Jasmonic Acid Enhances Al-Induced Root Growth Inhibition

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.

Figure 1.

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).

Figure 2.

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.

Figure 3.

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).

Figure 4.

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.

Figure 5.

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).

Figure 6.

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).

Figure 7.

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).

Figure 8.

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.

Figure 9.

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.