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. 2016 Dec 8;173(2):1420–1433. doi: 10.1104/pp.16.01756

Jasmonic Acid Enhances Al-Induced Root Growth Inhibition1,[OPEN]

Zhong-Bao Yang 1,2,2, Chunmei He 1,2,2, Yanqi Ma 1,2, Marco Herde 1,2, Zhaojun Ding 1,2,*
PMCID: PMC5291015  PMID: 27932419

Jasmonate mediates aluminum-induced root growth inhibition through regulation of microtubule polymerization and ALMT1-regulated malate exudation in an auxin-independent manner in Arabidopsis.

Abstract

Phytohormones such as ethylene and auxin are involved in the regulation of the aluminum (Al)-induced root growth inhibition. Although jasmonate (JA) has been reported to play a crucial role in the regulation of root growth and development in response to environmental stresses through interplay with ethylene and auxin, its role in the regulation of root growth response to Al stress is not yet known. In an attempt to elucidate the role of JA, we found that exogenous application of JA enhanced the Al-induced root growth inhibition. Furthermore, phenotype analysis with mutants defective in either JA biosynthesis or signaling suggests that JA is involved in the regulation of Al-induced root growth inhibition. The expression of the JA receptor CORONATINE INSENSITIVE1 (COI1) and the key JA signaling regulator MYC2 was up-regulated in response to Al stress in the root tips. This process together with COI1-mediated Al-induced root growth inhibition under Al stress was controlled by ethylene but not auxin. Transcriptomic analysis revealed that many responsive genes under Al stress were regulated by JA signaling. The differential responsive of microtubule organization-related genes between the wild-type and coi1-2 mutant is consistent with the changed depolymerization of cortical microtubules in coi1 under Al stress. In addition, ALMT-mediated malate exudation and thus Al exclusion from roots in response to Al stress was also regulated by COI1-mediated JA signaling. Together, this study suggests that root growth inhibition is regulated by COI1-mediated JA signaling independent from auxin signaling and provides novel insights into the phytohormone-mediated root growth inhibition in response to Al stress.

Aluminum (Al) is the third most abundant element in the Earth’s crust after oxygen and silicon and is the most abundant metal. When soil pH drops below 5.5, Al3+ is released into the soil solution and becomes one of the limiting factors for crop production in most acid soils (Kochian et al., 2004). Acid soils limit crop production on 30% to 40% of the world’s arable land and up to 70% of the world’s potentially arable land (Haug, 1983). Although the poor fertility of acid soils is due to a combination of mineral toxicities (Al and manganese) and deficiencies (phosphorus, calcium, magnesium, and molybdenum), Al toxicity is the single most important factor, being a major constraint for crop production on 67% of the total acid soil area (Eswaran et al., 1997).

Excess Al causes a rapid (minutes to few hours) inhibition of root growth, resulting in a reduced and damaged root system that limits mineral nutrient and water uptake. The inhibition of root growth has been widely used to evaluate the Al toxicity (Delhaize and Ryan, 1995; Kochian et al., 2004). Although much progress has been made during recent years in the understanding of Al resistance, the molecular and physiological mechanisms leading to Al-induced inhibition of root elongation are still not well understood. Particularly there is still much debate about whether the symplast or apoplast is the early target of Al toxicity. Root elongation is the result of division and elongation of the root cells. At the early stage of research on Al toxicity, the blockage of cell division was regarded as the primary mode of Al injury since the cessation of root elongation and the disappearance of mitotic figures was closely correlated (Clarkson, 1965). However, cell division is a slow process, while the inhibition of root elongation of Al-sensitive maize can occur within 30 min of Al treatment (Llugany et al., 1995). Recently, Kopittke et al. (2015) reported that 75 µm Al reduced root growth of soybean after only 5 min, with Al being toxic by binding to the walls of outer cells, which directly inhibited their loosening in the elongation zone, and concluded that the primary lesion of Al is apoplastic. Therefore, the rapid dynamics of root growth to Al stress indicates that Al first inhibits root cell expansion and elongation and later on also cell division (Horst et al., 2010; Kochian et al., 2015). And also, Horst et al. (1999) indicated that the cell wall-plasma membrane-cytoskeleton continuous system plays a role in the regulation of Al toxicity.

The root apex has been confirmed as the major site for Al injury (Ryan et al., 1993). In maize (Zea mays), Sivaguru and Horst (1998) specified that the distal transition zone (DTZ) in the root apex is the major perception site for Al stress, while in common bean (Phaseolus vulgaris), the elongation zone is also a toxic site for Al besides the DTZ (Rangel et al., 2007). The root apex has been thought to be an active site for phytohormone action (Baluška et al., 2010) and other signals such as nitric oxide, reactive oxygen species, etc. (Mugnai et al., 2014). Phytohormones such as auxin, ethylene, cytokinin, salycilic acid, and abscisic acid, etc. have been reported to regulate the Al-induced root growth inhibition (Kollmeier et al., 2000; Shen et al., 2004; Sun et al., 2010; Yang et al., 2014; Guo et al., 2014). In maize, the blockage of the auxin signal from the DTZ to the elongation zone has been revealed as the major reason for Al-induced inhibition of root elongation (Kollmeier et al., 2000), while in Arabidopsis (Arabidopsis thaliana), Trp aminotransferase 1 (TAA1)-mediated local auxin biosynthesis in the root apex transition zone (TZ) leads to the Al-induced root growth inhibition, and this process is controlled by ethylene signaling (Yang et al., 2014). Sun et al. (2007, 2010) found that Al stimulation enhanced the expression of genes involved in ethylene biosynthesis, such as ACSs (ACC synthase) and ACOs (ACC oxidase) and proposed that ACS and ACO play roles in Al-mediated inhibition of root growth. This ethylene-meditated Al-induced inhibition of root growth can be achieved by alteration of auxin distribution in root tips (Sun et al., 2010). In common bean, a cross-talk between abscisic acid, ethylene, and cytokinin has been proposed to regulate primary root growth under both Al and drought stress (Yang et al., 2012).

