to the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) protein kinase, resulting in the inhibition of the ability of CTR1 to phosphorylate EIN2 and causing the translocation into the nucleus of the C-terminal end of EIN2, which then leads to the stabilization of EIN3 and the activation of the EIN3/ETHYLENE-INSENSITIVE3-LIKE (EIL)-dependent transcriptional cascade (Mayerhofer et al., 2012; Wen et al., 2012; Dharmasiri et al., 2013; Merchante et al., 2013).

Ethylene is produced from Met through S-adenosyl-l-Met and 1-aminocyclopropane-1-carboxylic acid (ACC), which are catalyzed by 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), respectively (Kende, 1993). ACS controls the rate-limiting step in ethylene biosynthesis (Chae and Kieber, 2005). ACS and ACO are encoded by multigene families and regulated by many biotic and abiotic factors (Wang et al., 2002). When subjected to exogenously supplied ACC, Arabidopsis seedlings exhibit reduced root elongation (Růzicka et al., 2007).

The ethylene and auxin pathways show cross talk at multiple levels (Muday et al., 2012; Robles et al., 2013). For example, auxin increases ACS transcription, thus stimulating ethylene biosynthesis (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), and WEI8/TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) in roots, leading to increased auxin production and inhibition of root growth (Stepanova et al., 2005, 2008). In addition, ethylene promotes basipetal auxin transport to affect root elongation (Růzicka et al., 2007).

Here, we report that alkaline stress inhibits primary root elongation of Arabidopsis seedlings by affecting the meristem zone through reduced meristematic cell division potential. Our data indicate for the first time, to our knowledge, that ethylene modulates this alkaline stress-mediated inhibition of root growth by increasing auxin accumulation by stimulating expression of auxin biosynthesis-related genes and AUX1.

RESULTS

Alkaline Stress Inhibits Root Elongation by Affecting Meristem, Elongation, and Differentiation Zones

Previous reports indicated that root elongation is inhibited in media of pH 8.0 to pH 8.4 (Fuglsang et al., 2007; Xu et al., 2012). To further explore how alkaline stress modulates primary root growth, we grew Arabidopsis seedlings on one-half-strength Murashige and Skoog medium (MS) at the standard pH of 5.8 and then, subjected 5-d-old seedlings to one-half-strength MS at pH 8.0 or pH 9.0 as alkaline stress or pH 5.8 as control for 12 h. Then, the treated seedlings were transferred to one-half-strength MS (pH 5.8) for continued growth for 2 d, and the lengths of newly grown primary roots were measured. Our results showed that alkaline stress inhibited primary root elongation (Fig. 1A), consistent with previous reports (Fuglsang et al., 2007; Xu et al., 2012). Our data also indicated that the inhibition was positively associated with alkalinity (Fig. 1B). The primary root length was inhibited by 47.1% 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 zones were assayed in the alkaline stress-treated roots. Both root meristem and elongation zones were reduced in response to pH 8.0 or pH 9.0 compared with pH 5.8 (Fig. 1, C–E). Alkaline stress also decreased the cell length of the differentiation zone (Fig. 1F).

Figure 1.

Alkaline stress inhibits primary root growth by reducing the lengths of meristem and elongation zones and the cell lengths 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 for 12 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 newly grown roots were measured after the alkaline stress-treated plants were transferred to one-half-strength MS of pH 5.8 for another 2 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 indicate QCs. Bars = 100 μm. *, Proximal boundary of the root meristem. D to F, Root meristem zone lengths (D), root elongation zone lengths (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 for 12 h. Data shown are means ± sem. Different letters indicate significant differences between treatments (P < 0.05 by one-way ANOVA with Tukey’s multiple comparison test).

A decrease in root meristematic size can be caused by a reduction in stem cell niche activity, the loss of division potential of meristematic cells in the proximal meristem, or the acceleration of the elongation and differentiation of meristematic cells in the transition zone (Dello Ioio et al., 2007; Baluška et al., 2010). Thus, we first analyzed whether the reduction in meristem size was caused by possible changes in stem cell niche activity using both quiescent center (QC)25::GUS and QC46::GUS (Sabatini et al., 2003). GUS staining showed that both reporter constructs were expressed in the roots of these two lines treated with pH 8.0 or pH 9.0 (Fig. 2A), indicating that stem cell niche activity is not responsible for the reduced meristematic zone of plant roots treated with alkaline stress.

Figure 2.

