Advances in Bioscience and Biotechnology, 2010, 1, 384-390 ABB
doi:10.4236/abb.2010.15051 Published Online December 2010 (http://www.SciRP.org/journal/abb/).
Published Online December 2010 in SciRes. http://www.scirp.org/journal/ABB
Determining the transcriptional regulation pattern of PgTIP1
in transgenic Arabidopsis thaliana by constructing gene
coexpression networks*
Haiying Chen1, Lu Ying1, Jing Jin1, Qi Li2, We iming Cai1*
1Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Graduate School of Chinese Academy of
Sciences, Chinese Academy of Sciences, Shanghai, China;
2Genminix Informatics Co., Ltd.
Received 29 October 2010; revised 1 November 2010; accepted 1 November 2010.
ABSTRACT
The seed size, seed mass, and growth rate of trans-
genic Arabidopsis plants containing PgTIP1, a gin-
seng tonoplast aquaporin gene, are significantly
higher than those of wild-type Arabidopsis plants.
Whole genome expression and bioinformatics analy-
sis, including analysis of co-expression networks and
transcription factors (Tfscan), were used to deter-
mine the key genes that are activated after the ex-
pression of PgTIP1 and the transcription factors that
play important roles in the regulation of the genes
controlling growth of Arabidopsis thaliana seeds by
using transgenic Arabidopsis plants containing
PgTIP1. Differential gene analysis showed that
transformation of exogenous PgTIP1 to Arabidopsis
induced endogenous gene expression changes. Analy-
sis of gene co-expression networks revealed 2 genes,
PIP1 (plasma membrane aquaporin 1 gene) and
RD26 (responsive to desiccation 26 gene; a NAC
transcription factor), that were localized in the core
of the networks. Analysis of the transcriptional regu-
lation network of transgenic Arabidopsis plants con-
taining PgTIP1 showed that PIP1 and RD26 were
regulated via DNA binding with a finger domain on
transcription factor 2 (Dof2). In this study, we dem-
onstrated that Dof2 induces up-regulation of PIP1
and RD26 after transformation with PgTIP1. The
results of this study provide a new means for con-
ducting research into and controlling growth of
Arabidopsis thaliana seeds.
Keywords: Arabidopsis thaliana; Microarray;
Coexpression Network; Transcription Factor Analysis;
PgTIP1
1. INTRODUCTION
We screened differentially expressed genes using sup-
pression subtractive hybridization (SSH) between hor-
mone-autotrophic and hormone-dependent ginseng cal-
lus lines and isolated and characterized an aquaporin
gene PgTIP1 (GenBank accession number DQ237285)
that was specifically and highly expressed in hormone-
autotrophic ginseng cells [1]. We also demonstrated that,
when expressed in Arabidopsis thaliana, PgTIP1 sub-
stantially altered vegetative and reproductive growth and
development. Compared to wild-type (WT) Arabidopsis
plants, transgenic (Tg) Arabidopsis plants containing
PgTIP1 showed significantly increased seed size, seed
mass, and growth rates. Moreover, the fatty acid content
of seeds from the Tg Arabidopsis plants was 1.85-fold
higher than that of seeds from the wild-type control.
These results demonstrated that PgTIP1 is important in
the growth and development of plant cells.
In this study, we determined the key genes that were
activated after the expression of PgTIP1, promoted seed
growth, and activated transcription factors, which play
important roles in the regulation of the genes related to
growth control of Arabidopsis thaliana seeds. Whole
genome expression and bioinformatics analyses, include-
ing coexpression network and transcription factor
analyses, were conducted using Arabidopsis plants ex-
pressing PgTIP1.
2. METHOD AND MATERIALS
2.1. Plant Materials and Growth Conditions
Wild type and Tg PgTIP1 Arabidopsis seeds (ecotype:
*Sponsors: the National High Technology Research and Development
Program of China(2006AA10Z112); the National Transgenic Program
(2009ZX08004-008B); the Knowledge Innovation Program of the
Chinese Academy of Sciences (KJCX2-YW-L08); the China Manned
Space Flight Technology Project and the National Natural Science
Foundation of China (30770197, 90917009, 31070237).

