Download Jasmonic Acid Enhances Al-Induced Root Growth

Jasmonic Acid Enhances Al-Induced Root
Growth Inhibition1[OPEN]
Zhong-Bao Yang 2, Chunmei He 2, Yanqi Ma, Marco Herde, and Zhaojun Ding*
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, College of
Life Science, Shandong University, Jinan 250100, People’s Republic of China (Z-B.Y., C.H., Y.M., Z.D.); and
Department of Molecular Nutrition and Biochemistry of Plants, Institute of Plant Nutrition, Leibniz University
Hannover, 30419 Hannover, Germany (M.H.)
ORCID IDs: 0000-0003-0744-0853 (C.H.); 0000-0003-2804-0613 (M.H.); 0000-0003-0218-5136 (Z.D.).
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 COI1mediated 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
1
This work was supported by grants from the Ministry of Science
and Technology of China (2015CB942901), the National Natural Science Foundation of China (Projects 31470371, 31670275, 31400227,
and 31670259), Shandong Provincial Funds for Distinguished Young
Scholars (2014JQ201408), and the Shandong Provincial Natural Science Foundation project (ZR2014CQ021).
2
These authors contributed equally to the article.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Zhaojun Ding ([email protected]).
Z.D. and Z.-B.Y. conceived the study and designed and supervised
the experiments; Z.-B.Y., C.H., Y.M., and M.H. performed most of the
experiments and analyzed the data; Z.-B.Y. and Z.D. wrote the article;
all authors discussed the results and commented on the article.
[OPEN]
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www.plantphysiol.org/cgi/doi/10.1104/pp.16.01756
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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 mM Al reduced root growth of
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Jasmonate Controls Al-Regulated Root Growth
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 ethylenemeditated 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 ethyleneenhanced apical hook curvature, while, conversely,
EIN3 interacts with and represses MYC2 to inhibit JAinduced 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 (PINFORMED) 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 mM 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).
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
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Yang et al.
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 mM 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).
In addition, the expression of MYC2 was also strongly
induced under 10 mM 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 mM 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).
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 mM 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 mM AlCl3 or 1 mM LaCl3, 3.5 mM CdCl2, 2 mM 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 mM 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 6 SD (n $ 30).
the root tips was examined in response to Al stress
using the transgenic line pCOI1:COI1-VENUS. Either
10 or 25 mM 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
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-1carboxylic 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).
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
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Jasmonate Controls Al-Regulated Root Growth
Figure 2. Al enhances JA signaling in the root
apex. A and B, Expression of pCOI1:COI1VENUS 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 mM AlCl3 for
3 h. Bar = 100 mm. 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 mM AlCl3 for 0,
3, and 6 h. Bar = 100 mm. 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 mM AlCl3 for 0, 1, 3, and 6 h. Values
in E to H represent means 6 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.
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 a-(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).
NAA application significantly enhanced while PEOIAA 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
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Yang et al.
Figure 3. The Al-induced expression of JA signaling in the root apex and the JA-mediated rootgrowth 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 mM AlCl3 for 3 h in the presence or absence of 1 mM ACC or 1 mM AVG, respectively.
Bar = 100 mm. 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 6 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 mM 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 mM 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 ACCenhanced root growth inhibition in response to Al
stress. The wild-type seedlings and coi1-2 mutant
were exposed to 0 and 6 mM 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 6 SD (n = 30).
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).
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 downregulated, 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 downregulated 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
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Jasmonate Controls Al-Regulated Root Growth
the functional categories cellular protein modification, RNA processing, MT-based process, and
transport (Supplemental Fig. S11B; Supplemental Tables
S2 and S3).
JA Mediates Root Growth Sensitivity to Al Stress through
Stabilizing the Cortical Microtubule in Root Apex TZ
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:COI1VENUS 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 mM AlCl3 with or without 100 nM NAA, or 100 nM
L-kynurenine, or 10 mM PEO-IAA for 3 h. Bar = 100 mm. 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 6 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 mM AlCl3 for 3 h. Bar =
100 mm.