Alkaline stress reduces cell division potential. A and B, Images of GUS staining of 5-d-old QC25::GUS and QC46::GUS (A) and CYCB1;1::GUS (B) seedlings exposed to pH 5.8, 8.0, or 9.0 for 12 h. Bars = 100 μm. C and D, Flow cytometric 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 stained with 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 ± sem. Asterisks indicate significant differences with respect to each control (Student’s t test): *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Then, we assayed meristematic cell division potential with CYCLIN B1;1 (CYCB1;1)::GUS, an excellent marker to monitor cell cycle progression (Colón-Carmona et al., 1999). The percentage of GUS-stained cells in the root meristem of this reporter line was significantly reduced in alkaline stress-treated roots compared with that in control roots (Fig. 2B), suggesting that alkaline stress reduces the competence of meristematic cells to divide. This was further supported by flow cytometry analysis of the nuclear DNA content in the cells of alkaline stress-treated or untreated root tips. Indeed, alkaline stress-treated roots had 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-related genes, including A-TYPE CYCLIN-DEPENDENT KINASE1 (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 cell cycle-related transcription factor genes E2Fa and E2Fb, all of which promote cell cycle progression and stimulate cell division (De Veylder et al., 2002; Masubelele et al., 2005; Kono et al., 2006; Sozzani et al., 2006; Cruz-Ramírez et al., 2012). Quantitative real-time (qRT)-PCR analysis indicated that alkaline stress down-regulated the expression of all of the assayed genes, except for CDKA;1 and E2Fa (Fig. 2E). By contrast, E2Fc and RETINOBLASTOMA-RELATED (RBR), which repress cell division (del Pozo et al., 2002; Wildwater et al., 2005), were significantly up-regulated under alkaline stress (Fig. 2E).

Taken together, our data indicated that alkaline stress inhibits primary root growth by affecting the lengths of the meristem and elongation zones and cell lengths in the differentiation zone. Alkaline stress-induced reduction of meristem size is caused by inhibition of cell division in the meristem.

Ethylene Is Involved in Alkaline Stress-Mediated Inhibition of Primary Root Elongation

Ethylene is often associated with stress responses (Potters et al., 2009) and plays a role in the inhibition of primary root growth by affecting cell elongation (Swarup et al., 2007). Because our above data showed that alkaline stress substantially inhibited root elongation, we analyzed the possible involvement of ethylene signaling in alkaline stress-mediated root inhibition using Ag⁺, an antagonist of ethylene perception. Although Ag⁺ at 10 µm slightly inhibited root elongation, alkaline stress-induced inhibition of root elongation was markedly recovered in wild-type plants exposed to pH 8.0 and Ag⁺ together (Fig. 3A), suggesting that ethylene perception participates in the inhibition of primary root elongation by alkaline stress. Alkaline stress also induced the expression of ETR1, EIN2, and EIN3, pivotal components of ethylene signaling (Fig. 3B). When the ethylene reporter line EIN3-Binding Site (EBS)::GUS, in which the GUS reporter gene is driven by a synthetic EIN3-responsive promoter (Stepanova et al., 2007), was analyzed, a marked increase in the activity of EBS::GUS in the root apices was observed in the seedlings exposed to alkaline stress or ACC compared with that in untreated roots (Fig. 3, C and D). These data suggest that the ethylene signaling pathway is involved in alkaline stress-mediated inhibition of root growth. To examine this idea further, we used a genetic approach using the ethylene-insensitive mutants etr1-3, ein2-1, and ein3-1, which were used extensively in deciphering the physiological functions of ethylene (Alonso et al., 1999; Binder et al., 2006; Stepanova et al., 2007). When grown at pH 8.0 and pH 9.0, the etr1-3, ein2-1, and ein3-1 mutants showed less-pronounced inhibition of root growth compared with wild-type plants (Fig. 3, E and F). Collectively, these results indicate that ethylene signaling participates in alkaline stress-mediated inhibition of root elongation.

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 μm AgNO₃ 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 5.8 for another 2 d. Values are relative to plants 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 plants treated 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 of alkaline stress and ACC on the expression of the ethylene reporter EBS::GUS. Five-day-old seedlings were treated with pH 8.0 for 6 and 12 h or 10 μm ACC for 12 h. Bars = 100 μm. D, Relative GUS activity of EBS::GUS treated with pH 8.0 for 6 and 12 h or 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 measured after the treated plants were transferred to one-half-strength MS of pH 5.8 for another 2 d (E). 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 (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 condition (E and F): *, P < 0.05; and ***, P < 0.001.