H. Y. Chen et al. / Advances in Bioscience and Biotechnology 1 (2010) 384-390 385
Columbia) were germinated and grown in soil at a pho-
ton flux density of 150 µmol m-2 s
-1, 60-80% relative
humidity (RH), and a 16/8 h day/night (D/N) cycle at
20-22℃ in a phytotron. All experiments were performed
with the seedlings in Figure 1. Whole plant samples
were quickly removed from soil, washed with distilled
water, frozen in liquid nitrogen, and stored at −70℃ un-
til RNA extraction.
2.2. Generation of PgTIP1-Overexpressing
Arabidopsis Plants
The Tg plants containing PgTIP1 were generated as de-
scribed in Lin et al. [1]. Briefly, the ORF of PgTIP1 was
cloned into the pHB vector [2] using the HindIII and an
XbaI restriction sites to generate a double 35S:PgTIP1
transgene. Six-week-old Arabidopsis (ecotype Columbia)
plants were transformed with Agrobacterium using the
Xoral dip method [3,4]. Seeds were screened in 0.8%
selection medium containing 50 µg/ml hygromycin for 7
days and were then transferred to a 1.0% selection me-
dium for an additional 7 days. Resistant plants were
transferred to soil and analyzed further. Positive T0
(a)
(b)
Figure 1. (a) Wild type and transgenic Arabi-
dopsis thaliana at time 1 and time 2.Time 1 is
the bolting time and time 2 is 10 days after time
1; (b) Mature dried seeds from wild type and
transgenic Arabidopsis plants. (Bar = 0.5 mm).
plants were self-pollinated and T1 seeds were collected.
Individual T1 plants were tested for expression of PgTIP1
using RT-PCR. T1 plants expressing the PgTIP1 gene
were self-pollinated to produce a homozygous genera-
tion which was subsequently used for chip analysis.
2.3. RNA Isolation and Microarray
Hybridization
Total RNA was extracted using a QIAGEN RNAeasy
mini kit (Qiagen, CA) according to the manufacturer’s
instructions and incubated with oligo dT/T7 primers and
reverse-transcribed into double-stranded cDNA. In vitro
transcription of the purified cDNA was performed with
T7 RNA polymerase at 42℃ for 6 h. The amplified RNA
was purified and subjected to a second round of ampli-
fication and biotin labeling with Affymetrix’s IVT label-
ing kit. Biotin-labeled RNA was fragmented and hybrid-
ized to whole-genome Arabidopsis GeneChips (Affy-
metrix) for 16 h, washed, stained, and scanned.
2.4. Differential Gene Expression Analysis
Two-Factor Analysis of Variance is used to filter differ-
entially expressed genes according to two factors (here,
the factors are time and transgene). A random variant
model corrected (RVM) t-test [5] and f-test was used to
filter significant differentially expressed genes using the
time factor, the transgene factor, and the union of these 2
factors. The RVM can raise the degree of freedom to
effectively decrease deviation due to small sample size
[6,7].
2.5. Construction and Topological Attributes of
Coexpression Networks
We built gene coexpression networks to identify gene
interactions [8]. Gene coexpression networks were built
according to the normalized signal intensity of different-
tially expressed genes. For each pair of genes, we calcu-
lated the Pearson correlation and chose significant cor-
relation pairs with which to construct the network [9].
The purpose of network structure analysis is to locate
core regulatory factors (genes). In one network, these
factors connect most adjacent genes and have the highest
degrees, or connectivity values. For different networks,
core regulatory factors were determined by degree dif-
ferences between 2 class samples[10].
In network analysis, degree centrality is the simplest
and most important measure of gene centrality within a
network built for determining relative importance. De-
gree centrality is defined as the link numbers one node
has to another. Moreover, to study various properties of
networks, k-cores were introduced in the graph theory as
a method of simplifying graph topology analysis. A
k-core of a network is a sub-network in which all nodes

H. Y. Chen et al. / Advances in Bioscience and Biotechnology 1 (2010) 384-390 386
are connected to at least k other genes in the sub-net-
work. A k-core of a protein-protein interaction network
usually contains cohesive groups of proteins [11,12].
