Accepted Manuscript Title: Genome-wide expression analysis of rice ABC transporter family across spatio-temporal samples and in response to abiotic stresses Author: Nguyen Ngoc Tuyet Van Sunok Moon Ki-Hong Jung PII: DOI: Reference:
S0176-1617(14)00141-2 http://dx.doi.org/doi:10.1016/j.jplph.2014.05.006 JPLPH 51949
To appear in: Received date: Revised date: Accepted date:
19-10-2013 28-4-2014 13-5-2014
Please cite this article as: Van NNT, Genome-wide expression analysis of rice ABC transporter family across spatio-temporal samples and in response to abiotic stresses, Journal of Plant Physiology (2014), http://dx.doi.org/10.1016/j.jplph.2014.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Genome-wide expression analysis of rice ABC transporter family across spatio-temporal
2
samples and in response to abiotic stresses
3
Nguyen Ngoc Tuyet Van1, Sunok Moon1, and Ki-Hong Jung1*
4
1
5
University, Yongin 446-701, Korea
6
*
7
Nguyen Ngoc Tuyet Van ([email protected])
8
Sunok Moon ([email protected])
9
Ki-Hong Jung ([email protected])
ip t
Department of Plant Molecular Systems Biotechnology & Crop Biotech Institute, Kyung Hee
an
us
cr
Correspondence: [email protected]
Ac ce p
te
d
M
10
Page 1 of 36
10
Genome-wide expression analysis of rice ABC transporter family across spatio-temporal
12
samples and in response to abiotic stresses
13
ABSTRACT
14
Although the super family of ATP-binding cassette (ABC) proteins plays key roles in the
15
physiology and development of plants, the functions of members of this interesting family
16
mostly remain to be clarified, especially in crop plants. Thus, systematic analysis of this family
17
in rice (Oryza sativa), a major model crop plant, will be helpful in the design of effective
18
strategies for functional analysis. Phylogenomic analysis that integrates anatomy and stress meta-
19
profiling data based on a large collection of rice Affymetrix array data into the phylogenic
20
context provides useful clues into the functions for each of the ABC transporter family members
21
in rice. Using anatomy data, we identified 17 root-preferred and 16-shoot preferred genes at the
22
vegetative stage, and 3 pollen, 2 embryo, 2 ovary, 2 endosperm, and 1 anther-preferred gene at
23
the reproductive stage. The stress data revealed significant up-regulation or down-regulation of
24
47 genes under heavy metal treatment, 16 genes under nutrient deficient conditions, and 51 genes
25
under abiotic stress conditions. Of these, we confirmed the differential expression patterns of 14
26
genes in root samples exposed to drought stress using quantitative real-time PCR. Network
27
analysis using RiceNet suggests a functional gene network involving nine rice ABC transporters
28
that are differentially regulated by drought stress in root, further enhancing the prediction of
29
biological function. Our analysis provides a molecular basis for the study of diverse biological
30
phenomena mediated by the ABC family in rice and will contribute to the enhancement of crop
31
yield and stress tolerance.
32
Keywords
33
ABC transporters; abiotic stress responses; meta-analysis of tissue specific expression profiles;
34
rice; phylogenomic analysis
35
List of abbreviations used
36
ABA: Abscisic acid, ABC transporter: ATP-binding cassette transporter, As: Arsenate
Ac ce p
te
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an
us
cr
ip t
11
Page 2 of 36
Cd: Cadmium, COV: Coefficient of Variation, Cr: Chromium, NBD: nucleotide binding domain,
38
Pb: Lead, RT-PCR: Reverse transcription polymerase chain reaction, TMD: trans-membrane
39
domain
Ac ce p
te
d
M
an
us
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ip t
37
Page 3 of 36
40
Introduction
42
ATP-binding cassette transporter family (ABC-transporter) constitutes one of the largest protein
43
families with transporter activity and is conserved in all organisms. This family plays roles in the
44
export or import of various substrates across biological membranes. Minimal structure of ABC
45
transporter family requires highly conserved nucleotide-binding domains (NBD) and a less
46
conserved trans-membrane domain (TMD). The former consists of Walker A and Walker B
47
motifs for ATP binding and H- and Q-loops for the hydrolysis. The latter forms the ligand
48
binding sites by several α-helices and determines the specificity of substrates. A typical
49
membrane-bound ABC transporter contains four domains: two TMDs and two NBDs. ABC
50
transporter is formed from single gene or two genes (heterodimers or homodimers) (Kang et al.,
51
2011).