The enhanced ethylene signaling in alkaline stress-induced inhibition of root elongation could be caused by changes in ethylene biosynthesis. Thus, we first assayed the primary root elongation of the seedlings treated with ACC. We found that, although the root length was reduced by 35.2%, 45.9%, and 67.1% after ACC treatment for 6, 12, and 24 h, respectively, a similar but more severe inhibition of root elongation by alkaline stress was observed (39.3%, 51.4%, and 84.8% after alkaline stress treatment for 6, 12, and 24 h, respectively; Fig. 4A). Next, an antagonist of ethylene biosynthesis (Co²⁺) was used to further test whether ethylene biosynthesis was involved in alkaline stress-mediated inhibition of root elongation. Although Co²⁺ at 10 µm had no effect on root elongation compared with untreated seedlings, Co²⁺ substantially ameliorated the alkaline stress-mediated inhibition of root elongation (Fig. 4B), suggesting a role of ethylene biosynthesis in alkaline stress-mediated root elongation. Furthermore, we analyzed the expression of four genes (ACS2, ACS5, ACS6, and ACS8) involved in ethylene biosynthesis in roots (Kende, 1993) by qRT-PCR. As expected, the expression of these genes was dramatically induced under alkaline stress treatment (Fig. 4C). Finally, we directly measured ethylene levels in the roots using gas chromatography (GC) and found that the ethylene level was significantly higher in alkaline stress-treated roots than that in the untreated control (Fig. 4D), suggesting that increased ethylene could be responsible for reduced primary root length under alkaline stress. To further verify this, we examined an ethylene overproducer1-1 (eto1-1) mutant, in which ACS5 stability is increased, leading to overproduction of ethylene (Chae et al., 2003). When exposed to pH 8.0 and pH 9.0, primary root elongation was inhibited by 52.9% and 77.8%, respectively, in eto1-1 seedlings but only 46.6% and 63.2%, respectively, in wild-type plants compared with the controls, indicating that the mutant is more sensitive to alkaline stress (Fig. 4, E and F). Consistent with this, ethylene accumulation in eto1-1 roots was higher than that in wild-type roots (Fig. 4G).

Figure 4.

Ethylene biosynthesis participates in alkaline stress-induced inhibition of primary root elongation. A, The effect of ACC and alkaline stress on primary root elongation. Five-day-old wild-type seedlings were treated with 10 μm ACC of pH 8.0 for 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 newly grown roots were measured and statistically analyzed. Data are presented as relative root elongation compared with control values. B, The effect of CoCl₂ 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 μm CoCl₂ 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 5.8 for another 2 d. Values are relative to plants treated with pH 5.8 as the control. C, qRT-PCR analysis of ACS2, ACS5, ACS6, and ACS8 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 seedlings subjected 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 or 12 h or 10 μm 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 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 5.8 for another 2 d (E). Data are relative to control values obtained from wild-type and eto1-1 seedlings, respectively, treated with 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 upon exposure to pH 8.0 for 12 h. Error bars represent sem of three independent experiments. Different letters indicate significant differences between treatments (P < 0.05 by one-way ANOVA with Tukey’s multiple comparison test; A). Asterisks indicate significant differences based on Student’s t test (C and D) or two-way ANOVA with Bonferroni posttests compared with related control (B) or the wild type at each pH condition (E–G): FW, fresh weight; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Alkaline Stress Represses Root Elongation through Ethylene-Induced Auxin Accumulation

Ethylene stimulates auxin accumulation in root meristem and elongation zones (Růžička et al., 2007), and alkaline stress increases auxin distribution in root tips (Xu et al., 2012). Auxin, as a critical phytohormone for root growth, may also be involved in ethylene-mediated root inhibition of the seedlings exposed to alkaline stress. Accordingly, we visualized auxin signaling using a domain II (DII)-VENUS line, which expresses the VENUS fast-maturing form of yellow fluorescent protein fused with the Aux/indole-3-acetic acid (IAA) auxin interaction domain. DII-VENUS is rapidly degraded in response to auxin (Brunoud et al., 2012). Our results showed that the DII-VENUS signal decreased in the roots subjected to alkaline stress (pH 8.0 for 6 and 12 h or pH 9.0 for 12 h) compared with the untreated control (Fig. 5, A and B), suggesting an increase in auxin abundance under alkaline stress. This was further supported by measurements of IAA content using GC-mass spectrometry. The IAA concentrations in the roots treated with either pH 8.0 for 6 and 12 h or pH 9.0 for 12 h were 135.84 ± 13.54 and 194.21 ± 15.04 ng g⁻¹ of fresh weight or 236.96 ± 15.02 ng g⁻¹ of fresh weight, respectively, whereas IAA concentration of untreated roots was 107.81 ± 9.03 ng g⁻¹ of fresh weight (Fig. 5C).

Figure 5.