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 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 JAregulated 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 mM AlCl3 for 6 h obviously depolymerized MTs in root apex TZ of wildtype plants, while a reduced depolymerization of
MTs was found in the coi1-2 mutant. Consistent
with the Al effects, both MeJA and oryzalin, a MTdepolymerizing 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.
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+
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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 mM AlCl 3
with or without 2.5 n M NAA or 1 mM 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 6 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 mM AlCl 3 in the presence or absence of 0.5 n M 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 6 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 wildtype and mutant seedlings were exposed to 0 to 8 mM AlCl 3 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 6 SD (n = 30).
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 coi12 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).
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,
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Jasmonate Controls Al-Regulated Root Growth
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
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. Sixday-old wild-type and coi1-2 seedlings were exposed to 0 and
10 mM AlCl 3 for 6 h. UBQ1 was used as the reference, and a nontreated wild-type was used as the sample control. Values represent
means 6 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).
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;
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 mM AlCl 3 , 100 n M MeJA, or 100 n M oryzalin
for 6 h. Bar = 100 mm. 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 mM
AlCl 3 in the presence of 0 to 70 n M 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 6 SD
(n = 30).
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Yang et al.
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 mMAlCl3, 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 mMAlCl3 for 6 (A) or 24 h (C–E).
Values represent means 6 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.
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
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Jasmonate Controls Al-Regulated Root Growth
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 membranecytoskeleton 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 (MICROTUBULEASSOCIATED 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 COI1mediated 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. 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, ALMT1mediated 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.
Plant Physiol. Vol. 173, 2017
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Yang et al.
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
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.
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
mM, 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 mM AlCl3.
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.
Confocal Microscopy
JA and JA-Ile Analysis
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 mM 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.
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-(6 )-jasmonic acid and N-[D6-(6 )-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 mL 70% methanol and 0.1% formic acid, and subsequently centrifuged at
20,000g. The supernatant (5 mL) 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.
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 mg 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 mM 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 39-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
GUS Staining
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-mm 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 280°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
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Jasmonate Controls Al-Regulated Root Growth
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 mM 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).
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.
Received November 16, 2016; accepted December 6, 2016; published December
8, 2016.
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.
Supplemental Figure S1. Root growth of wild-type, myc2-2, and p35S:
MYC2 seedlings in response to Al stress.
Supplemental Figure S2. Root growth of wild-type and coi1-2 mutant
seedlings in response to varied pH solutions.
Supplemental Figure S3. Root growth of wild-type plants under Al stress.
Supplemental Figure S4. Time response of pCOI1:COI1-VENUS in the
epidermis and cortex of root tips to Al.
Supplemental Figure S5. Expression of pCOI1:COI1-VENUS in the epidermis and cortex of root tips in response to different metal ions stress.
Supplemental Figure S6. Expression of pCOI1:COI1-VENUS in the epidermis and cortex of root tips to varied pH solutions.
Supplemental Figure S7. Tissue expression of pCOI1:GUS in response to
Al stress.
Supplemental Figure S8. Expression of pCOI1:GUS in the root apex in the
presence of ACC or AVG with or without Al treatment.
Supplemental Figure S9. Expression of pCOI1:COI1-VENUS in the epidermis of root tips of wild-type and ein3eil1-1 mutant seedlings.
Supplemental Figure S10. Expression of the TAA1:GFP and DR5rev:GFP
transgene in the root apex cortex of Al-subjected wild-type and coi1-2
seedlings.
Supplemental Figure S11. Transcriptomic analysis of the response to Al
exposure.
Supplemental Figure S12. Expression of ALMT1 in roots and malate exudation from roots in wild-type, myc2-2 mutant, and p35S:MYC2 transgenic line seedlings in response to Al.
Supplemental Table S1. Comparison of the differentially expressed genes
in roots of wild-type and coi1-2 exposed to Al stress.
Supplemental Table S2. Up-regulated genes in roots of Al-exposed coi1-2
mutant.
Supplemental Table S3. Down-regulated genes in roots of Al-exposed
coi1-2 mutant.
Supplemental Table S4. List of genes and specific primer pairs used for
qRT-PCR.
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