M
an
us
cr
ip t
41
By the difference in protein size, orientation (the organization of domain), and TMDs
53
sequence (idiotypic and/or linker), plant ABC transporter family is currently classified to eight
54
subfamilies. From ABCA to ABCD, subfamilies are characterized in forward TMD-NBD
55
organization, whereas ABCG subfamily contains reverse orientation NBD–TMD. The soluble
56
subfamilies E and F only encode two NBDs but no TMD, and the members of ABCI subfamily
57
retain single domain such as NBD or accessory domain. Even TMD sequences were selected as
58
criterion for the subfamily classification except soluble ABC transporters, high analogy among
59
their sequences do not entirely indicate similarity in their function. However, a few closely
60
linked members in their phylogenic tree are more likely to play similar or redundant functions.
61
For example, AtABCB1 and its closest homolog AtABCB19, participate in auxin transport
62
(Sidler, 1998), or AtABCC1 and AtABCC2 exhibit redundant function in the translocation of
63
plant vacuolar phytochelatin (Song et al., 2010). Beyond the range of species, functions of genes
64
with homology are frequently conserved. For example, Qin et al. (2013) provided evidence that
65
OsABCG15 is essential for post-meiotic anther and pollen exine development, like the homolog
66
of OsABCG15 in Arabidopsis, AtABCG26/WBC27 (Quilichini et al., 2010). Another example is
67
AtABCG32/PEC1 and OsABCG31 that are involved in the formation of the cuticular layer of the
68
cell wall to protect from losing water (Bessire et al., 2011, Chen et al., 2011a). Therefore,
69
phylogenetic analysis among different species provides us useful clues for the functional study
Ac ce p
te
d
52
Page 4 of 36
among homologous genes. Even if the structural feature has been well studied, the identification
71
of biological functions in plants is very limited. In case of Arabidopsis ABC transporters, there
72
are 128 family members and functions of 26 ABC transporters have been characterized (Kang et
73
al., 2011), and the number for rice ABC transporters with known functions is much smaller than
74
that of Arabidopsis. Until now, three ABC transporters related to water retention in leaf, pollen
75
exine development, and production of phytic acid in rice have been functionally characterized
76
(Chen et al., 2011b, Qin et al., 2013, Xu et al., 2009).
cr
ip t
70
Water scarcity causes a huge damage on crop production. Besides their developmental roles,
78
ABC transporters perform the function in rescuing plants which are exposed to various abiotic
79
stresses, especially drought. So far, the identification of both the importer (Kang et al., 2010) and
80
exporter (Kuromori et al., 2010) of ABA in Arabidopsis enhanced our knowledge about ABA
81
signaling which is one of the most important phytohormone pathways associated with drought
82
stress responses. ABC transporters also have functions related to guard cell responses (Klein et
83
al., 2004, Kuromori et al., 2011) or required for functional cuticle formation, associated with
84
water retention in the leaf like AtABCG32/PEC1 and OsABCG31 as mentioned earlier. Genome-
85
wide analysis of ABC transporters in rice associated with water scarcity has not been carried out
86
and will be required to provide novel insight of ABC transporter for rescuing water scarcity.