Alkaline stress modulates auxin accumulation in the roots. A, The effect of alkaline stress on auxin distribution and accumulation 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 μm. B, Quantification of the DII-VENUS fluorescence intensities in the root meristem zones of the DII-VENUS seedlings in A. Values are relative to those of seedlings 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 pH 9.0 for 12 h. FW, Fresh weight. D and E, qRT-PCR analysis of auxin biosynthesis-related gene expression in the roots of 5-d-old wild-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 analysis of 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. Values are 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, and wei8-1 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 plants were 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. Asterisks indicate significant differences based on Student’s t test (B–F) or two-way ANOVA with Bonferroni posttests compared with the wild type at each pH condition (G and H): *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Next, we examined the expression of genes encoding auxin biosynthesis-related proteins of the indole-3-acetaldoxime, tryptamine, and indole-3-pyruvic acid pathways (Cheng et al., 2007; Ikeda et al., 2009; Zhao and Hasenstein, 2009; Brumos et al., 2014; Gao et al., 2014). Upon treatment at pH 8.0 for 6 and 12 h, the transcript levels of the Trp biosynthetic genes (i.e. ASA1, ASB1, PHOSPHORIBOSYLANTHRANILATE TRANSFERASE1 [PAT1], PHOSPHORIBOSYLANTHRANILATE ISOMERASE1 [PAI1], and INDOLE-3-GLYCEROL PHOSPHATE SYNTHASE1 [IGPS1]) increased (Fig. 5D), and those of genes believed to be involved in Trp-dependent IAA biosynthesis (i.e. CYTOCHROME P450, FAMILY 79, SUBFAMILY B, POLYPEPTIDE2 [CYP79B2], YUCCA1, YUCCA6, TAA1, TRYPTOPHAN AMINOTRANSFERASE RELATED1 [TAR1], TAR2, and NITRILASE3 [NIT3]) also increased (Fig. 5E). The expression of SUPERROOT1 (SUR1) and SUR2, which are involved in indole glucosinolate biosynthesis, was also up-regulated by pH 8.0 for 12 h but not pH 8.0 for 6 h (Fig. 5E). These data suggest that the transcriptional promotion of the auxin biosynthetic genes could be responsible for the alkaline stress-induced increase in auxin levels. Additionally, we also assayed the expression of genes for auxin conjugation, because IAA can be released by hydrolytic cleavage of IAA conjugates, contributing to local auxin concentration (Ludwig-Müller, 2011; Korasick et al., 2013). One of the IAA-amino acid conjugate hydrolases, IAA-LEUCINE RESISTANT-LIKE2 (ILL2), which releases free IAA by cleaving IAA-amino conjugates, was up-regulated by alkaline stress, whereas genes encoding other hydrolases, such as ILL3 and IAA-ALANINE RESISTANT3, remained unchanged (Fig. 5F). Not surprisingly, the expression 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 alkaline stress (Fig. 5F).

Furthermore, we verified the role of auxin biosynthesis in alkaline stress-induced root inhibition with wei2-2 and wei8-1 mutants, in which auxin biosynthesis is impaired (Stepanova et al., 2005, 2008; Yang et al., 2014), because their expression is enhanced by alkaline stress as shown above. When exposed to pH 8.0 or pH 9.0, the inhibitions of root elongations of both wei2-2 and wei8-1 were less pronounced than those of wild-type plants (Fig. 5, G and H), suggesting involvement of both WEI2 (ASA1) and WEI8 (TAA1) in alkaline stress-induced inhibition of root growth. Taken together, these results reveal that alkaline stress represses root elongation by increasing auxin accumulation through increased expression of auxin biosynthesis genes.

To test whether ethylene is involved in alkaline stress-induced auxin accumulation and signaling for the inhibition of root elongation, we treated DII-VENUS with ACC and assayed the changes of the DII-VENUS signal. The fluorescence intensity decreased to 68.5% and 54.1% in the presence of ACC for 6 and 12 h, respectively (Fig. 6, A and B), indicating that ethylene increased auxin signaling as reported by Růzicka et al. (2007). Then, we investigated the effect of Ag⁺ and Co²⁺ on DII-VENUS expression in alkaline stress-treated roots and found that DII-VENUS fluorescence intensity increased by 21.2% or 22.1% in the roots subjected to alkaline stress in the presence of either Ag⁺ or Co²⁺, respectively, compared with that in the roots treated with alkaline stress alone (Fig. 6, C and D), implying the involvement of ethylene biosynthesis and signaling in alkaline stress-increased auxin signaling. This view was further verified using an auxin-insensitive mutant auxin resistant1-3 (axr1-3), which carries a mutation in AXR1, encoding a subunit of a heterodimeric RELATED TO UBIQUITIN1-activating enzyme, resulting in a failure to degrade Aux/IAA proteins (Lincoln et al., 1990; Cernac et al., 1997; Pozo et al., 1998). Our results showed that, although the eto1-1 mutant with higher ethylene level was more sensitive to alkaline stress (Fig. 6, E and F), eto1-1 did not enhance the sensitivity to alkaline stress displayed by axr1-3 in terms of relative root elongation of eto1-1, axr1-3, and eto1-1 axr1-3 under alkaline stress (Fig. 6, E and F). Ethylene-promoted auxin signaling could be because of the change of auxin accumulation in alkaline stress-mediated root inhibition. Accordingly, we directly measured auxin contents of the roots of wild-type, etr1-3, and ein3-1 plants exposed to alkaline stress. Our results revealed that auxin contents increased only by 32.1% and 41.6% in etr1-3 and ein3-1, respectively, compared with 80.1% in the roots of wild-type plants (Fig. 6G). Taken together with our above data for ethylene involvement in alkaline stress-induced inhibition of root elongation (Figs. 3, A, E, and F and 4, B, E, and F), these results indicate that ethylene plays its role in alkaline stress-mediated root inhibition by increasing auxin levels.

