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physiological importance of IAMT1 in plant growth and physio-
logical responses to environmental cues [13e16]. Qin et al. (2005)
demonstrated that the RNAi silencing mutants iamt1 (iamt1-RNAi)
showed high-auxin phenotypes. In contrast, the T-DNA insertion
mutants of iamt1 (iamt1-1, salk_072125) did not show obvious
developmental phenotypes. Therefore, they assumed that products
of a truncated transcript of IAMT1 may still have methyltransferase
activity [16]. Interestingly, recent studies demonstrated that iamt1-
1 mutants exhibited slightly faster opening of the apical hook,
impaired gravitropism of hypocotyls, and tolerance to high-
temperature male sterility [14,15], although no significant reduc-
tion in IAA levels was observed [15]. The reasons for the differences
between the phenotype of iamt1-RNAi and iamt1-1 are not clear.
The dramatic high-auxin phenotypes of iamt1-RNAi might be
caused by simultaneous repression of additional members of the
SABATH family, a group of carboxyl methyltransferase genes
Phenylacetic acid (PAA) is another naturally occurring auxin
widely distributed in plants [17,18], but its physiological signifi-
cance is yet to be demonstrated [18]. PAA is generally less effective
than IAA except for the promotion of lateral root formation in peas
[19], but endogenous amounts of PAA are higher than those of IAA
in various plant species [17,20]. PAA and IAA regulate various genes
via the same auxin signaling pathway and are assumed to share the
same regulatory roles as auxins [17,21]. A distinctive difference is
that PAA does not undergo polar transportation [17]. Based on this
evidence, we recently proposed a model wherein IAA acts as a cell
to cell communicator and PAA may play a role in the maintenance
of steady-state auxin levels required for the preservation of cellular
activity in plants [22]. More recently, we demonstrated that the
GH3 family alters the ratio of IAA and PAA in Arabidopsis [23].
These observations suggest that various IAA metabolic enzymes
may also regulate the endogenous levels of PAA in plants.
light for 6e8 h at 22 ꢀC, and were cultivated in the dark for 3 days
and then for 12 h after rotating the plate 90ꢀ.
2.2. IAMT1 protein preparation in E. coli
DNA fragments of IAMT1 and its homologs were amplified by
PCR using genomic DNA from Arabidopsis seedlings as a template
using gene-specific primer pairs listed in Table S1. A cDNA of the
IAMT1 gene was cloned into the pCold-TF DNA vector (Takara Bio,
Kusatsu, Japan) using the In-Fusion system. The E. coli strain BL21
star (DE3) was transformed with the pCold-TF:IAMT1 plasmid. The
transformants were incubated at 37 ꢀC in TB medium with 50
mL carbenicillin. Protein expression was induced by adding iso-
propyl -D- -thiogalactopyranoside at a final concentration of
mg/
b
L
1 mM. After incubation at 15 ꢀC for 24 h, the cells were collected by
centrifugation. The cells were resuspended in sodium phosphate
buffer (50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole,
pH 7.0) and sonicated to get the cell extract. The TF-fused IAMT1
proteins were purified from the cell extract using TALON metal
affinity resin (Takara Bio, Kusatsu, Japan). The TF-fused proteins of
IAMT1 homologs were also prepared in a similar manner. The
trigger factor region was removed by the Factor Xa following the
manufacturer’s instructions and purified proteins were condensed
by ultrafiltration using an Amicon Ultra centrifugal unit (Merck
Millipore, Burlington, MA).