te
d
M
an
us
77
Completion of the rice genome sequence has revealed a large number of ABC transporters
88
(IRGSP, 2005, Verrier et al., 2008). The main obstacle to their functional identification is the
89
functional redundancy within this supergene family and the lack of information guiding
90
functional analysis. Thus, a systematic overview of the ABC family in rice might be a useful
91
starting point for further functional analyses under diverse stress conditions as well as for unique
92
developmental process. Phylogenomics is a phylogenetic approach to predict the biological
93
functions of members of large gene families by assessing the similarity among gene products
94
(Cao et al., 2012, Jung et al., 2010a). The power of estimating functional redundancy is even
95
more enhanced by combining phylogenic analysis with a large amount of transcriptome data,
96
such as integrating analysis of gene expression data related to SUB1A-mediated submergence
97
tolerance with the phylogenic tree of the AP2 family (Jung et al., 2010b). Recently, we
98
demonstrated that selection of the predominant gene family member in light conditions has
Ac ce p
87
Page 5 of 36
99 100
better performance in identifying defective phenotypes through the related loss-of-function study than selecting non-predominant family members (Jung et al., 2008). In this study, we performed phylogenomic analysis of ABC transporter family members to
102
provide functional clues of individual family members. The anatomy or stress meta-expression
103
profiles based on a large collection of Affymetrix data were then integrated into the phylogenic
104
tree context of the ABC transporter family. As a result, we identified 38 genes that showed
105
tissues/organ preferred expression, 3 genes that were ubiquitously expressed, 47 genes that
106
responded to heavy metal, 16 genes associated with nutrient deficiency, and 51 genes associated
107
with abiotic stress. Of them, expression patterns of 14 genes in response to drought stress in root
108
are confirmed by quantitative RT-PCR analysis. We developed a functional gene network
109
associated with nine ABC transporter genes differentially expressed in root after drought
110
treatment and this network suggests a hypothetical model to elucidate the underlying molecular
111
mechanisms, which might in turn facilitate future agricultural applications in rice for enhancing
112
water use efficiency.
M
an
us
cr
ip t
101
d
113
Materials and methods
115
Multiple sequence alignment and phylogenetic tree building
116
To perform phylogenomic analysis of ABC transporters in rice and Arabidopsis thaliana, we
117
collected 125 family members with locus IDs from the Rice Genome Annotation Project
118
(RGAP, http://rice.plantbiology.msu.edu/) (Table S1) and 128 Arabidopsis family members
119
from a previous report (Verrier et al., 2008). We selected a representative transcript encoding the
120
ABC protein for each locus in rice as suggested in RGAP. Multiple amino acid sequences were
121
aligned using ClustalX program version 2.0.11 (Higgins et al., 1996). The phylogenetic analysis
122
was performed by using MEGA 5.2 and under the following parameters: neighbor-joining tree
123
method, complete deletion, and bootstrap with 500 replicates (Tamura et al., 2011). We
124
developed a phylogenic tree with rice and Arabidopsis ABC transporters. Rice ABC proteins are
125
currently classified into eight subfamilies (Fig.1, Fig. S1) in accordance with new nomenclature,
126
based on RGAP annotation release 7 (http://rice.plantbiology.msu.edu/downloads_gad.shtml).
Ac ce p
te
114
Page 6 of 36
We also generated a phylogenic tree for each subfamily except for ABCD and ABCE, which
128
have only 2 and 3 members, respectively (Fig. S1).
129
Meta-analysis of tissue specific expression profiles and stress responsive expression analysis
130
Integration of transcriptomes into the context of the phylogenic tree might be a good reference to
131
direct useful experimental strategies for further functional analysis. Therefore, we used meta-
132
analysis of tissue specific expression profiles based on 983 Affymetrix array data downloaded
133
from the NCBI gene expression omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) (Cao et al.,
134
2012). We then uploaded the log2 normalized intensity data in tab-delimited text format into
135
Multi Experiment Viewer (MEV, http://www.tm4.org/mev/) and illustrated them by using heat
136
maps (Figs. 2-4, Fig. S2). We separated the tissue samples into two groups according to
137
development stage: vegetative stage and reproductive stage. We then selected genes that have at
138
least 2-fold higher expression level in each tissue type relative to the other tissues with a p-value
139
less than 0.05. In addition, we generated a heat map image based on log2 fold-change data in
140
response to abiotic stresses and then integrated the heat map into the phylogenetic tree context.