Jasmonates (JAs) are key regulators for plant growth and development as well as in plant responses to biotic and abiotic stresses (Creelman and Mullet, 1995; Wasternack and Hause, 2013) through the interaction with other phytohormones such as ethylene, auxin, gibberellins, etc. (Wasternack and Hause, 2013; Zhu, 2014). For instance, ethylene can inhibit root growth through a CORONATINE INSENSITIVE1 (COI1) and light-dependent, but JA-independent pathway (Adams and Turner, 2010). JA enhances the transcriptional activity of EIN3 (ETHYLENE INSENSITIVE3)/EIL1 (EIN3-Like1) by removal of JA-Zim domain (JAZ) proteins, which physically interact with and repress EIN3/EIL1 during root-hair formation (Zhu et al., 2011). JA-activated transcription factor MYC2, on the one hand, promotes EIN3 degradation through binding to the promoter of an F-box gene, EIN3 BINDING F-BOX PROTEIN1, to induce its expression, and on the other, physically interact with EIN3 and inhibit its DNA binding activity. Similarly, Song et al. (2014) also observed that MYC2 interacts with EIN3 to attenuate the transcriptional activity of EIN3 and repress ethylene-enhanced apical hook curvature, while, conversely, EIN3 interacts with and represses MYC2 to inhibit JA-induced expression of wound-responsive genes and herbivory-inducible genes and attenuate JA-regulated plant defense against generalist herbivores. In roots, the JA-induced growth inhibition occurs via a cross-talk with auxin. MYC2 directly binds to the promoters of PLETHORA (PLT1 and PLT2), which are central regulators in auxin-mediated root meristem and root stem cell-niche development, and suppresses their expression (Chen et al., 2011). In addition, JA was reported to affect auxin transport via modulating the PIN2 (PIN-FORMED) protein (Sun et al., 2011). In spite of this, the role of JA in the Al-regulated inhibition of root growth is not yet known.

To better understand how phytohormones perceive and respond to the Al stress signaling, the role of JA and its interplay with ethylene and auxin in Al-induced root growth inhibition was studied.

RESULTS

JA Enhances the Al-Induced Root Growth Inhibition

To examine the role of JA in Al-induced inhibition of root growth, the phenotypes of loss-of-function mutants aos (allene oxide synthase), coi1-2, and myc2-2, which are involved in JA biosynthesis and signaling pathway (Park et al., 2002, Kazan and Manners, 2008), were examined under Al stress. The result showed that aos, coi1-2, and myc2-2 mutants all displayed a greatly reduced root growth inhibition in response to Al stress, while the p35S:MYC2 transgenic line showed only a slightly elevated root growth inhibition at 4 µm Al treatment (Fig. 1, A–C; Supplemental Fig. S1). However, there was no difference between wild type and the coi1-2 mutant when plants were exposed to solutions containing different toxic ions such as lanthanum (La3+), cadmium (Cd2+), copper (Cu2+), and sodium (Na+; Fig. 1D) or to various pH solutions (4.5–6.0; Fig. 1E; Supplemental Fig. S2). Exogenous applied methyl jasmonate (MeJA), a concentration (≤0.5 nm) that had no effect on root growth, significantly enhanced the Al-induced inhibition of root growth in wild-type seedlings. The cotreated seedlings with both MeJA and Al showed much shorter roots than seedlings treated with only Al (Fig. 1F).

Figure 1.

Figure 1.

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The Al-induced inhibition of root growth is regulated by JA. A to C, Root growth of seedlings in the wild type and mutants including aos, coi1-2, and myc2-2 subjected to Al stress. The wild-type and mutant seedlings were exposed to 0 to 8 µm AlCl3 for 7 d. D, Root growth of seedlings in the wild type and the coi1-2 mutant in response to different metal ions. The wild-type and coi1-2 mutant seedlings were exposed to 0 and 6 µm AlCl3 or 1 µm LaCl3, 3.5 µm CdCl2, 2 µm CuCl2, and 10 mm NaCl. E, Root growth of seedlings in the wild type and the coi1-2 mutant in response to varied pH solutions. *, **, and *** indicate a significant difference between the wild type and mutants in the presence of Al treatment at P < 0.05, < 0.01, and < 0.001, respectively. F, Effect of exogenous MeJA on the root growth of wild-type seedlings subjected to Al stress. The wild-type seedlings were exposed to 0 or 6 µm AlCl3 in the presence of 0 to 0.5 nm MeJA for 7 d. * and ** indicate a significant difference between MeJA and non-MeJA treatments in the presence of Al stress at P < 0.05 and < 0.01, respectively. Values in A to F represent means ± sd (n ≥ 30).

Al Stress Induces the Up-Regulation of JA Signaling in Root Tip

To address how JA regulates Al-induced inhibition of root growth, the expression of the JA receptor COI1 in the root tips was examined in response to Al stress using the transgenic line pCOI1:COI1-VENUS. Either 10 or 25 µm AlCl3 treatment, which caused ∼80 and 90% of inhibition of root growth after 7-d exposure (Supplemental Fig. S3), respectively, for 3 h highly induced the expression of pCOI1:COI1-VENUS in the root tips especially in the root apex TZ (Fig. 2, A and B). Time course analysis showed that the up-regulation pCOI1:COI1-VENUS in the root tips occurred after 2-h exposure to Al stress (Supplemental Fig. S4), and the response was specific to Al in comparison with other metal ions or various pH changes (Supplemental Figs. S5 and S6). This result was also confirmed with the pCOI1:GUS transgenic line. The expression of pCOI1:GUS in the root tips including both the meristem zone and TZ was highly induced under 10 µm AlCl3 treatment (Fig. 2C). In addition, the enhanced expression of pCOI1:GUS was also observed in the zone of root after apex and leaves, but with a much less extent (Supplemental Fig. S7).

Figure 2.

Figure 2.

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Al enhances JA signaling in the root apex. A and B, Expression of pCOI1:COI1-VENUS in the cortex (A) and epidermis (B) of root tips after exposure to Al stress. The 6-d-old seedlings of pCOI1:COI1-VENUS transgenic line were exposed to 0, 10, and 25 µm AlCl3 for 3 h. Bar = 100 µm. C and D, Expression of pCOI1:GUS and pMYC2:GUS in the root tips after exposure to Al stress. The 6-d-old seedlings of pCOI1:GUS and pMYC2:GUS transgenic lines were exposed to 10 µm AlCl3 for 0, 3, and 6 h. Bar = 100 µm. E and F, qRT-PCR analysis of the expression of genes in Al-exposed roots of wild-type seedlings. G and H, Total JA and JA-Ile concentration in roots after exposure to Al. The 6-d-old seedlings of wild-type plants were exposed to 10 µm AlCl3 for 0, 1, 3, and 6 h. Values in E to H represent means ± sd (n = 3 in E and F and n = 4 in G and H). *, **, and *** indicate a significant difference between the Al and non-Al exposed seedlings at P < 0.05, P < 0.01, and P < 0.001, respectively.