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 μm ACC for 6 or 12 h. Bars = 100 μm. B, Quantification of the DII-VENUS fluorescence intensities in the meristem zones of the DII-VENUS roots in A. Values are relative to those of the seedlings subjected 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 10 μm AgNO₃ or 10 μm CoCl₂ for 12 h. Bars = 100 μm. D, Quantification of the DII-VENUS fluorescence intensities in the meristem zones of DII-VENUS roots in C. Values are relative to those of the seedlings subjected to pH 5.8. E and F, The seedlings of 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 5.8 for 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 ± sem. Asterisks indicate significant differences based on Student’s t test (B) or two-way ANOVA with Bonferroni posttests compared with related control (D) or the wild type at each pH condition (E–G): FW, fresh weight; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

AUX1 Is Required in Alkaline Stress-Induced Inhibition of Primary Root Elongation

The polar transport of auxin is critical for auxin distribution and accumulation in roots and mediated by influx carriers of the AUX/LAX family and efflux carriers of the PIN family (Blilou et al., 2005; Overvoorde et al., 2010). The auxin efflux carrier PIN2 is involved in alkaline stress-induced inhibition of root elongation (Xu et al., 2012). The auxin influx carrier AUX1 also plays a crucial role in root elongation (Růžička et al., 2007; Stepanova et al., 2007). We explored the possible role of AUX1 in alkaline stress-induced inhibition of primary root elongation. We found that the expression of AUX1 was up-regulated in the roots exposed to alkaline stress with 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, the AUX1-Yellow Fluorescent Protein (YFP) signal was enhanced to 157.5%, 200.6%, or 254.8% in the roots of AUX1::AUX1-YFP seedlings treated with pH 8.0 for 6 and 12 h or pH 9.0 for 12 h compared with that of untreated control (Fig. 7, B and C), showing that AUX1 expression is induced by alkaline stress. Then, the aux1-7 mutant was used to assay the role of AUX1 in alkaline stress-mediated root inhibition. For this purpose, both wild-type and aux1-7 seedlings were exposed to alkaline stress, and the root lengths were statistically analyzed as above. We found that, when the seedlings were subjected to pH 8.0 and pH 9.0, root elongation in wild-type plants was reduced to 53.1% and 36.8%, respectively, whereas root elongation of aux1-7 seedlings was reduced only to 85.6% and 67.4%, indicating that root elongation in aux1-7 was relatively insensitive to alkaline stress (Fig. 6, E and F). Taken together, our data reveal that AUX1 participates in alkaline stress-mediated inhibition of primary root elongation.

Figure 7.

AUX1 plays a role in alkaline stress-mediated inhibition of the primary root elongation. A, qRT-PCR analysis of AUX1 expression 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 relative to 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 or pH 9.0 for 12 h. Bars = 100 μm. C, Quantification of AUX1-YFP fluorescence intensities in the root meristem zones of the AUX1::AUX1-YFP seedlings in B. Values are relative to those of the seedlings subjected to pH 5.8. Error bars represent sem of three independent experiments. ***, Significant differences based on Student’s t test (P < 0.001).