141
We selected genes showing up-regulation under diverse stresses with log2 changes greater than
142
1.5-fold or lower -1.5-fold for down-regulation with a coefficient of variation (COV) less than 1,
143
compared with normal conditions (Fig. 5, Tables S2-3).
144
Promoter analysis
145
To analyze the promoter regions for four loci (OsABCG30, OsABCG41, OsABCG47, and
146
OsABCG48) differentially expressed between japonica and indica, we extracted 2kb upstream
147
sequences from start codon of four loci in both japonica and indica genomes from Gramene
148
(http://plants.ensembl.org/Oryza_sativa/Info/Index)
149
(http://ensembl.gramene.org/Oryza_indica/Info/Index). Comparison of transcription factor
150
binding sites (TFBSs) was processed by Plant promoter analysis navigator, Plantpan
151
(http://plantpan.mbc.nctu.edu.tw/) (Chang et al., 2008). Figure S3 showed the results of
152
compared TFBSs and similarity alignment between japonica and indica promoter regions for 4
153
loci. Yellow colored regions indicate high homologs between compared japonica and indica
154
sequences and the rest was not indicated by colored lines or regions.
155
Plant material and drought treatment
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and
EMBL
Page 7 of 36
Rice (Oryza sativa) seeds (Dongjin and IR64 varieties) were germinated on Murashige Skoog
157
medium under controlled conditions of 28°C day/25°C night temperatures, 8-h light/16-h dark
158
cycle, and 78% relative humidity after sterilization with 50% (w/v) commercial bleach for 30
159
minutes with gentle shaking. After 7 days of germination, the seedlings were washed with
160
sterilized water to discard agar and incubated in water for a day, then completely removed water
161
and air-dried at 28°C. Roots were harvested at 2 and 4 h after exposure to stress treatment,
162
frozen in liquid nitrogen, and stored at -70°C for further analysis (Jeong et al., 2010, Oh et al.,
163
2009).
164
RNA extraction, semi-quantitative RT-PCR, and real-time PCR
165
Roots and shoots of rice seedlings were frozen in liquid nitrogen and ground with a Tissue Lyser
166
II (Qiagen; Hilden, Germany). RNAs were extracted with the RNAiso Plus Kit according to the
167
manufacturer’s protocol (Takara Bio; Kyoto, Japan). For tissue specific expression patterns by
168
RT-PCR, we used two control primer sets for rice ubiquitin 5 (OsUbi5, LOC_Os01g22490) and
169
rice elongation factor-1α (OseEF-1α, LOC_Os03g08020) (Jain et al., 2006). The PCR cycle
170
conditions used were 95°C for 30s, 57°C for 30s, and 72°C for 1 min 30s for 22-38 cycles. For
171
real-time PCR, we used cycling conditions of 95°C for 15s, 57°C for 30s and 72°C for 60s. To
172
validate the significant expression changes of genes in response to drought stress, we carried out
173
real-time PCR analysis with three independent biological replicates. Relative transcript levels
174
and fold change were calculated by 2-∆Ct and 2-∆∆Ct method (Schmittgen and Livak, 2008),
175
respectively. Student t-test indicates the significant change under less than 0.05 and 0.01 p-
176
values as asterisk (*) and double asterisk (**), respectively, in Figures 7-8.