In addition, the expression of MYC2 was also strongly induced under 10 µm AlCl3 treatment with the pMYC2:GUS transgenic line (Fig. 2D). Accordingly, the Al stress induced up-regulation of MYC2 and COI1 was also observed by quantitative real-time PCR (qRT-PCR) analysis that showed a significantly increased expression of MYC2 and COI1 under 10 µm AlCl3 treatment for 6 h (Fig. 2, E and F). The lack of a significant response after 3-h Al treatment might be due to the fact that the local induction in the root apical zone was masked when analyzing the whole roots.

Al exposure for 3 h also up-regulated the expression of JA biosynthesis related genes such as AOS, ALLENE OXIDE CYCLASE3 (AOC3), and OPDA REDUCTASE3 (OPR3) in roots. However, different from COI1 and MYC2, the expression of AOS, AOC3, and OPR3 was declined with Al exposure up to 6 h (Fig. 2F). The expression of other JA synthesis related genes such as LIPOXYGENASE2 (LOX2), AOC1, and AOC2 was not affected under Al stress. Consistently, Al exposure within 3 h elevated JA-Ile but not total JA levels in roots, while both total JA and JA-Ile concentration in roots was reduced with the prolonged Al exposure time to 6 h (Fig. 2, G and H).

Al-Induced JA Signaling in Root-Apex TZ and the Resultant Root-Growth Inhibition Is Regulated by Ethylene

JA signaling has been reported to regulate root growth and development through the cross-talk with ethylene (Wasternack and Hause, 2013). To address if the Al-induced up-regulation of COI1 in the root apex is dependent on ethylene signaling, we examined the local induction of pCOI1:COI1-VENUS or pCOI1:GUS in response to Al stress when cotreated with 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene biosynthesis, or aminoethoxyvinyl-Gly (AVG), the inhibitor of ethylene biosynthesis. The Al-induced up-regulation of pCOI1:COI1-VENUS and pCOI1:GUS in the root apex especially in root TZ was clearly enhanced by ACC application, while it was strongly repressed by AVG cotreatment (Fig. 3, A and B; Supplemental Fig. S8). Consistently, the Al-induced up-regulation of pCOI1:COI1-VENUS in root apex was eliminated in the ein3 eil1-1 double mutant that has defects in ethylene signaling (Fig. 3C; Supplemental Fig. S9).

Figure 3.

Figure 3.

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The Al-induced expression of JA signaling in the root apex and the JA-mediated root-growth inhibition in response to Al stress are regulated by ethylene. A, Expression of pCOI1:COI1-VENUS in the root apex in the presence of ACC or AVG with or without Al treatment. The 6-d-old transgenic seedlings were exposed to 0 and 25 µm AlCl3 for 3 h in the presence or absence of 1 µm ACC or 1 µm AVG, respectively. Bar = 100 µm. B, Quantitative analysis of the fluorescent intensity indicated in A in the root tips. *, and *** indicate the significant difference between Al and ACC or AVG treatment at P < 0.05 and < 0.001, respectively. Values represent means ± sd (n = 10). C, Al-induced expression of COI1 in root tips was eliminated in ein3eil1-1 mutant. The 6-d-old transgenic seedlings were exposed to 0 and 25 µm AlCl3 for 3 h. D, JA acts as the down-stream signaling of ethylene to regulate the Al-induced inhibition of root growth. The wild-type seedlings and ein3eil1-1 mutant were exposed to 0 and 6 µm AlCl3 in the presence or absence of 0.5 nm MeJA for 7 d. E, The blockage of JA signaling could not fully reduce the ACC-enhanced root growth inhibition in response to Al stress. The wild-type seedlings and coi1-2 mutant were exposed to 0 and 6 µm AlCl3 in the presence or absence of 50 nm ACC for 7 d. In E and F, ** and *** indicate the significant difference between Al treatment and Al plus MeJA (D) or Al plus ACC (E) treatment at P < 0.01 and P < 0.001, respectively. Values in D and E represent means ± sd (n = 30).

To further clarify if JA enhanced Al-induced inhibition of root growth downstream of ethylene, the phenotype of ethylene defective mutant ein3 eil1-1 was examined. Results showed that the JA-enhanced root growth inhibition under Al stress was observed in both wild-type seedlings and the ein3 eil1-1 mutant (Fig. 3D). On the other hand, though the ACC-enhanced root growth inhibition under Al stress was also observed in the coi1-2 mutant, the extent of inhibition was relatively lower than in wild-type seedlings (Fig. 3E). These results indicated that JAs act downstream of ethylene in Al-induced root growth inhibition, there might be alternative signaling pathway in parallel with JA signaling to mediate ethylene signaling in this process.

JA and Auxin Independently Regulate the Al-Induced Root Growth Inhibition

According to these above observations and the previous reports (Yang et al., 2014), both JA and auxin act downstream of ethylene to regulate root-growth inhibition under Al stress. Therefore, we further investigated the relationship of JA and auxin signaling in response to Al stress. Neither blocking auxin signaling with the auxin antagonist α-(phenylethyl-2-one)-indole-3-acetic acid (PEO-IAA; Hayashi et al., 2008) nor auxin biosynthesis with TAA1/TAR inhibitor l-kynurenine (He et al., 2011) affected the Al-induced up-regulation of COI1 in the root apex (Fig. 4, A and B). Exogenously applied 1-naphthaleneacetic acid (NAA) also did not influence the Al-induced up-regulation of COI1 in the root apex (Fig. 4, A and B). On the other hand, the mutation of COI1, which disrupts JA signaling, neither affected the Al-induced auxin maximum shown by the expression of the DR5rev:GFP transgene (Fig. 4C; Supplemental Fig. S10A), nor influenced the Al-induced up-regulation of TAA1:GFP in the root apex TZ (Fig. 4D; Supplemental Fig. S10B).