We next examined whether ethylene was involved in alkaline stress-induced AUX1 expression. Alkaline stress-enhanced AUX1 expression was dramatically repressed in the roots of wild-type plants by additional application of either Ag⁺ or Co²⁺ (Fig. 8A). This repression of AUX1 expression was further verified by its protein level, which was indicated by reduced AUX1-YFP fluorescence intensity 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-promoted AUX1 expression was also inhibited in etr1-3, ein2-1, and ein3-1 (Fig. 8D). Furthermore, eto1-1 did not affect the sensitivity of aux1-7 to alkaline stress in terms of root elongation of eto1-1, aux1-7, and eto1-1 aux1-7 subjected to pH 8.0 or pH 9.0 (Fig. 6, E and F). Collectively, our data suggest that alkaline stress-promoted AUX1 expression is mediated by ethylene in alkaline stress-mediated inhibition of primary root elongation.

Figure 8.

Alkaline stress-increased AUX1 expression is regulated by ethylene. A, qRT-PCR analysis of AUX1 expression in the roots treated with pH 5.8 or pH 8.0 with additional application of 10 μm AgNO₃ or 10 μm CoCl₂. Values are relative to those of seedlings subjected to pH 5.8 as the control. B, The effect of AgNO₃ or CoCl₂ on alkaline stress-increased expression of AUX1-YFP. The AUX1-YFP fluorescence is shown in the roots of 5-d-old AUX1::AUX1-YFP seedlings incubated in one-half-strength MS of pH 5.8 or 8.0 with additional application of 10 μm AgNO₃ or 10 μm CoCl₂ for 12 h. Bars = 100 μm. C, Quantification of AUX1-YFP fluorescence intensities in the meristem zones of AUX1::AXU1-YFP roots in B. Values are relative to those of the seedlings subjected 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, and ein3-1 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 independent experiments. Asterisks indicate significant differences based on two-way ANOVA with Bonferroni posttests compared with related control (A and C) or the wild type at each pH condition (D): *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

DISCUSSION

Alkaline pH serves as an important factor in plant growth and environmental responses by modulating numerous metabolic and physiological processes, including photosynthesis, microfilament depolymerization, ionic homeostasis, reactive oxygen species balance, and selectivity of the membrane system, and controlling plant growth, such as root hair formation and primary root elongation (Shi and Sheng, 2005; Monshausen et al., 2007; Zhang and Mu, 2009; Zhou et al., 2010; Liu and Guo, 2011). When subjected to alkaline stress, plants can respond by modifying auxin distribution, plasma membrane H⁺-ATPase activity, and microfilament stabilization, resulting in shorter roots (Fuglsang et al., 2007; Yang et al., 2010; Zhou et al., 2010; Liu and Guo, 2011; Xu et al., 2012, 2013). Alkaline stress inhibits primary root elongation (Fuglsang et al., 2007; Xu et al., 2012). Here, additional detailed examination indicated that the shorter root length was caused by reduced meristematic cell division. This phenomenon is also observed in plant responses to other stresses, such as excess copper, phosphate starvation, and toxic levels of chromium (Sánchez-Calderón et al., 2005; Castro et al., 2007; Yuan et al., 2013). However, the root meristematic cell division potential is not affected during Glc-mediated inhibition of root meristem growth (Yuan et al., 2014). The reduced meristematic cell division could be caused by the change in the expression level of cell cycle-related genes. Indeed, alkaline stress repressed CYCA2;1, CYCB1;1, CYCB3;1, CYCD1;1, CYCD2;1, CYCD3;1, CYCD4;1, CYCD4;2, CYCD6;1, and E2Fb, which are positive regulators of cell cycle progression, and enhanced the expression of E2Fc and RBR, which are negative regulators of cell proliferation. Considering that ethylene negatively regulates cell division 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 alkaline stress increases ethylene level in the roots. In addition, the increased auxin accumulation in plant response to alkaline stress may also involve this process. Růžička et al. (2009) showed that 2,4-dichlorophenoxyacetic acid reduces the root meristem size and the mitotic activity in the root meristem. It is also reported that aluminum stress can result in increased accumulation of both ethylene and auxin in the roots (Sun et al., 2010) and reduce primary root growth by inhibiting cell cycle progression (Herrera and Bucio, 2013), suggesting that aluminum-promoted accumulation of these two phytohormones 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 boron supply (Sun et al., 2007, 2010; Lei et al., 2011; Martín-Rejano et al., 2011; Khan et al., 2013; Habben et al., 2014; Ludwików et al., 2014). Excess aluminum and low boron can elicit rapid production of ethylene, leading to marked inhibition of root elongation (Sun et al., 2007, 2010; Martín-Rejano et al., 2011). However, whether ethylene is involved in plant response to alkaline stress had not been examined. Our results here, using physiological, pharmacological, and genetic approaches, provided solid evidence for the involvement of ethylene in alkaline stress-mediated inhibition of root elongation. Although root elongation in the etr1-3, ein2-1, and ein3-1 mutants was less sensitive to alkaline stress, the eto1-1 mutant, which overproduces ethylene, was hypersensitive to alkaline stress. Furthermore, an ethylene perception antagonist (Ag⁺) and an ethylene biosynthesis inhibitor (Co²⁺) alleviated alkaline stress-mediated inhibition of root elongation. Staal et al. (2011) reported that exogenous ACC reduces cell elongation of the elongation zone of Arabidopsis roots by changes in apoplastic alkalinization by modulating plasma membrane H⁺-ATPase. Thus, although both ethylene biosynthesis and signaling are promoted in the roots sensing alkaline stress, ethylene can also affect apoplastic pH of the roots. We also noted more severe inhibition of primary root elongation in alkaline stress-treated seedlings compared with that in ACC-treated plants. Taken together with the data that ethylene levels in ACC-treated roots were significantly higher than those in alkaline stress-treated roots, these data suggest that another factor(s) other than ethylene is also involved in alkaline stress-mediated inhibition of primary root elongation. This view is consistent with our data that Ag⁺ and Co²⁺ only partially alleviate root elongation inhibition under alkaline stress.