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178
In silico network analysis
179
A hypothetical functional gene network mediated by rice ABC transporters was developed from
180
RiceNet (http://www.functionalnet.org/ricenet/) (Lee et al., 2011) by querying the MSU locus
181
IDs of 14 ABC transporter genes with confirmed drought response in Fig. 7. We uploaded the sif
182
file from RiceNet to Cytoscape version 2.8.1 software. For whole genes in network, we also
183
generated log2 fold-change data in response to abiotic stresses. We then integrated log2 fold-
184
change data in response to abiotic stresses into nodes by using “File>Import>Attribute from
Page 8 of 36
Table” function. We assigned node color with criteria >1.5 and
S0176-1617(14)00141-2 http://dx.doi.org/doi:10.1016/j.jplph.2014.05.006 JPLPH 51949
To appear in: Received date: Revised date: Accepted date:
19-10-2013 28-4-2014 13-5-2014
Please cite this article as: Van NNT, Genome-wide expression analysis of rice ABC transporter family across spatio-temporal samples and in response to abiotic stresses, Journal of Plant Physiology (2014), http://dx.doi.org/10.1016/j.jplph.2014.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Genome-wide expression analysis of rice ABC transporter family across spatio-temporal
2
samples and in response to abiotic stresses
3
Nguyen Ngoc Tuyet Van1, Sunok Moon1, and Ki-Hong Jung1*
4
1
5
University, Yongin 446-701, Korea
6
*
7
Nguyen Ngoc Tuyet Van ([email protected])
8
Sunok Moon ([email protected])
9
Ki-Hong Jung ([email protected])
ip t
Department of Plant Molecular Systems Biotechnology & Crop Biotech Institute, Kyung Hee
an
us
cr
Correspondence: [email protected]
Ac ce p
te
d
M
10
Page 1 of 36
10
Genome-wide expression analysis of rice ABC transporter family across spatio-temporal
12
samples and in response to abiotic stresses
13
ABSTRACT
14
Although the super family of ATP-binding cassette (ABC) proteins plays key roles in the
15
physiology and development of plants, the functions of members of this interesting family
16
mostly remain to be clarified, especially in crop plants. Thus, systematic analysis of this family
17
in rice (Oryza sativa), a major model crop plant, will be helpful in the design of effective
18
strategies for functional analysis. Phylogenomic analysis that integrates anatomy and stress meta-
19
profiling data based on a large collection of rice Affymetrix array data into the phylogenic
20
context provides useful clues into the functions for each of the ABC transporter family members
21
in rice. Using anatomy data, we identified 17 root-preferred and 16-shoot preferred genes at the
22
vegetative stage, and 3 pollen, 2 embryo, 2 ovary, 2 endosperm, and 1 anther-preferred gene at
23
the reproductive stage. The stress data revealed significant up-regulation or down-regulation of
24
47 genes under heavy metal treatment, 16 genes under nutrient deficient conditions, and 51 genes
25
under abiotic stress conditions. Of these, we confirmed the differential expression patterns of 14
26
genes in root samples exposed to drought stress using quantitative real-time PCR. Network
27
analysis using RiceNet suggests a functional gene network involving nine rice ABC transporters
28
that are differentially regulated by drought stress in root, further enhancing the prediction of
29
biological function. Our analysis provides a molecular basis for the study of diverse biological
30
phenomena mediated by the ABC family in rice and will contribute to the enhancement of crop
31
yield and stress tolerance.
32
Keywords
33
ABC transporters; abiotic stress responses; meta-analysis of tissue specific expression profiles;
34
rice; phylogenomic analysis
35
List of abbreviations used
36
ABA: Abscisic acid, ABC transporter: ATP-binding cassette transporter, As: Arsenate
Ac ce p
te
d
M
an
us
cr
ip t
11
Page 2 of 36
Cd: Cadmium, COV: Coefficient of Variation, Cr: Chromium, NBD: nucleotide binding domain,
38
Pb: Lead, RT-PCR: Reverse transcription polymerase chain reaction, TMD: trans-membrane
39
domain
Ac ce p
te
d
M
an
us
cr
ip t
37
Page 3 of 36
40
Introduction
42
ATP-binding cassette transporter family (ABC-transporter) constitutes one of the largest protein
43
families with transporter activity and is conserved in all organisms. This family plays roles in the
44
export or import of various substrates across biological membranes. Minimal structure of ABC
45
transporter family requires highly conserved nucleotide-binding domains (NBD) and a less
46
conserved trans-membrane domain (TMD). The former consists of Walker A and Walker B
47
motifs for ATP binding and H- and Q-loops for the hydrolysis. The latter forms the ligand
48
binding sites by several α-helices and determines the specificity of substrates. A typical
49
membrane-bound ABC transporter contains four domains: two TMDs and two NBDs. ABC
50
transporter is formed from single gene or two genes (heterodimers or homodimers) (Kang et al.,
51
2011).