Figure 4.

Figure 4.

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The Al-induced JA signaling in the root apex is independent of auxin signaling. A, Effect of exogenous NAA and antagonists of auxin biosynthesis or signaling on Al-induced expression of pCOI1:COI1-VENUS in the root tips. Four-day-old transgenic lines of the pCOI1:COI1-VENUS were pretreated with 0 and 100 nm NAA for 2 d and then subjected to 0 and 25 µm AlCl3 with or without 100 nm NAA, or 100 nm l-kynurenine, or 10 µm PEO-IAA for 3 h. Bar = 100 µm. B, Quantitative analysis of the fluorescent intensity in the root tip shown in A. ** and *** indicate a significant difference between treatment and nontreated control at P < 0.01 and P < 0.001, respectively. Values represent means ± sd (n = 10). C and D, Expression of the TAA1:GFP and DR5rev:GFP transgene in the root apex epidermis of Al-treated wild-type and coi1-2 seedlings. Six-day-old seedlings of the TAA1:GFP and DR5rev:GFP transgenic lines were exposed to 0 and 25 µm AlCl3 for 3 h. Bar = 100 µm.

NAA application significantly enhanced while PEO-IAA reduced the Al-induced inhibition of root growth in wild-type plants (Fig. 5A). However, neither the NAA-enhanced nor PEO-IAA cotreatment alleviated root growth inhibition in response to Al stress was affected in the coi1-2 mutant (Fig. 5A). In addition, the JA-enhanced root growth inhibition in response to Al stress was also observed in the arf7arf19 mutant, a loss-of-function mutant of auxin response factors ARF7 and ARF19, and slr-1 (solitary root1), a gain-of-function mutant of IAA14 (Fig. 5B), which has been reported to have a reduced level of auxin signaling (Fukaki et al., 2005). Together, these results suggest that JA and auxin act in parallel to regulate Al-induced root growth inhibition, which further supported additive effects in the arf7arf19coi1-2 triple mutant under Al stress. Compared to the wild type, the arf7arf19 coi1-2 triple mutant displayed much longer roots compared with the coi1-2 single mutant and the arf7arf19 double (Fig. 5D).

Figure 5.

Figure 5.

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The JA-mediated Al-induced root growth inhibition is independent of auxin signaling. A, Exogenous application of NAA and auxin signaling antagonist PEO-IAA affect the Al-induced inhibition of root growth at the same level in wild-type and coi1-2 mutant seedlings. The wild-type and coi1-2 mutant seedlings were exposed to 0 and 25 µm AlCl3 with or without 2.5 nm NAA or 1 µm PEO-IAA for 7 d. ** and *** indicate a significant difference between Al treatment and Al plus NAA or Al plus PEO-IAA treatment in either wild-type or coi1-2 mutant seedlings at P < 0.01 and P < 0.001, respectively. Values represent means ± sd (n = 30). B, Exogenous application of MeJA enhanced the Al-induced root growth inhibition at the same level in wild-type, arf7arf19, and slr-1 mutant seedlings. The wild-type, arf7arf19, and slr-1 mutant seedlings were exposed to 0 and 25 µm AlCl3 in the presence or absence of 0.5 nm MeJA for 7 d. ** indicates a significant difference between Al treatment and Al plus MeJA treatment in either wild-type, arf7arf19, or slr-1 mutant seedlings at P < 0.01. Values represent means ± sd (n = 30). C and D, The root growth phenotype (C) and relative primary root growth (D) in wild-type, coi1-2, arf7arf19, and arf7arf19coi1-2 mutant seedlings in response to Al stress. The wild-type and mutant seedlings were exposed to 0 to 8 µm AlCl3 for 7 d. ** and *** indicate a significant difference between wild-type and mutant seedlings in response to Al stress at P < 0.01 and P < 0.001, respectively. Values represent means ± sd (n = 30).

Transcriptional Analysis of the JA-Regulated Genes in Response to Al Stress

To investigate how JA regulates Al-induced inhibition of root growth, a transcriptional analysis through RNA-seq was performed by comparing the coi1-2 mutant and wild-type plants in the presence or absence of Al (Supplemental Table S1). In the absence of Al, 149 and 147 genes were up- and down-regulated, respectively, at least 2-fold in the roots of the coi1-2 mutant compared to wild-type plants. In the presence of Al, 1,747 and 5,838 genes were up- and down-regulated, respectively, in the roots of wild-type plants, while 1,449 and 3,773 genes were up- and down-regulated, respectively, in the coi1-2 mutant. While the comparison of the Al-exposed coi1-2 mutant and wild-type plants reveals that a total 1,187 genes were up-regulated and only 197 genes were down-regulated at least 2-fold (Supplemental Fig. S11A; Fig. 6A). Functional analysis of the differentially expressed genes displaying more than 4-fold changes in Al-exposed coi1-2 mutant and wild-type plants revealed that the functions of 28% of the up-regulated and 35% down-regulated genes could not be clarified. Among the up-regulated genes, 14% were involved in cellular protein modification, 12% in stress/defense response, 7% in each of secondary metabolism and RNA processing, 6% in each of microtubule (MT)-based process and primary metabolism, 5% in each of regulation of transcription and cell wall organization/modification, and 4% in signal transduction. The down-regulated genes were functionally annotated in stress/defense response (23%), primary metabolism (11%), transport (11%), secondary metabolism (6%), regulation of transcription (4%), cell wall organization/modification (4%), signal transduction (4%), etc. Major differences existed between the up- and down-regulated genes in the functional categories cellular protein modification, RNA processing, MT-based process, and transport (Supplemental Fig. S11B; Supplemental Tables S2 and S3).

Figure 6.

Figure 6.

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A, Cluster analysis of the expressed genes with more than 2-fold changes in wild-type and coi1-2 mutant seedlings in response to Al stress. The cluster analysis of 27 MT-associated genes was particularly listed. RPKM (reads per kilobase per million reads) represents the gene expression level. B, Validation of the expression of several MT-associated genes listed in A by qRT-PCR. Six-day-old wild-type and coi1-2 seedlings were exposed to 0 and 10 µm AlCl3 for 6 h. UBQ1 was used as the reference, and a nontreated wild-type was used as the sample control. Values represent means ± sd (n = 3). Asterisks indicate that wild-type and coi1-2 mutant means differ significantly at *P < 0.05, **P < 0.01, and ***P < 0.001 (t test).

