Download Identification of a Cis-Acting Element of ART1, a

Identification of a Cis-Acting Element of ART1,
a C2H2-Type Zinc-Finger Transcription Factor
for Aluminum Tolerance in Rice1[OA]
Tomokazu Tsutsui, Naoki Yamaji, and Jian Feng Ma*
Institute of Plant Science and Resources, Okayama University, Chuo 2–20–1, Kurashiki 710–0046, Japan
Rice (Oryza sativa) is one of the most aluminum (Al)-tolerant species among small-grain cereals. Recent identification of a
transcription factor AL RESISTANCE TRANSCRIPTION FACTOR1 (ART1) revealed that this high Al tolerance in rice is
achieved by multiple genes involved in detoxification of Al at different cellular levels. ART1 is a C2H2-type zinc-finger
transcription factor and regulates the expression of 31 genes in the downstream. In this study, we attempted to identify a cisacting element of ART1. We used the promoter region of SENSITIVE TO AL RHIZOTOXICITY1, an Al tolerance gene in the
downstream of ART1. With the help of gel-shift assay, we were able to identify the cis-acting element as GGN(T/g/a/C)V(C/
A/g)S(C/G). This element was found in the promoter region of 29 genes among 31 ART1-regulated genes. To confirm this cisacting element in vivo, we transiently introduced this element one or five times tandemly repeated sequence with 35S minimal
promoter and green fluorescent protein reporter together with or without ART1 gene in the tobacco (Nicotiana tabacum) mesophyll
protoplasts. The results showed that the expression of green fluorescent protein reporter responded to ART1 expression.
Furthermore, the expression increased with repetition of the cis-acting element. Our results indicate that the five nucleotides
identified are the target DNA-binding sequence of ART1.
Ionic aluminum (mainly Al3+) inhibits root elongation
at low concentrations by damaging the root cells functionally and structurally (Kochian et al., 2004; Ma, 2007;
Poschenrieder et al., 2008). However, some plant species
or cultivars have developed strategies to cope with Al
both internally and externally. Internal detoxification
is achieved by sequestration of Al into the vacuoles
and chelation with organic acids including citrate and
oxalate, which is seen in some Al-accumulating plants
such as hydrangea (Hydrangea macrophylla) and buckwheat (Fagopyrum esculentum; Ma, 2007). Several mechanisms for the external detoxification have been
proposed, but the most-studied one is the secretion of
organic acid anions from the roots in response to Al
stress in both monocots and dicots (Ma, 2005, 2007). The
organic acid anions secreted include oxalate, citrate,
and malate, depending on plant species. All of them are
able to chelate toxic Al, thereby detoxifying Al in the
rhizosphere (Ryan et al., 2001; Kochian et al., 2004; Ma,
1
This work was supported by a grant from the Ministry of
Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation, IPG–0006 to J.F.M.) and a Grant-in-Aid for Scientific Research (grant nos. 21780057, 21248009, and 22119002 to N.Y.,
J.F.M., and J.F.M., respectively) on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
* Corresponding author; e-mail [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:
Jian Feng Ma ([email protected]).
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.175802
2007; Poschenrieder et al., 2008). Genes responsible
for Al-induced secretion of malate (AL-ACTIVATED
MALATE TRANSPORTER1) have been identified in
wheat (Triticum aestivum; Sasaki et al., 2004), Arabidopsis (Arabidopsis thaliana; Hoekenga et al., 2006), and rape
(Brassica napus; Ligaba et al., 2006). Recently, the genes
involved in Al-induced secretion of citrate have also
been identified in barley (Hordeum vulgare; Furukawa
et al., 2007), sorghum (Sorghum bicolor; Magalhaes
et al., 2007), Arabidopsis (Liu et al., 2009), and maize
(Zea mays; Maron et al., 2010). All these genes encode a
citrate efflux transporter that belongs to the multidrug
and toxic compound extrusion family. So far, transporter for Al-induced oxalate secretion has not been
identified.