In addition to ethylene, auxin functions in responses to various environmental stimuli by modifying root development (Overvoorde et al., 2010). Exposure to excess Cu, Cd, Al, or As(III) or high Glc or salt stresses disturbs normal auxin accumulation and distribution, reducing root growth (Wang et al., 2009; Sun et al., 2010; Hu et al., 2013; Krishnamurthy and Rathinasabapathi, 2013; Yuan et al., 2013, 2014). Auxin efflux or influx carriers are involved in these changes in auxin accumulation and distribution (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 alkaline stress-mediated root inhibition (Xu et al., 2012). Our results suggested that AUX1 mediates the alkaline stress-induced accumulation of auxin for the inhibition of root elongation. Furthermore, auxin biosynthesis was also concluded to be involved in this process, because the expression of auxin biosynthesis-related genes was elevated and the inhibitions of root elongations of both wei2-2 and wei8-1 were less pronounced than those of wild-type plants when subjected to alkaline stress.

Ethylene and auxin act synergistically to control root elongation (Rahman et al., 2001; Chilley et al., 2006; Swarup et al., 2007). The possibility that ethylene and auxin function together in plant responses to alkaline stress was explored in our study, which verified the cross talk between ethylene and auxin. The increase in auxin level was reduced in the roots of the mutants etr1-3 and ein3-1 or Ag⁺/Co²⁺-treated wild-type plants, and eto1-1 did not strengthen the sensitivity to alkaline stress displayed by axr1-3 and aux1-7. However, auxin may not be involved in alkaline-induced ethylene accumulation, because alkaline stress can efficiently increase ethylene level to about 3-fold in the roots of the wild type (287.91%), axr1-3 (303.11%), and aux1-7 (281.87%; Supplemental Fig. S1, A and B). Also, the EBS::GUS expression in the roots of both axr1-3 EBS::GUS and aux1-7 EBS::GUS was significantly promoted by alkaline stress, although to a slightly lower degree than in EBS::GUS (Supplemental Fig. S1, C and D).

Taken together, our results suggest that ethylene modulates alkaline stress-mediated inhibition of root growth by increasing auxin accumulation by stimulating 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 published transgenic and mutant lines used in this study are QC25::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 (Chae et al., 2003). The EBS::GUS reporter line originally generated by Anna Stepanova in the laboratory of Joe Ecker (Stepanova et al., 2005) was provided by Jose Alonso (Department of Genetics, North Carolina State University); eto1-1 aux1-7 and eto1-1 axr1-3 were provided by Bonnie Bartel (Department of Biochemistry and Cell Biology, Rice University) and Lucia C. Strader (Department of Biology, Washington University, St. Louis; Strader et al., 2010; Thole et al., 2014), aux1-7 EBS::GUS and axr1-3 EBS::GUS were provided by Anna Stepanova (Department of Genetics, North Carolina State University, Raleigh; Stepanova et al., 2007), etr1-3 was provided by W.H. Zhang (Institute of Botany, Chinese Academy of Sciences; Sun et al., 2010), and wei8-1 was provided by Z.J. Ding (School of Life Sciences, 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) were obtained 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 planted on 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 adjusted with 1 m KOH. Seedlings were grown at 23°C and 100 μmol m⁻² s⁻¹ of illumination under 16-h-light/8-h-dark conditions.