JA Mediates Root Growth Sensitivity to Al Stress through Stabilizing the Cortical Microtubule in Root Apex TZ

The crucial role of the microtubular cytoskeleton in regulation of Al-induced inhibition of root growth has been reported (Sivaguru et al., 1999, 2003). In response to Al stress, 27 MT-related genes were up-regulated at least 2-fold in roots of the coi1-2 mutant compared with the wild type (Fig. 6A). This result was further confirmed by qRT-PCR analysis with nine randomly selected MT-related genes except the gene AT3G47690 (Fig. 6B). To study if JA-regulated root-growth inhibition in response to Al stress is related to the change of MT organization, we crossed the coi1-2 mutant with GFP-MAP4, which is a mammalian MT-associated protein fused with the GFP gene and has been used as a MT reporter gene to visualize cortical MT arrangement in living plant cells (Marc et al., 1998). Considering the importance of the root apex TZ in Al-induced root growth inhibition and phytohormone-modulated Al stress signaling transduction (Yang et al., 2014), we focused on the analysis of cortical microtubules in the root apex TZ. An exposure to 10 μm AlCl3 for 6 h obviously depolymerized MTs in root apex TZ of wild-type plants, while a reduced depolymerization of MTs was found in the coi1-2 mutant. Consistent with the Al effects, both MeJA and oryzalin, a MT-depolymerizing agent, strongly depolymerized the MTs (Fig. 7A). Moreover, oryzalin at concentrations from 0 to 70 nm dramatically inhibited root growth in both wild-type and coi1-2 seedlings, but the extent of the inhibition was less in coi1-2 than in the wild-type plants (Fig. 7B). In the presence of Al stress, increasing oryzalin application enhanced the Al-induced root growth inhibition in the wild-type seedlings but not in the coi1-2 mutant, with the exception of the highest oryzalin concentration of 70 nm. These results strongly suggest that Al stress induces depolymerization of cortical MT in the root apex TZ via JA signaling, and the coi1-2-reduced root growth sensitivity to Al stress is related to the maintenance of MT polymerization.

Figure 7.

Figure 7.

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The Al-induced depolymerization of cortical MTs in the root apex TZ and MT-regulated Al-induced root growth inhibition is mediated by JA signaling. A, The cortical MTs of the root apex TZ cells of 6-d-old GFP-MAP4 and coi1-2GFP-MAP4 seedlings upon treatment with 0 and 10 µm AlCl3, 100 nm MeJA, or 100 nm oryzalin for 6 h. Bar = 100 µm. B, Root growth of wild-type seedlings and coi1-2 mutant in response to Al stress and oryzalin treatment. The wild-type seedlings and coi1-2 mutant were exposed to 0 and 10 µm AlCl3 in the presence of 0 to 70 nm oryzalin for 7 d. * and ** indicate a significant difference between Al treatment alone and Al plus oryzalin treatment in either wild-type or coi1-2 mutant seedlings at P < 0.05 and P < 0.01, respectively. Values represent means ± sd (n = 30).

JA Regulates Al-Induced Malate Exudation and Al Accumulation in Root Tips

It is well known that ALMT1 and MATE mediate malate and citrate exudation from roots, respectively, and play key roles in the detoxification of Al by chelating Al3+ and thus excluding it from roots (Hoekenga et al., 2006; Kobayashi et al., 2007; Liu et al., 2009). ALS3 is thought to be responsible for redistributing toxic Al3+ from the Al-sensitive to the -unsensitive root zones (Larsen et al., 2005), while ALS1 may regulate the transport of Al3+ into vacuoles where it is finally sequestrated (Larsen et al., 2007). The transcription factor STOP1 has been identified to regulate ALMT1, MATE, and ALS3 and to participate into the regulation of Al resistance (Liu et al., 2009; Sawaki et al., 2009). However, among all these Al-resistance/tolerance-related genes, only ALMT1 showed 2-fold higher expression in Al-exposed coi1-2 mutant roots than wild-type control (Supplemental Table S1). Further qRT-PCR analysis showed the expression of ALMT1 was slightly but significantly higher in coi1-2 mutant compared to wild-type seedlings in response to Al stress, while no difference was detected in the expression of MATE, ALS3, ALS1, and STOP1 (Fig. 8A). The malate exudation from roots in response to Al stress in wild-type seedlings and the coi1-2 mutant was also examined. The result showed that malate exudation was significantly higher in coi1-2 than that in the wild-type control at 0- to 6-h Al exposure period (Fig. 8B). The increased malate exudation in coi1-2 resulted in reduced Al accumulation in the coi1-2 mutant roots, which was confirmed by morin staining or direct Al content measurement using the intact roots or factional Al analysis (Fig. 8, C–E). However, neither the ALMT1 expression nor malate exudation from roots of Al-treated seedlings was affected in the myc2-2 mutant or p35S:MYC2 transgenic line (Supplemental Fig. S12).

Figure 8.

Figure 8.

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ALMT1-mediated malate exudation from roots participate in the COI1-mediated Al exclusion from root tips. A, Expression of Al resistance-related genes in Al-exposed roots of wild-type and coi1-2 mutant seedlings. B, Malate exudation from Al-exposed roots of wild-type seedlings and the coi1-2 mutant. The 5-d-old seedlings were exposed to 0 and 10 µmAlCl3, and the exudates were collected at 6 h and 24 h after exposure of roots to AlCl3. C, Morin staining of the Al accumulation in the root tips of wild-type and coi1-2 mutant seedlings. Bar = 100 nm. D, Al content in Al-exposed roots of wild-type and coi1-2 mutant seedlings. E, Fractional analysis of Al concentration in the cell wall and cell sap (symplast) of Al-exposed roots of wild-type and coi1-2 mutant seedlings. A and C to E, the 6-d-old seedlings were exposed to 0 and 10 µmAlCl3 for 6 (A) or 24 h (C–E). Values represent means ± sd (A, n = 3; D and E, n = 4). A, UBQ1 was used as the reference, and a nontreated wild type was used as the sample control. *, **, and *** in A, B, D, and E indicate a significant difference between wild-type seedlings and the coi1-2 mutant seedlings at P < 0.05, P < 0.01, and P < 0.001, respectively.