Rice (Oryza sativa) shows higher tolerance to Al than
other gramineous crops including maize, wheat, barley,
and sorghum (Ma, 2007). However, different from these
crops, which employ secretion of organic acid anions
as a main mechanism of Al tolerance, organic acid anion
secretion is not the major mechanism for high Al tolerance in rice because the amount of secretion is very
small (Ma et al., 2002). Recent identification of a transcription factor, AL RESISTANCE TRANSCRIPTION
FACTOR1 (ART1), revealed that high Al tolerance in
rice is achieved by multiple genes involved in detoxification of Al (Yamaji et al., 2009). ART1 is a C2H2-type
zinc-finger transcription factor, which is localized in the
nucleus of all root cells (Yamaji et al., 2009). ART1
regulates 31 genes, which are implicated in both internal
and external detoxification of Al at different cellular
levels. Several downstream genes regulated by ART1
have been functionally characterized. For example,
Plant PhysiologyÒ, June 2011, Vol. 156, pp. 925–931, www.plantphysiol.org Ó 2011 American Society of Plant Biologists
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Tsutsui et al.
SENSITIVE TO AL RHIZOTOXICITY1 (STAR1) and
STAR2 encode an ATP-binding and a transmembrane
domain of a bacterial-type ATP-binding cassette transporter, respectively. The complex between STAR1 and
STAR2 transports UDP-Glc, which is supposed to be
used for modification of the cell wall although the
exact mechanism remains unknown (Huang et al.,
2009). Recently, Nrat1 (Nramp Al transporter 1), belonging to Nramp (natural resistance-associated macrophage protein) family, was reported to be required
for Al tolerance in rice (Xia et al., 2010). Nrat1 is
localized at the plasma membranes of all root cells and
functions as a transporter for uptake of trivalent Al ion
in rice, which is required for a prior step of final Al
detoxification through sequestration of Al into vacuoles (Xia et al., 2010). The expression of all these ART1regulated genes is specifically up-regulated by Al
(Yamaji et al., 2009; Xia et al., 2010). However, most
downstream genes have not been functionally characterized.
In this study, we investigated a cis-acting element of
ART1 by using gel-shift assay and transient expression
analysis. We have been successful in identification of cisacting element of ART1, which is present in the promoter
regions of 29 genes out of 31 genes regulated by ART1.
RESULTS AND DISCUSSION
Identification of a Cis-Acting Element on the STAR1
Promoter with Gel-Shift Assay
STAR1 is one of the downstream genes regulated by
ART1 (Yamaji et al., 2009). Previously, a yeast (Saccharomyces cerevisiae) one-hybrid assay showed that the
ART1 protein interacted with promoter regions of
STAR1 between 2436 and 2298 from the translation
start site (Yamaji et al., 2009), indicating the presence of
cis-acting element(s) in this region. To establish the
gel-shift assay system, we produced recombinant
ART1 protein using wheat germ cell-free protein synthesis system (Takai et al., 2010), and designed a probe
covering the region between 2446 and 2288 (STAR1
full) from start codon of STAR1 (Fig. 1A). A gel-shift
assay (in vitro) yielded a band at the size of ART1/
digoxigenin (DIG)-labeled probe complex (Fig. 1B,
lane 1). This band disappeared in the presence of
competitor (100- or 500-fold of non-DIG-labeled
STAR1 full probe; Fig. 1B, lanes 2 and 3). To confirm
that the band shift was caused by binding of ART1
protein, super shift assay with an anti-ART1 antibody
was performed. As a result, a band was detected with
larger Mr, which is thought to be the formation of
ART1/anti-ART1 antibody/DIG-labeled probe complex (Fig. 1B, lane 4). Presence of non-DIG-labeled
probe also weakened the band abundance with increasing concentration of the competitor (Fig. 1B, lanes
5 and 6). These results indicate that the cis-acting
sequence recognized by the ART1 protein is present in
the region between 2446 and 2288 from translation
start site of STAR1 gene, which is consistent with
Figure 1. Schematic diagram of the probes used for gel-shift assay and
identification of the DNA-binding region of the ART1 transcription
factor. A, Schematic diagram of the eight probes on the promoter of
STAR1 for the gel-shift assay. B, Identification of DNA-binding region of
ART1 on STAR1 promoter region. Lanes 1 to 3, Binding of ART1 protein
to STAR1 full probe (2446 to 2288) in the absence (lane 1) and
presence of 100 ng (lane 2) and 500 ng (lane 3) non-DIG-labeled probe
as a competitor. Lanes 4 to 6, Super gel-shift assay. The gel-shift assay
was performed in the presence of anti-ART1 antibody and with 0 (lane
4), 100 ng (lane 5), and 500 ng (lane 6) of non-DIG-labeled probe as a
competitor. The black and gray arrowheads indicate the position of
shifted band of ART1/DIG-labeled probe complex and ART1/DIGlabeled probe/anti-ART1 antibody complex, respectively. The white
asterisks indicate free probe signals.