M
an
us
cr
ip t
41
By the difference in protein size, orientation (the organization of domain), and TMDs
53
sequence (idiotypic and/or linker), plant ABC transporter family is currently classified to eight
54
subfamilies. From ABCA to ABCD, subfamilies are characterized in forward TMD-NBD
55
organization, whereas ABCG subfamily contains reverse orientation NBD–TMD. The soluble
56
subfamilies E and F only encode two NBDs but no TMD, and the members of ABCI subfamily
57
retain single domain such as NBD or accessory domain. Even TMD sequences were selected as
58
criterion for the subfamily classification except soluble ABC transporters, high analogy among
59
their sequences do not entirely indicate similarity in their function. However, a few closely
60
linked members in their phylogenic tree are more likely to play similar or redundant functions.
61
For example, AtABCB1 and its closest homolog AtABCB19, participate in auxin transport
62
(Sidler, 1998), or AtABCC1 and AtABCC2 exhibit redundant function in the translocation of
63
plant vacuolar phytochelatin (Song et al., 2010). Beyond the range of species, functions of genes
64
with homology are frequently conserved. For example, Qin et al. (2013) provided evidence that
65
OsABCG15 is essential for post-meiotic anther and pollen exine development, like the homolog
66
of OsABCG15 in Arabidopsis, AtABCG26/WBC27 (Quilichini et al., 2010). Another example is
67
AtABCG32/PEC1 and OsABCG31 that are involved in the formation of the cuticular layer of the
68
cell wall to protect from losing water (Bessire et al., 2011, Chen et al., 2011a). Therefore,
69
phylogenetic analysis among different species provides us useful clues for the functional study
Ac ce p
te
d
52
Page 4 of 36
among homologous genes. Even if the structural feature has been well studied, the identification
71
of biological functions in plants is very limited. In case of Arabidopsis ABC transporters, there
72
are 128 family members and functions of 26 ABC transporters have been characterized (Kang et
73
al., 2011), and the number for rice ABC transporters with known functions is much smaller than
74
that of Arabidopsis. Until now, three ABC transporters related to water retention in leaf, pollen
75
exine development, and production of phytic acid in rice have been functionally characterized
76
(Chen et al., 2011b, Qin et al., 2013, Xu et al., 2009).
cr
ip t
70
Water scarcity causes a huge damage on crop production. Besides their developmental roles,
78
ABC transporters perform the function in rescuing plants which are exposed to various abiotic
79
stresses, especially drought. So far, the identification of both the importer (Kang et al., 2010) and
80
exporter (Kuromori et al., 2010) of ABA in Arabidopsis enhanced our knowledge about ABA
81
signaling which is one of the most important phytohormone pathways associated with drought
82
stress responses. ABC transporters also have functions related to guard cell responses (Klein et
83
al., 2004, Kuromori et al., 2011) or required for functional cuticle formation, associated with
84
water retention in the leaf like AtABCG32/PEC1 and OsABCG31 as mentioned earlier. Genome-
85
wide analysis of ABC transporters in rice associated with water scarcity has not been carried out
86
and will be required to provide novel insight of ABC transporter for rescuing water scarcity.