DISCUSSION

Phytohormones are key regulators of root growth and development and root growth plasticity under various environmental stress conditions (Satbhai et al., 2015), including Al stress (Yang et al., 2014). For instance, ethylene, auxin, and cytokinin have been reported to regulate the Al-induced inhibition of root growth (Massot et al., 2002; Sun et al., 2010; Yang et al., 2014). Ethylene can affect either auxin biosynthesis (Yang et al., 2014) or auxin transport (Sun et al., 2010) in the root apex in response to Al stress. JA as one of the key phytohormones has been realized as a defense and growth hormone, and as such modulates numerous processes related to development and stress responses (Wasternack and Hause, 2013). Here we found that Al stress induced the up-regulation of JA receptor COI1 in root tips after 2-h exposure (Fig. 2, A–C and E; Supplemental Fig. S4). After 1-h Al treatment, an increased JA-Ile level, which has been shown to be necessary for COI1-mediated degradation of JAZ1 and JA signaling (Wasternack and Hause, 2013), was observed in roots (Fig. 2, G and H). However, both JA-Ile and total JA levels in roots decreased after 6-h Al exposure (Fig. 2, G and H). These results strongly suggested that the Al-induced JA-Ile production was involved in the early stage of COI1-mediated JA signaling to control root growth under Al stress.

In short-term Al exposure, Al first targets the epidermis and cortex of the root and rapidly (within a few minutes) binds to the negatively charged carboxyl groups of pectin in cell wall (Horst et al., 2010). The initial effect of toxic Al is a deleterious effect on cell expansion resulting in a decrease in the elemental elongation rate, one possibility is through affecting the loosening of the cell walls (Kopittke et al., 2015). Here we observed that the Al-induced JA signaling increase in root tips occurred after 2-h Al exposure (Supplemental Fig. S4), suggesting that JA signaling might not mediate the fast response of root growth inhibition under Al stress.

In plants, the JA signal generally acts cooperatively with other plant hormones. For example, JA and ethylene can act either antagonistically or synergistically in controlling stress responses or development (Zhu, 2014; Zhu and Lee, 2015). The JA-ethylene interaction has been linked mainly through ethylene-activated transcription factors EIN3 and its close homolog, EIL1 (Lehman et al., 1996; Chang et al., 2013; Song et al., 2014; Zhang et al., 2014b). Zhang et al. (2014a) reported that under cadmium or salt stresses, ethylene and JA affect the crosstalk between stress-initiated nitrate allocation to roots, which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. In this study, ethylene was also found, as an upstream signal, to participate into the regulation of Al-induced JA signaling in the root apex and JA regulated root growth inhibition in response to Al stress (Fig. 3). Therefore, this study provides further evidence to show JA regulates various bio- or abiotic stresses through the interaction with ethylene.

JA-regulated plant growth and development through the interaction with auxin has been reported. For instance, JA repressed hook formation by repressing HOOKLESS1 expression (Zhang et al., 2014b), which has been identified as a key modulator of auxin distribution and responses to regulate hook development and as a direct target gene of EIN3/EIL1 (Roman et al., 1995; Lehman et al., 1996; Chang et al., 2013). In roots, JA was shown to reduce primary root growth and promote lateral root density, while this response was impaired in the mutant of yuc8 and yuc9, two members of the YUCCA (YUC) gene family involved in auxin biosynthesis. This provides evidence that the JA signaling pathway is linked to auxin homeostasis through the modulation of YUC8 and YUC9 gene expression (Hentrich et al., 2013). However, auxin signaling does not regulate JA signaling to control Al-induced root growth, nor is JA involved in auxin-regulated root growth in response to Al stress, suggesting auxin and JA act independently under Al stress.

Furthermore, auxin was found to mediate Al-induced root-growth inhibition independent of the ALMT1-mediated malate exudation in roots (Yang et al., 2014). While COI1-mediated JA signaling was involved in the regulation of ALMT1-mediated malate exudation and thus exclusion of Al from roots (Fig. 8). However, MYC2 itself seems not to directly regulate ALMT1 expression and malate exudation from roots under Al stress (Supplemental Fig. S12), suggesting a redundant role of MYC2 with other JA signaling components. In addition, the possibility of COI1 in the regulation of ALMT1 through affecting its protein activity cannot be ambiguously ruled out, since the ALMT1 expression was only mildly affected in coi1-2.

Alterations of cellular growth processes by Al often induce swellings of root apices and root-hair tips (Jones and Kochian, 1995). This phenomenon has been attributed to interactions of Al with the cytoskeleton, supposedly interfering with its structure and function (Delhaize and Ryan, 1995; Jones and Kochian, 1995). Several studies have revealed that Al can cause MT depolymerization (Macdonald et al., 1987; Sasaki et al., 1997; Blancaflor et al., 1998; Sivaguru et al., 1999, 2003). In maize, Sivaguru et al. (1999) reported that short-term (1 h) Al treatment completely depolymerized MTs in cells of the outermost cortex file of DTZ, the most sensitive maize root region to Al toxicity (Sivaguru and Horst, 1998), and this lesion expanded to most of the epidermis cells and to many root periphery cells of the proximal part of the TZ after longer exposures of root apices to Al. Similarly, in Arabidopsis, the cortical MTs in the distal elongation zone were rapidly (30 min) depolymerized by Al treatment (Sivaguru et al., 2003). Considering the rapid influence of Al on the MTs, the question remains as to how Al affects the MTs. It has been speculated by Horst et al. (1999) that Al affect root growth of maize through interaction with the cell wall- plasma membrane-cytoskeleton continuum. The rapid binding of Al to cells of the epidermis and outer cortex may simultaneously induce the transfer of putative signals. It has been suggested that the rapid Al-induced changes to cytosolic Ca2+ can mediate the polymerization of MTs (Sivaguru et al., 2003). In maize root apices, ethylene was found to depolymerize MTs in the cells of the inner cortex (Baluška et al., 1993). To our knowledge, this study is the first reporting that JA depolymerizes MTs, while it appears that MTs in the coi1-2 mutant were more bundled compared to the wild-type seedlings (Fig. 7A). Al obviously depolymerized the MTs in the DTZ of the wild-type plants while this depolymerization was less in the coi1-2 mutant (Fig. 7A), providing evidence that JA is involved in Al-induced depolymerization of MTs. This was also supported by the transcriptome and qRT-PCR analysis of expression of several MT organization-related genes, which was higher in coi1-2 mutant than wild type in response to Al stress, including MAP65-2 (MICROTUBULE-ASSOCIATED PROTEIN) and MAP65-3 (Fig. 6). MAP65-2 and MAP65-3 have been reported to play important roles in organizing cortical MT array (Caillaud et al., 2008; Lucas et al., 2011; Lucas and Shaw, 2012). The disruption drug oryzalin dramatically reduced root growth in wild-type plants, while this inhibition was less in the coi1-2 mutant. This phenomenon became more obvious in Al-exposed plants, where oryzalin clearly enhanced the Al-induced root growth inhibition in wild-type plants but not in coi1-2 mutant unless its concentration reached 70 nm (Fig. 7B). Taken together, these results strongly suggest that COI1-regulated JA signaling participates in the regulation of Al-induced root growth inhibition through depolymerizing the MTs in the root apex TZ.