previous findings obtained by yeast one-hybrid assay
(Yamaji et al., 2009).
To narrow this region, we designed two probes;
STAR1-1 (2507 to 2358 from the start codon) and
STAR1-2 (2368 to 2208) covering the region identified
above (Fig. 1A). Gel-shift assay showed that ART1 protein
was bound with STAR1-2 probe, but not with STAR1-1
(Fig. 2A). This is also supported by the presence of self
competitor of the STAR1-2 probe that inhibited the interaction between ART1 protein and STAR1-2 probe (Fig.
2A). These results indicate that the cis-acting sequence
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Plant Physiol. Vol. 156, 2011
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Identification of the ART1 Cis-Element
the candidate region should be between 2368 and
2288. To confirm this region, gel-shift assay with a
probe (STAR1-3, 2368 to 2288) was performed. STAR1-3
was able to be bound with ART1 protein and super
shifted in the presence of anti-ART1 antibody (Fig. 2B). To
further narrow the target region of STAR1 promoter, we
divided STAR1-3 region into three parts (Fig. 1A, probes
1–3). Gel-shift assay showed that only probe 2 (2358 to
2319) was able to bind ART1 protein, whereas probe
1 (2386 to 2347) and probe 3 (2330 to 2291) was not
able to bind ART1 protein (Fig. 2B). These results indicate
that the region between 2358 and 2319 in the STAR1
promoter contains cis-acting element of ART1. This is
further confirmed by competition and super shift experiments (Fig. 2C, lanes 5–8).
The candidate region was further narrowed by gelshift assay with a new probe (probe 4, 2368 to 2329).
A complex between probe 4 and ART1 protein was
detected at the similar size as that of probe 2 (Fig. 2C,
lane 1). Presence of non-DIG-labeled probe 4 inhibited
the formation of this complex (Fig. 2C, lane 2). Presence of ART1 antibody caused super shift of this
complex (Fig. 2C, lane 3). This result combined with
that from probes 1 and 2 indicated that the cis-element
Figure 2. Further identification of the DNA-binding site of the ART1. A,
Binding of ART1 protein to STAR1-1 probe (2368 to 2208) and STAR1-2
probe (2507 to 2358) on the STAR1 promoter in the absence or
presence of 500 ng non-DIG-labeled probes as a competitor. B, Binding
of ART1 protein to STAR1-3 probe (2368 to 2288), probe 1 (2386 to
2347), probe 2 (2358 to 2319), probe 3 (2330 to 2291) on the STAR1
promoter in the absence or presence of 500 ng non-DIG-labeled probes
as a competitor and/or anti-ART1 antibody. C, Binding of ART1 protein to
probe 4 (2368 to 2329), probe 2 (2358 to 2319) on the STAR1
promoter in the absence or presence of 500 ng non-DIG-labeled probes
as a competitor and/or anti-ART1 antibody. The black and gray arrowheads indicate the position of shifted band of ART1/DIG-labeled probe
complex and ART1/DIG-labeled probe/anti-ART1 antibody complex,
respectively. The white asterisks indicate free probe signals.
recognized by ART1 protein is present in the region
between 2368 and 2208 of STAR1 promoter region.
Since both STAR1 full (2446 to 2288) and STAR1-2
(2368 to 2208) probes were able to bind ART1 protein,
Figure 3. DNA-binding affinity of the ART1 protein to a 21-bp fragment
(2352 to 2329) of the STAR1 promoter. A, Scheme of probes with two
nucleotide substations each. Lowercase shows nucleotides substituted.