te
d
M
an
us
77
Completion of the rice genome sequence has revealed a large number of ABC transporters
88
(IRGSP, 2005, Verrier et al., 2008). The main obstacle to their functional identification is the
89
functional redundancy within this supergene family and the lack of information guiding
90
functional analysis. Thus, a systematic overview of the ABC family in rice might be a useful
91
starting point for further functional analyses under diverse stress conditions as well as for unique
92
developmental process. Phylogenomics is a phylogenetic approach to predict the biological
93
functions of members of large gene families by assessing the similarity among gene products
94
(Cao et al., 2012, Jung et al., 2010a). The power of estimating functional redundancy is even
95
more enhanced by combining phylogenic analysis with a large amount of transcriptome data,
96
such as integrating analysis of gene expression data related to SUB1A-mediated submergence
97
tolerance with the phylogenic tree of the AP2 family (Jung et al., 2010b). Recently, we
98
demonstrated that selection of the predominant gene family member in light conditions has
Ac ce p
87
Page 5 of 36
99 100
better performance in identifying defective phenotypes through the related loss-of-function study than selecting non-predominant family members (Jung et al., 2008). In this study, we performed phylogenomic analysis of ABC transporter family members to
102
provide functional clues of individual family members. The anatomy or stress meta-expression
103
profiles based on a large collection of Affymetrix data were then integrated into the phylogenic
104
tree context of the ABC transporter family. As a result, we identified 38 genes that showed
105
tissues/organ preferred expression, 3 genes that were ubiquitously expressed, 47 genes that
106
responded to heavy metal, 16 genes associated with nutrient deficiency, and 51 genes associated
107
with abiotic stress. Of them, expression patterns of 14 genes in response to drought stress in root
108
are confirmed by quantitative RT-PCR analysis. We developed a functional gene network
109
associated with nine ABC transporter genes differentially expressed in root after drought
110
treatment and this network suggests a hypothetical model to elucidate the underlying molecular
111
mechanisms, which might in turn facilitate future agricultural applications in rice for enhancing
112
water use efficiency.
M
an
us
cr
ip t
101
d
113
Materials and methods
115
Multiple sequence alignment and phylogenetic tree building
116
To perform phylogenomic analysis of ABC transporters in rice and Arabidopsis thaliana, we
117
collected 125 family members with locus IDs from the Rice Genome Annotation Project
118
(RGAP, http://rice.plantbiology.msu.edu/) (Table S1) and 128 Arabidopsis family members
119
from a previous report (Verrier et al., 2008). We selected a representative transcript encoding the
120
ABC protein for each locus in rice as suggested in RGAP. Multiple amino acid sequences were
121
aligned using ClustalX program version 2.0.11 (Higgins et al., 1996). The phylogenetic analysis
122
was performed by using MEGA 5.2 and under the following parameters: neighbor-joining tree
123
method, complete deletion, and bootstrap with 500 replicates (Tamura et al., 2011). We
124
developed a phylogenic tree with rice and Arabidopsis ABC transporters. Rice ABC proteins are
125
currently classified into eight subfamilies (Fig.1, Fig. S1) in accordance with new nomenclature,
126
based on RGAP annotation release 7 (http://rice.plantbiology.msu.edu/downloads_gad.shtml).
Ac ce p
te
114
Page 6 of 36
We also generated a phylogenic tree for each subfamily except for ABCD and ABCE, which
128
have only 2 and 3 members, respectively (Fig. S1).
129
Meta-analysis of tissue specific expression profiles and stress responsive expression analysis
130
Integration of transcriptomes into the context of the phylogenic tree might be a good reference to
131
direct useful experimental strategies for further functional analysis. Therefore, we used meta-
132
analysis of tissue specific expression profiles based on 983 Affymetrix array data downloaded
133
from the NCBI gene expression omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) (Cao et al.,
134
2012). We then uploaded the log2 normalized intensity data in tab-delimited text format into
135
Multi Experiment Viewer (MEV, http://www.tm4.org/mev/) and illustrated them by using heat
136
maps (Figs. 2-4, Fig. S2). We separated the tissue samples into two groups according to
137
development stage: vegetative stage and reproductive stage. We then selected genes that have at
138
least 2-fold higher expression level in each tissue type relative to the other tissues with a p-value
139
less than 0.05. In addition, we generated a heat map image based on log2 fold-change data in
140
response to abiotic stresses and then integrated the heat map into the phylogenetic tree context.