Root Length Measurement

To study the inhibitory effect of alkaline stress on root elongation, 5-d-old Arabidopsis seedlings were exposed to one-half-strength MS of pH 5.8, 8.0, or 9.0 for 12 h and then transferred to one-half-strength MS of pH 5.8 for another 2 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 exposed to 10 μm ACC for 6, 12, and 24 h and then grown in one-half-strength MS of pH 5.8 for an additional 2 d. To assay the effect of AgNO₃ and CoCl₂ on root elongation 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 μm AgNO₃ or 10 μm CoCl₂ for 12 h and then transferred to one-half-strength MS of pH 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 to pH 5.8, 8.0, or 9.0, and root elongation was measured 2 d after the seedlings were transferred and grown in one-half-strength MS of pH 5.8. The results are expressed as the mean ± sem (n > 30 roots). Each measurement was repeated with 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-β-d-glucuronide). The GUS staining time was dependent on the transgenic marker lines: 6 h for QC25::GUS, 12 h for QC46::GUS and CYCB1;1::GUS, and 4 h for EBS::GUS. The 4-methyl ketone of umbrella glucoside 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 described method (Zhao et al., 2014). Briefly, the samples were chopped with nucleus isolation buffer (10 mm MgSO₄, 50 mm KCl, 5 mm HEPES, 1 mg mL⁻¹ dithiothreitol [Sigma], and 0.2% [v/v] Triton X-100) and filtered through a 33-μm nylon 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 μg mL⁻¹. The nuclear DNA content was analyzed using flow cytometry (BeckMan Coulter) with a 488-nm solid-state laser (100 mW) for excitation, and emission data were collected after a 590-nm long-pass filter.

Microscopic Analysis

For phenotypic analysis of root or GUS staining microexamination, seedlings 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 examined under differential interference contrast optics (Olympus BX63; Olympus) and photographed using a CCD camera (Olympus DP72; Olympus).

Confocal microscopy was performed using an Olympus FluoView 1000 Confocal Laser-Scanning Microscope according to the manufacturer’s instructions. The YFP lines were mounted onto microscope slides with 20 μg mL⁻¹ PI. At least 24 seedlings were analyzed per treatment. The signal intensity was measured using Photoshop CS5 (Adobe), and error bars were obtained based on measurements of more than 20 seedlings per treatment. The intensity ratio was obtained by comparing DII-VENUS or AUX1::AUX1-YFP fluorescence intensity with 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 with RQ1 RNase-free DNase I (Promega), first strand complementary DNA synthesis was carried out using Superscrip II Reverse Transcriptase (Invitrogen) or M-MLV Reverse Transcriptase (NOVA; LCP Biomed Co.) according to the manufacturer’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-well plates according the following protocol: 3 min at 95°C followed by 40 cycles of denaturation for 15 s at 95°C, annealing for 15 s at 58°C, and extension for 20 s at 72°C. Primers were designed using Beacon Designer 7.0 (Premier Biosoft International). ACTIN2 (AT3G18780) and EUKARYOTIC TRANSLATION INITIATION FACTOR 4A (EIF4A; AT3G13920) were used as internal controls using GeNorm (Czechowski et al., 2005). All experiments were performed with three independent biological replicates and three technical repetitions. The genes analyzed and the corresponding specific primers are listed in Supplemental Table S1 (Sun et al., 2010; Yan et al., 2013; Gao et al., 2014; Hong et 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 the comparative ΔΔCT method with ACTIN2 and ELF4A as internal standards. The related expression of specific genes was calculated by the formulas ΔCT = CT-specific gene − CT reference gene and ΔΔCT = ΔCT alkaline-treated sample − ΔCT untreated sample. The related level was calculated as 2−ΔΔCT. 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 immediately frozen in liquid nitrogen, extracted, and purified for endogenous IAA. The purified samples were methylated by a stream of diazomethane gas, resuspended in 100 µL of ethyl acetate, and analyzed by GC-mass spectrometry. A Shimadzu GCMS-QP2010 Plus equipped with an HP-5MS Column (30-m long, 0.25-mm i.d., 0.25-lm Film; Agilent) was used to determine the level of IAA.

Quantification of Ethylene by GC

The ethylene level was measured according to the previously described method (Sun et al., 2010; Du et al., 2014). Whole-root samples (200 mg) were excised 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 injected into a gas chromatograph equipped with a flame ionization detector column packed with activated aluminum at 100°C. Ethylene was detected by an ionization detector and recorded by an integrator. The sample injected temperature 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 with Tukey’s multiple comparison test, two-way ANOVA with Bonferroni posttests, or Student’s t test using GraphPad Prism version 5.0 (GraphPad Software; 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.

Glossary

ACC

1-aminocyclopropane-1-carboxylic acid

DII

domain II

GC

gas chromatography

IAA

indole-3-acetic acid

MS

Murashige and Skoog medium

PI

propidium iodide

qRT

quantitative real time