B, Binding of ART1 protein to STAR1 cis1 probes in the absence or
presence of 500 ng non-DIG-labeled mutated probes (STAR1 cis1 and
STAR1 M1-M9) as a competitor. Black arrowhead indicates the position
of shifted band of ART1/DIG-labeled STAR1-cis1 probe complex. The
white asterisks indicate free probe signals.
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Tsutsui et al.
probes with single mutation at this part (PM1–PM18;
Fig. 4A) and performed binding assay with ART1
protein (Fig. 4B). All substitutions of the nucleotide at
the position of 2343 and 2342 (probes PM1–6) resulted
in no binding with ART1 protein, indicating that these
nucleotides (GG) are critical for ART1 binding (Fig. 4B).
By contrast, substitution of T to C (probe PM9) at the
position of 2341 did not affect the binding to ART1
protein although substitution to G and A (probes PM7
and 8) at the same position resulted in weakened signal.
Substitution of C to A and G (probes PM10 and 11) at
the position of 2340 did not affect the binding to the
ART protein, whereas that to T (probe PM12) resulted
in loss of the binding (Fig. 4B). Probes PM13 and 14
Figure 4. Identification of cis-acting element of ART1. Characterization of the DNA-binding affinity of the ART1 protein to the STAR1
promoter with various single-nucleotide substitutions at the 6-bp region
(position 2343 to 2338). A, Probes with single nucleotide substitution
used for gel-shift assay. Lowercase shows mutation point. B, Binding of
ART1 protein to different mutated DIG-labeled probes (PM1–PM18).
Probe STAR1-cis1 was used as a positive control. Black arrowhead
indicates the position of shifted band of ART1/DIG-labeled probe
complex. The white asterisks indicate free probe signals.
region recognized by ART1 is located between 2358
and 2329 of STAR1 promoter region.
To identify the cis-acting element within this region,
we prepared the narrowest 30-bp probe STAR1-cis1
(2358 to 2329) and a series of probes (STAR1-M1 to
-M9) by substitution of two or six bases at the position
between 2352 and 2329 in STAR1-cis1 (Fig. 3A). The
binding ability of ART1 protein with these substituted
probes was examined by the competition assay.
STAR1-M1, -M2, or -M9 as a competitor was able to
inhibit the interaction of ART1 protein and STAR1-cis1
probe (Fig. 3B). In contrast, presence of probes
(STAR1-M5 and -M6) was not able to inhibit the
binding to ART1 protein (Fig. 3B). Probes of STAR1M3, -M4, -M7, and -M8 showed weak inhibitory effect
on ART1 binding. These results indicate that ART1
protein is mainly bound to the core region (GTCC)
between 2342 and 2339 of STAR1 promoter.
Since two substitutions for each probe were made in
above experiment, there is a possibility that the nucleotides of two sides are also included in the cis-acting
element. We therefore consider the putative cis-acting
element to be GGTCCT. We produced DIG-labeled
Figure 5. Transient assay of ART1 cis-acting element in tobacco
protoplasts. A, Schematic diagram of the reporter, effector, and internal
control plasmids used in transient expression analysis. B and C,
Expression level of effector gene (ART1) and reporter gene (GFP)
normalized by internal control gene (DsRed) in tobacco protoplasts
expressing no [p35S(246)], one (13), or five-repeated (53) ART1 cisacting element. The reporter plasmid was transfected with internal
control plasmid with or without effector plasmid. The expression level
of each gene was determined by quantitative RT-PCR. Expression level
relative to p35S(246) with ART1 effector is shown. Data are means 6
SD of three biological replicates.
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Identification of the ART1 Cis-Element
Table I. The number and position of ART1 cis-element in ART1 regulated genes
Position of ART1 cis-element indicate 2-kb promoter region from start codon of these genes.