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We selected genes showing up-regulation under diverse stresses with log2 changes greater than
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1.5-fold or lower -1.5-fold for down-regulation with a coefficient of variation (COV) less than 1,
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compared with normal conditions (Fig. 5, Tables S2-3).
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Promoter analysis
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To analyze the promoter regions for four loci (OsABCG30, OsABCG41, OsABCG47, and
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OsABCG48) differentially expressed between japonica and indica, we extracted 2kb upstream
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sequences from start codon of four loci in both japonica and indica genomes from Gramene
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(http://plants.ensembl.org/Oryza_sativa/Info/Index)
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(http://ensembl.gramene.org/Oryza_indica/Info/Index). Comparison of transcription factor
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binding sites (TFBSs) was processed by Plant promoter analysis navigator, Plantpan
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(http://plantpan.mbc.nctu.edu.tw/) (Chang et al., 2008). Figure S3 showed the results of
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compared TFBSs and similarity alignment between japonica and indica promoter regions for 4
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loci. Yellow colored regions indicate high homologs between compared japonica and indica
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sequences and the rest was not indicated by colored lines or regions.
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Plant material and drought treatment
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Rice (Oryza sativa) seeds (Dongjin and IR64 varieties) were germinated on Murashige Skoog
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medium under controlled conditions of 28°C day/25°C night temperatures, 8-h light/16-h dark
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cycle, and 78% relative humidity after sterilization with 50% (w/v) commercial bleach for 30
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minutes with gentle shaking. After 7 days of germination, the seedlings were washed with
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sterilized water to discard agar and incubated in water for a day, then completely removed water
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and air-dried at 28°C. Roots were harvested at 2 and 4 h after exposure to stress treatment,
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frozen in liquid nitrogen, and stored at -70°C for further analysis (Jeong et al., 2010, Oh et al.,
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2009).
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RNA extraction, semi-quantitative RT-PCR, and real-time PCR
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Roots and shoots of rice seedlings were frozen in liquid nitrogen and ground with a Tissue Lyser
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II (Qiagen; Hilden, Germany). RNAs were extracted with the RNAiso Plus Kit according to the
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manufacturer’s protocol (Takara Bio; Kyoto, Japan). For tissue specific expression patterns by
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RT-PCR, we used two control primer sets for rice ubiquitin 5 (OsUbi5, LOC_Os01g22490) and
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rice elongation factor-1α (OseEF-1α, LOC_Os03g08020) (Jain et al., 2006). The PCR cycle
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conditions used were 95°C for 30s, 57°C for 30s, and 72°C for 1 min 30s for 22-38 cycles. For
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real-time PCR, we used cycling conditions of 95°C for 15s, 57°C for 30s and 72°C for 60s. To
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validate the significant expression changes of genes in response to drought stress, we carried out
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real-time PCR analysis with three independent biological replicates. Relative transcript levels
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and fold change were calculated by 2-∆Ct and 2-∆∆Ct method (Schmittgen and Livak, 2008),
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respectively. Student t-test indicates the significant change under less than 0.05 and 0.01 p-
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values as asterisk (*) and double asterisk (**), respectively, in Figures 7-8.
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In silico network analysis
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A hypothetical functional gene network mediated by rice ABC transporters was developed from
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RiceNet (http://www.functionalnet.org/ricenet/) (Lee et al., 2011) by querying the MSU locus
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IDs of 14 ABC transporter genes with confirmed drought response in Fig. 7. We uploaded the sif
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file from RiceNet to Cytoscape version 2.8.1 software. For whole genes in network, we also
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generated log2 fold-change data in response to abiotic stresses. We then integrated log2 fold-
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change data in response to abiotic stresses into nodes by using “File>Import>Attribute from
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Table” function. We assigned node color with criteria >1.5 and