2.3. Enzyme assays
Enzymatical assays with IAMT1 and homolog proteins were
performed in a 50
protein, 50 mM Tris-HCl at pH 7.5, 1 mM IAA or PAA, and 1 mM S-
adenosyl-
-methionine (SAM). Enzymes were boiled at 100 ꢀC and
used as controls. The assays were initiated by the addition of SAM,
maintained at 25 ꢀC for 4 h, and stopped by the addition of 80
L of
mL volume containing 10 mg purified recombinant
L
In this study, we investigated the role of Arabidopsis IAMT1 in
auxin metabolism. We demonstrated that IAMT1 can convert not
only IAA to MeIAA but also PAA to PAA-methyl ester (MePAA)
in vitro. However, overexpression of IAMT1 decreased the amounts
of IAA, but not PAA, in root tips of Arabidopsis. We found that
neither alteration of auxin levels nor obvious phenotypes were
observed in the CRISPR/Cas9-based null mutants of iamt1 under
our growth conditions. Our results suggest that IAMT1 exclusively
regulates the levels of IAA in vivo and that members of the SABATH
family, a group of carboxyl methyltransferases, may also regulate
IAA levels in Arabidopsis.
m
n-hexane. MeIAA and MePAA were extracted by mixing the liquid
and the upper layers were collected. These samples were analyzed
using the LECO Pegasus HT gas chromatography (GC)-mass spec-
trum system (LECO, St. Joseph, MI). An SGE BPX35 column (Shi-
mazu, Kyoto, Japan) was used with helium as the carrier gas at a
flow rate of 1 mL minꢁ1. The GC program was as follows: 50 ꢀC for
2 min, ramp to 320 ꢀC at 30 ꢀC minꢁ1, followed by a 3 min hold at
320 ꢀC. Reaction products were determined by comparison of GC
retention time and mass spectrum patterns with those of authentic
standards. For the calculation of specific activities, 10
recombinant protein was reacted with 100 M IAA or PAA, 1 mM
SAM, and 50 mM Tris-HCl, at pH 7.5 and 40 ꢀC for 30 min at a 50
volume. The reactions were initiated by the addition of SAM and
stopped by the addition of 50 L acetonitrile. The reaction products
were extracted with 100 L of n-hexane containing 1 ng/ L ethyl
mM of IAMT1
m
2. Materials and methods
m
L
2.1. Plant materials and growth conditions
m
m
m
Arabidopsis thaliana ecotype Col-0 was used as the wild type
(WT) plant. Arabidopsis seeds were stratified at 4 ꢀC for 2 d in the
dark. Seedlings were grown vertically on Murashige and Skoog
(MS) agar medium with 1% sucrose under 16 h light condition at
23 ꢀC. Transgenic Arabidopsis pMDC7:IAMT1 were used for exper-
PAA (EtPAA) as an internal standard and were analyzed by GC-MS.
2.4. Generation of IAMT1ox plants
iments. For
b
-estradiol (ER) treatment, seeds were germinated and
The full-length IAMT1 cDNA was provided by RIKEN BioResource
Research Center, Japan. The ORF of IAMT1 was amplified by adapter
PCR from the RAFL cDNA clone using gene-specific primers (IAMT1-
F and IAMT1-R) and adaptor attB primers (adaptor attB1 and
adaptor attB2). The IAMT1 fragment was inserted into the pDONR/
Zeo vector using the BP clonase II reaction and then this entry clone
was transferred into the pMDC7 vector [24] by the LR clonase II
reaction (Invitrogen, Waltham, MA). Arabidopsis WT plants were
transformed with the resulting construct, pMDC7:IAMT1, using the
floral dip method with the Agrobacterium tumefaciens GV3101
pMP90 strain.
grown on MS agar medium containing ER (2
mM) for 4 days. MS
medium with 0.1% DMSO was used as a mock treatment. For
quantitative RT-PCR (qRT-PCR) and auxin metabolite analysis,
seedlings were grown vertically on MS agar medium for 10 days,
transferred to MS media, underwent shaken cultured at 100 rpm
for 2 d, and were then cultured for another 2 days in the presence of
ER (2
mM final concentration) or the mock solution. After ER
treatment, plants were collected, freeze-dried, and kept at ꢁ80 ꢀC
until use. For the gravitropic response test, the WT and iamt1-c
seeds were stratified at 4 ꢀC for 2 days in the dark, exposed to
Please cite this article as: E. Takubo et al., Role of Arabidopsis INDOLE-3-ACETIC ACID CARBOXYL METHYLTRANSFERASE 1 in auxin metabolism,
Takubo, Eiko