Arabidopsis
Homolog
No. of ART1
Cis-Element
Position of ART1 Cis-Element
3
4
2178, 2584, 21,878
272, 21,212, 21,271, 21,345
3
2
2235, 2392, 21,038
2878, 21,499
4
2137, 21,094, 21,633, 21,894
Subtilisin-like Ser protease
7
SDD1/
At1g04110
Subtilisin-like
Ser protease
4
2296, 2653, 21,295,
21,330, 21,447, 21,890, 21,996
2215, 2564, 21,348, 21,964
At3g19640
At1g80830
At1g22400
Putative Mg2+ transporter
Nramp/Nrat1
UDP-glucuronosyl/
UDP-glucosyltransferase
OsALS1
3
3
5
2930, 2947, 21,773
2490, 21,364, 21,382
2313, 2330, 2509, 21,141, 21,845
2
2490, 21,679
STAR2
STAR1
GLOSSY1-like
2
5
4
2211, 21,168
2343, 2711, 2735, 2972
2387, 2442, 2486, 2699
MATE/OsFRDL2
LrgB-like
4
7
2556, 2805, 2968, 21,831
2138, 2225, 2589, 2638,
21,020, 21,225, 22,000
SAM-dependent
methyltransferase
Cytochrome P450
family protein
Nitrate reductase
3
239, 21,395, 21,659
1
2225
1
2218
Allyl alcohol
dehydrogenase
4
21,052, 21,288, 21,643, 21,700
2456, 2576, 22,003
2286, 2645, 21,109, 21,144
21,136, 21,303, 21,966
288, 2141, 21,591
2157, 2347, 2470, 21,146
2385, 2611, 21,655, 21,828
21,969
2187, 2543, 2702
2735, 21,018, 21,144,
21,559, 21,673, 21,860
21,463, 21,518, 21,549,
21,564, 21,787
21,949, 21,969
RAP-DB
Cell wall maintenance
and root elongation
Os01g0178300
Os01g0652100
None
At1g29050
Os01g0860500
Os03g0760800
At5g24090
At2g39540
Os04g0583500
EXP14/
At3g03220
At5g59810
Os09g0479900
Os10g0524600
Membrane protein
Os01g0869200
Os02g0131800
Os02g0755900
Os03g0755100
Os05g0119000
Os06g0695800
Os09g0426800
Os10g0206800
Os10g0578800
ALS1/
At5g39040
At2g37330
At1g67940
WAX2/
At5g57800
At1g51340
At1g32080
Description
OsCDT3
PMR5-like DUF231 domain
containing protein
Chitinase
Gibberellin-regulated
cystein-rich protein family
Expansin-A10
Metabolism and
detoxification
Os01g0716500
At5g10830
Os02g0186800
At2g30750
Os02g0770800
Os12g0227400
NIA1/
At1g77760
At3g03080
Unknown
Os01g0731600
Os01g0766300
Os01g0919200
Os03g0126900
Os03g0304100
Os04g0419100
Os04g0494900
Os07g0493100
Os07g0587300
At1g78780
None
None
None
At1g56320
None
At5g11420
None
None
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Unknown function DUF642 family
Nonprotein coding transcript
Hypothetical protein
3
4
3
3
4
4
1
3
6
Os11g0488100
None
Hypothetical protein
5
Os11g0490100
At1g67330
Uncharacterized plant-specific
DUF579 family
2
with substitution of C to A and T at the position of 2339
did not show ability to bind ART protein (Fig. 4B), but
probe PM15 with substitution to G showed strong
binding to ART1 protein. Finally, substitution of T to
A, C and G (probes PM16 to 18) resulted in stronger
signal of ART1/DIG-labeled probe complex compared
with STAR1-cis1 probe (Fig. 4B), indicating that this
nucleotide is not important for the recognition. Taken
together, all these results indicate that the cis-acting core
element of ART1 is GGN(T/g/a/C)V(C/A/g)S(C/G)
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Tsutsui et al.
sequences, localized between 2343 and 2339 from
translation start site of STAR1 gene. However, the
ART1-binding affinity of nucleotides with small character is weaker than those with large characters.
In Vivo Confirmation of ART1 Cis-Acting Element
To confirm the cis-acting element identified by gelshift assay, we introduced single or five repeated cisacting element fused with cauliflower mosaic virus
(CaMV) 35S minimal (246) promoter (Fang et al.,
1989) and GFP as a reporter gene (Fig. 5A) into tobacco
(Nicotiana tabacum) protoplasts. As an effector, ART1
genomic DNA fragment including 2-kb-long own
promoter was coexpressed as well as red fluorescent
protein gene DsRed-monomer under the control of
CaMV 35S promoter as an internal control (Fig. 5A).