In conclusion, the role of COI1-mediated JA signaling in the regulation of Al-induced root growth inhibition is presented as a schematic model depicted in Figure 9. According to this model, the COI1-mediated JA signaling controls the Al-induced inhibition of root growth through the regulation of MT polymerization. This process is controlled by ethylene independent of auxin signaling affecting root growth via cell wall modification. The Al-induced JA signaling in the root apex also participates in the regulation of ALMT1-mediated malate exudation from roots and thus Al exclusion by modulating ALMT1.

Figure 9.

Figure 9.

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Schematic representation of COI1-mediated JA signaling in the root apex modulating root growth inhibition in response to Al stress. COI1-mediated JA signaling is involved in the Al-induced root-growth inhibition through regulation of cortical MT polymerization in the root apex. This process is controlled by ethylene and independent of auxin signaling, which has been shown to control Al-induced root growth inhibition through the regulation of cell wall modification-related genes (Yang et al., 2014). In addition, ALMT1-mediated malate exudation from roots and thus Al exclusion from roots in response to Al stress are regulated by COI1-mediated JA signaling but not MYC2.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Col-0; mutant lines aos (SALK_017756), coi1-2 (Chen et al., 2011), myc2-2 (Chen et al., 2011), yuc1D (Zhao et al., 2001), slr-1 (Fukaki et al., 2005; Vanneste et al., 2005), eto1-2 (He et al., 2011), ein3-1eil1-1 (Alonso et al., 2003; He et al., 2011), and arf7arf19 (Okushima et al., 2007); and transgenic lines p35S:MYC2 (Zhai et al., 2013), pCOI1:COI1-VENUS (Larrieu et al., 2015), DR5rev:GFP (Ding and Friml, 2010), TAA1:GFP-TAA1 (Stepanova et al., 2008), pCOI1:GUS (Chen et al., 2011), pMYC2:GUS (Chen et al., 2011), and GFP-MAP4 (Rosero et al., 2013) were used in this study. Seedlings were grown hydroponically as described by Yang et al. (2014) at pH 5.0 in a growth chamber with a 16-h-light/8-h-dark cycle at 22°C.

Treatments and Root Growth Analysis

For root growth experiments, the seeds were sown onto mesh floating on MGRL nutrient solution (Fujiwara et al., 1992) containing Al (total [AlCl3] 0–8 µM, pH 5.0) or Al plus MeJA, ACC, NAA, PEO-IAA (gifts from Ken-ichiro Hayashi), or oryzalin (pH 5.0) for 7 d. The solution was renewed every 2 d. At day 7, the roots were scanned and primary root length was calculated with ImageJ software.

For short-term treatments, the seedlings were pregrown in nutrient solution for 6 d and then transferred to different treatment solutions containing either 10 or 25 µm AlCl3.

Confocal Microscopy

Imaging was performed on an LSM-700 or LSM-780 laser-scanning confocal microscope (Zeiss). For visualization of the expression of pCOI1:COI1-VENUS, DR5rev:GFP, TAA1:GFP, or GFP-MAP4 in roots, after treating the 6-d-old plants with chemicals for 3 or 6 h, roots were mounted with propidium iodide. For visualization of morin-stained Al in the root tips, the excised roots were stained with 100 μm morin in MES buffer (pH 5.5) for 1 h with gentle shaking; after washing with MES buffer, the green Al-morin fluorescence signal was observed.

RNA Isolation and qRT-PCR

Approximately 500 6-d-old seedlings were treated with AlCl3 and shock frozen in liquid nitrogen after harvest. Total RNA was isolated using the RNeasy PlantMini Kit (Qiagen) following the manufacturer’s protocol, and first-strand cDNA was synthesized from 1 μg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) following the manufacturer’s protocol. qRT-PCR was performed using the CFX Connect Real-Time System (Bio-Rad) with FastStart Universal SYBR Green Master (Rox; Roche). Samples for qRT-PCR were run in three biological replicates and two technical replicates. For the normalization of gene expression, the ubiquitin gene UBQ1 (AT3G52590) was used as an internal standard and the nontreated wild type was used as a sample control. Primers were designed using Primer 5 software, and the specifications of the primers of the genes studied are given in Supplemental Table S4.

RNA-Seq Analysis

The RNA-seq analysis was performed by BGI Tech. Approximately 500 seedlings (6 d old) of both the wild-type Col-0 and coi1-2 mutant line were exposed to a 2% MGRL solution containing 0 or 10 μm AlCl3 (pH 5.0). After 6 h, the roots were sampled and RNA was isolated. The total RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads (for eukaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments (about 200 bp). Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H, and DNA polymerase I were added to synthesize the second strand. The double-strand cDNA is purified with magnetic beads. End reparation and 3′-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR amplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library. The library products are ready for sequencing via Illumina HiSeqTM 2000 or other sequencer when necessary. The raw reads were obtained by transferring the original image data produced by the sequencer into sequences by base calling. The raw reads were then cleaned by removing the low-quality reads and/or adaptor sequences. The clean reads with high-quality sequences were mapped to reference sequences and/or the reference gene set using SOAPaligner/ SOAP2 (Li et al., 2009). No more than two mismatches were allowed in the alignment.