Transient expression assay showed no expression of
ART1 in the protoplasts not introducing ART1 effector
plasmid, but showed similar expression level of ART1
in the protoplasts introducing ART1 effector plasmid,
irrespectively of repetition of the cis-acting element on
the reporter plasmid (Fig. 5B). In the absence of ART1
effector, the expression of GFP as a reporter gene was
at the level of background (Fig. 5C). In the presence of
ART1 effector, the expression of GFP was higher in
protoplasts expressing pentameric cis-acting element
than monomeric one (Fig. 5C). These results demonstrated that the ART1 interacts with the cis-acting
element and the transcriptional activation potential is
enhanced by the repetition of the cis-acting element.
Search of Cis-Acting Element in All Downstream Genes
Regulated by ART1
We used promoter region of STAR1 for identification
of cis-acting element of ART1 in above experiments.
Since ART1 regulates 31 genes (Yamaji et al., 2009), we
therefore, examined whether the cis-acting element
found in STAR1 also exists in other genes by searching
the promoters (up to 2 kb from start codon) of all these
genes. The cis-acting element was found in the promoter region of 29 genes out of 31 genes regulated by
ART1 (Table I). Furthermore, the cis-acting element is
present in the multiple positions of the same promoter
region. For example, in the promoter region of STAR1,
the cis-acting element was found in 2343, 2711, 2735,
and 2972. In STAR2 promoter, two copies of the cisacting element are present in the region 2211, 21,168,
and in Nrat1 promoter, there are three copies in the
2490, 21,364, and 21,382. These results suggest that
ART1 activates the expression of these genes by binding to the same target sequence.
The expression of ART1 is not induced by Al (Yamaji
et al., 2009), but the expression of the downstream
genes is up-regulated by Al. For example, the expression of STAR1 and STAR2 was induced by approximately 6- to 10-fold by a short exposure (2 h) to Al
(Huang et al., 2009). The expression of Nrat1 is also upregulated by approximately 8 times by 3 h exposure to
Al (Xia et al., 2010). These findings suggest that activation of ART1 by Al is required in vivo to induce the
expression of downstream genes. ART1 is localized at
the nuclei and this localization is unaffected by Al
(Yamaji et al., 2009). It would be an interesting topic to
next elucidate the signal transduction pathway from
Al perception to activation of ART1.
MATERIALS AND METHODS
Preparation of Recombinant ART1 Protein
Recombinant ART1 protein was produced by using cell-free protein
expression system. Protein synthesis of ART1 using wheat (Triticum aestivum)
germ extract was performed essentially according to the method described by
ENDEXT technology protocol (CellFree Sciences Co., Ltd). For construction of
ART1 vector for the cell-free system, the open reading frame of ART1 was
amplified by PCR from rice (Oryza sativa cv ‘Koshihikari’) root cDNA. Primer pairs used for amplification and introduction of restriction sites were
5#-ACTAGTATGGATCGCGACCAGATGACGAACA-3# and 5#-CCATGGTCACTTGTCACCATTCTCCTCCTG-3#. The plasmid was constructed with
PCR-amplified DNA fragments containing ART1 coding region cloned into
the SpeI and NcoI site of the wheat germ expression vector pEU3b (Sawasaki
et al., 2002). Two microliters of the high-purity plasmid DNA (1 mg/mL) was
added to a tube containing the transcription premix solution (CellFree
Sciences Co., Ltd) for transcription reaction. The mixture was incubated
at 37°C for 6 h in an incubator. After cooled down to the room temperature,
the mRNA mixture was resuspended and 10 mL of the mixture was added into
10 mL of WEPRO 3240 (wheat germ extract solution for translation reaction).
The mixed solution (20 mL) was then transferred to the bottom of the singlebreak strip well containing SUB-AMIX (206 mL) to form bilayers. After incubated at 15°C for 20 h, the mixture is used as recombinant ART1 protein for
gel-shift assay.
Gel-Shift Assay
Gel-shift assay was performed essentially according to the method described by DIG gel shift kit, second generation protocol (Roche Applied
Science). DNA-binding reaction was carried out in a 20-mL volume containing
25 mM HEPES-KOH (pH 7.6), 40 mM KCl, 0.1% (w/v) Nonidet P-40, 10 mM
ZnCl2, 1 mg Poly(d[I-C]), 1 ng DIG-labeled probe, and 63 ng recombinant
ART1 protein as prepared above. For super shift assay, 1 mL of anti-ART1
antibody (Yamaji et al., 2009) was added to the above solution. After incubating for 30 min at 20°C, the mixture was subjected to electrophoresis with an 8%
native PAGE.
The transfer of above DIG-labeled probes from native PAGE to a positivechanged nylon membrane was performed with a semidry blotting system (30
min at 144 mA). The transferred probes were then fixed to the membrane by
cross linking with UV light for 3 min at 120 mJ. The membrane was placed in a
washing buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% [w/v] Tween 20, pH 7.5)
for 5 min at room temperature. The washed membrane was slowly shaken for
30 min in 50 mL of blocking solution (1% [w/v] blocking reagent, 0.1 M maleic
acid, 0.15 M NaCl, pH 7.5). The membrane was then transferred to 10 mL of
anti-DIG antibody solution (0.75 mU/mL anti-DIG-AP, 1% [w/v] blocking
reagent, 0.1 M maleic acid, 0.15 M NaCl, pH 7.5) and slowly shaken for 30 min.
The membrane was washed twice each for 15 min in 100 mL of washing
buffer and equilibrated in 10 mL of detection buffer (0.1 M Tris-HCl, 0.1 M
NaCl, pH 9.5) for 5 min. The membrane was placed with DNA side facing up
on a hybridization bag and applied 1 mL of disodium 3-(4-methoxyspiro
{l,2-dioxetane-3,2#-(5#-chloro)tricyclo[3.3.1.13,7]decan}-4-yl) phenyl phosphate
working solution [1 mg/mL disodium 3-(4-methoxyspiro {l,2-dioxetane-3,2#(5#-chloro)tricyclo[3.3.1.13,7]decan}-4-yl) phenyl phosphate solution, 0.1 M TrisHCl, 0.1 M NaCl, pH 9.5] and incubated for 5 min at room temperature and
for a further 10 min at 37°C to enhance the luminescent reaction. Chemiluminescence signal was detected using LAS-1000 (FUJIFILM Corporation).
Isolation of Tobacco Leaf Protoplasts
The seeds of tobacco (Nicotiana tabacum cv ‘Petit Havana SR1’) were sown
on filter paper moistened with deionized water in a petri dish, and germinated
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Identification of the ART1 Cis-Element
at 25°C under a 16-h/8-h light/dark cycle. After 10 d, the seedlings were
transferred to a plastic mesh floating on a one-fifth Hoagland culture solution
in a 1.5-L plastic container. After a further 14-d growth, the plants were
transferred to a 3.5-L plastic pot (six plants per pot) containing one-fifth
Hoagland solution. The plants were grown in a growth chamber at 25°C under
a 16-h/8-h light/dark cycle.
For isolation of protoplast, young tobacco leaves were cut into small pieces
with razor and incubated in an enzyme mix solution (1% [w/v] cellulase
onozuka RS, 0.3% [w/v] macerozime R-10, 20 mM MES-KOH, 20 mM CaCl2,
400 mM mannitol, pH 5.6) with shaking at a low speed (20 rpm) in the dark at
20°C. After 15 h, the digested leaves were filtered through kimwipe in a funnel
into a falcon tube, followed by centrifugation at 100g for 5 min. After the
supernatant was removed, the pellet was resuspend in a 20-mL washing
solution (10 mM MES-KOH, 20 mM CaCl2, 400 mM mannitol, pH 5.6) and
washed twice with the washing solution. The protoplasts were finally collected by centrifugation at 100g for 5 min and resuspended in a MaMg
solution (10 mM MES-KOH, 30 mM MgCl2, 400 mM mannitol, pH 5.6; Negrutiu
et al., 1987) and used for following transient expression experiment.