The gene expression levels were calculated using the reads per kilobase per million reads method (Mortazavi et al., 2008). Differentially expressed genes (DEGs) within samples were screened with a modified method according to Audic and Claverie (1997). Statistical analysis of DEGs was conducted using the P value and the false discovery rate (FDR). The P value corresponds to the differential gene expression test. FDR is a method used to determine the threshold of P values in multiple tests (Benjamini and Yekutieli, 2001). FDR ≤ 0.001 and absolute value of log2 ratio ≥ 1 were used as the threshold to judge the significance of gene expression differences. The functional categories of the DEGs were BLASTed against the nonredundant GenBank, KEGG Pathway, and UniProt protein databases by the Gene Ontology annotation.

GUS Staining

For histochemical analysis of GUS activity, the seedlings were immersed into the staining solution consisting of 0.1 m sodium phosphate buffer (pH 7.0), 2 mm potassium ferri- and ferrocyanide, 0.1% Triton X-100, and 2 mm X-glucuronide at 37°C overnight. The samples were observed and photographed with an Olympus BX53 microscope equipped with an Olympus DP72 camera system.

JA and JA-Ile Analysis

Approximately 200 mg of freeze-dried root materials from 6-d-old seedlings were used for JA and JA-Ile analysis by LC-MS/MS. Four biological replicates were measured for the analysis. The freeze-dried roots were ground to a fine powder with a bead mill and 1 mL of pure methanol containing the labeled internal standards (ISTD) for D6-(± )-jasmonic acid and N-[D6-(± )-jasmonyl]-(l)-Ile (HPC Standards) was added and samples were shaken additionally at 30 L/s for 1 min. The samples were centrifuged at 20,000g (4°C), the supernatant was nearly dried with a vacuum concentrator and resuspended in 300 µL 70% methanol and 0.1% formic acid, and subsequently centrifuged at 20,000g. The supernatant (5 µL) was separated with a 1200 SL HPLC (Agilent Technologies) on a Accucore Phenyl Hexyl column (Thermo Fisher Scientific) with mobile phase A consisting of water with 0.1% formic acid and mobile phase B consisting of methanol with 0.1% formic acid. With a flow rate of 0.3 mL/min the gradient was 0 to 4 min 40% to 55% B, 4 to 6 min 55% to 100% B, 6 to 7 min 100% B, and 7 to 14 min 40%B. With this gradient JA and JA-Ile elute at 7 and 9 min with well-defined peaks, respectively. The masses specific for JA, JA-Ile, and the respective internal standard were quantified on a 6460 Triple Quad mass spectrometer (Agilent Technologies). Quantification was performed with the following transitions (JA 209.1–59.1, JA ISTD 215.2–59.2, JA-Ile 322.2–130, JA-Ile ISTD 328.2–130.1), and absolute amounts were calculated by comparison with the respective internal standard of known concentration.

Al Content Analysis

After treatment, roots were washed at least three times with double distilled water and excised and then transferred into a 0.45-μm unit of centrifugal filter (PALL) and centrifuged at 3,000g for 10 min at 4°C to remove apoplastic solution. The roots were then frozen at −80°C overnight. The root cell sap was obtained by thawing the samples at room temperature and then centrifuging at 22,000g for 10 min. The pellet was washed with 70% (v/v) ethanol three times and designated as the cell wall fraction. For the determination of Al in roots or root fractions, the samples were digested with concentrated 65% ultra-pure HNO3, and after approximate dilution, the Al concentration was determined by GFS-AAS.

Root Exudate Collection and Malate Analysis

The collection of root exudates and determination of malate were performed according to Kobayashi et al. (2007) with minor modifications. Briefly, the seeds were sown onto 10-mm squares of plastic mesh (25 plants) and allowed to grow for 4 d in 100% MGRL solution with 1% (w/v) Suc at pH 5.0. Then the 4-d-old seedlings were pretreated with 2% MGRL nutrient solution for 1 h and subsequently transferred to a 2-mL root exudation collection-medium containing 2% MGRL nutrients without or with 10 μm AlCl3. The root exudates were collected after 6-h and 24-h treatment. The amount of malate in the exudates was determined with the NAD/NADH cycling-coupled enzymatic method as described previously (Takita et al., 1999).

Statistical Analysis

Statistical analysis was performed using SAS 9.2 (SAS Institute). Means were compared using Student’s t test. Asterisks in the figures denote significant differences as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative database and the GenBank/EMBL databases under the following accession numbers: COI1 (AT2G39940, NM_129552), MYC2 (AT1G32640, NM_102998), SLR (AT4G14550, AF334718), YUC1 (AT4G32540, NM_119406), TAA1 (AT1G70560, NM_105724), EIN3 (AT3G20770, NM_112968), EIL1 (AT2G27050, BT003344), ARF7 (AT5G20730, NM_122080), ARF19 (AT1G19220, NM_101780), ALMT1 (AT1G08430, NM_100716), MATE (At1g51340, NM_104012), STOP1 (AT1G34370, NM_103160), ALS3 (AT2G37330, NM_129289), ALS1 (AT5G39040, NM_123266), and STAR1 (AT1G67940, NM_105464). RNA sequencing data analyzed in this study are available in the Gene Expression Omnibus database under accession number GSE83361.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Ken-ichiro Hayashi for PEO-IAA chemicals. We also thank Jiri Friml, Chuanyou Li, Jose Alonso, Joanne Chory, Marco Herde, and Laurent Laplaze for published materials.

Glossary

ACC

1-aminocyclopropane-1-carboxylic acid

DEG

differentially expressed gene

DTZ

distal transition zone

FDR

false discovery rate

ISTD

internal standard

JA

jasmonate

MeJA

methyl jasmonate

MT

microtubule

NAA

1-naphthaleneacetic acid

PEO-IAA

a-(phenylethyl-2-one)-indole-3-acetic acid

qRT-PCR

quantitative real-time PCR

TZ

transition zone

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