DNA Transfer to Protoplasts
For transient assay in tobacco protoplasts, the CaMV 35S minimal promoter (246) was used (Fang et al., 1989). GFP was used as a reporter gene.
One- or five-repeated cis-acting element fused with 35S minimal promoter
was synthesized and incubated for 10 min at 95°C for synthesis of the doublestrand DNA fragments. The fragments generated were then inserted into
upstream of GFP and the NOS terminator in pBluescript vector.
For construction of a translational ART1 with the ART1 promoter as an
effector, genomic fragment containing a 2-kb upstream region and the coding region of ART1 (have no intron) was amplified by PCR from rice (cv
‘Nipponbare’) genomic DNA. Primer pairs used for amplification were
5#-AAAGCTTAGGGCTCCTTGAGATTGA-3# and 5#-GAATTCTCACTTGTCACCATTCTCCTCCTG-3#. The amplified fragment was cloned into pCRTOPO-XL vector using TOPO-XL PCR kit (Invitrogen).
DsRed was used as an internal standard. The DsRed-monomer coding region
was excised from the pDsRed-monomer vector (Takara Bio Inc.) by using SalI
and NotI and then inserted between CaMV 35S promoter and NOS terminator
in pBluescript vector.
Transfer of plasmids prepared above to the protoplasts was performed
according to Negrutiu et al. (1987). The suspended protoplasts were mixed with
20 mg each of plasmids containing reporter (GFP) and DsRed plasmids with or
without effector (ART1) plasmid. An equal amount of the polyethylene glycol
(PEG) solution (40% [w/v] PEG-4000, 100 mM Ca[NO3]2, 400 mM mannitol, pH
5.6) was added and mixed slowly. After incubating at room temperature for 30
min, the protoplasts/plasmid/PEG mixture was slowly diluted to 10 mL with
the washing solution. The protoplasts were then collected by centrifugation at
100g for 5 min at room temperature. After removing the supernatant, the pellet
was resuspended in a 1.3-mL Murashige and Skoog medium (0.22% [w/v]
Murashige and Skoog, 400 mM mannitol, 10 mM MES-KOH, pH 5.4) and
incubated in the dark for 17 h at 22°C, followed by exposure to Al at 100 mM
AlCl3 in the dark. After 3 h, the protoplasts were collected by centrifugation at
100g for 5 min at room temperature and the pellet was sampled using liquid
nitrogen for RNA extraction as described below.
RNA Isolation and Quantitative Real-Time PCR
Total RNA was isolated from transformed tobacco protoplasts using the
RNeasy plant mini kit (Qiagen). The RNA quality was assessed on agarose
gels and with the NanoDrop ND-1000 (Thermo Fisher Scientific). Reverse
transcription (RT) reaction was performed using SuperSript II reverse transcriptase (Invitrogen) and Oligo(dT) primers. The quantitative RT-PCR was
performed on an Eppendorf MasterCycler ep realplex real-time PCR (Eppendorf) using the specific primers described as follows: GFP, 5#-AGGAGCGCACCATCTTCTTCAA-3# and 5#-GCTGTTGTAGTTGTACTCCAGC-3#; DsRed,
5#-GGACAACACCGAGGACGTCATC-3# and 5#-CGCCCTTGGTCACCTGCAGCTT-3#; ART1, 5#-CAGTGCTTCTCGTGGGTCTT-3# and 5#-CCTGTGCGTGAAGAACCACT-3#. One-tenth dilutions of the cDNAs were used as
template for the quantitative RT-PCR in a total volume of 20 mL as follows;
10 mL of SYBR Premix Ex Taq (TaKaRa Bio Inc.), 0.4 mL of 503 ROX reference
dye, 1.2 mL primer mix (50:50 mix of forward and reverse primers at 10 pmol
mL21 each), 6.8 mL of distilled water, and 2 mL template. The reaction conditions
were: 30 s at 95°C followed by 40 cycles of 30 s at 95°C, 20 s at 57°C, and 35 s at
72°C. The DsRed was used as an internal control. Relative expression levels were
calculated by the comparative Ct method. Three independent biological replicates were made for each treatment.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession number AB379846 (ART1).
Received March 3, 2011; accepted April 16, 2011; published April 18, 2011.
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