2.6. Transcription Factor Analysis (Tfscan)
Transcription factor analysis (Tfscan) reveals how tran-
scription factors regulate genes. First, the sequences of
differentially expressed genes are searched, and then,
using the Jemboss software the relationship between
genes and transcription factors is determined by count-
ing the correlation between the gene sequence and tran-
scription factor sequence. Next, we built a transcription
factor regulation network (TF-Gene-Network) with the
interactions between genes and transcription factors. The
network’s core transcription factor is the most important
center and has the largest degree [9,13]. Pearson correla-
tion analysis [9] is used to measure the regulatory ability
of transcription factors by calculating the correlation
between transcription factors and the genes they regulate
and the correlations between the genes regulated by the
same factors.
3. RESULTS
3.1. Important Roles of PIP1 and RD26 in the
Coexpression Netw o r k
Results indicated that Tg Arabidopsis thaliana grow
faster and stronger than the WT and have more plump
seeds “Figure 1”. The most intriguing phenotype of Tg
Arabidopsis is the size of mature seeds, which is sig-
nificantly larger than that of WT seeds “Figure 1(b)”.
After two-factor RVM analysis with a threshold of p <
0.05, 5796 genes were identified according to time factor
(group A), 391 differentially expressed genes were se-
lected with the Tg factor (group B), and 388 differen-
tially expressed genes were chosen taking both Tg and
time factors into account (group C). The union of group
B and group C was used to build coexpression networks.
One particular coexpression network was constructed
using the signal intensity of wild type of Arabidopsis
thaliana “Figure 2(a)” and the other was constructed
using the signal intensity of Tg Arabidopsis thaliana
“Figure 2(b)”. The correlation significance level, or in-
teraction between genes in coexpression networks, was
calculated by Pearson correlation analysis and found to
be greater than 0.99 with a correlation significance of
less than 0.0001.
To further understand the effect of the water channel
gene PgTIP1 transfer into Arabidopsis thaliana, the Tg
coexpression network was simplified to a sub-network
containing only core regulatory factors and their interac-
tions “Figure 3”. Core regulatory factors have large de-
grees and k-core in the Tg coexpression network and
have the biggest degree distance in the wild type and Tg
(a)
(b)
Figure 2. Coexpression networks of wild type (a) and trans-
genic (b) Arabidopsis thaliana. Colors represent the same
sub-network with similar k-core values. Comparing network
complexity, transgenic species are clearly more complex than
wild type.
coexpression networks “Figure 4”.
In the concentration area of genes related to the water
channel and growth “Figure 3”, there are two important
genes, PIP1 and RD26. These genes are up-regulated
core regulatory factors in Tg and both related with
water channel. Additionally, these genes regulate several
genes related to growth according to the Tg sub-network
“Figure 3”.
3.2. Transcription Factor Dof2 and Dof3
Transcription factor analysis (Tfscan) was enlisted for
the genes in WT and Tg coexpression networks. In the
Tfscan of genes in the Tg sub-network, two important
transcription factors, Dof2 and Dof3, were found in the
transcription regulation network (TF-Gene-Network)
“Figure 5”. Both Dof2 and Dof3 were co-expressed

H. Y. Chen et al. / Advances in Bioscience and Biotechnology 1 (2010) 384-390 387
Figure 3. Transgenic (Tg) sub-network. Red node represents an up-regulated gene, blue node represents
a down-regulated gene; regular node represents a gene related to water channel, and diamond-shaped
node represents a gene related to growth; solid line represents positive correlation between 2 genes, and
dashed line represents negative correlation between 2 genes.
Figure 4. Degree polygon of core regulatory factors in wild type (WT) and transgenic (Tg)
coexpression networks. Horizontal axis represents gene name. Vertical axis represents degree value of
genes in WT and Tg types. Degree (Tg-WT) = degree (Tg) – degree (WT).

H. Y. Chen et al. / Advances in Bioscience and Biotechnology 1 (2010) 384-390 388
with other genes in the network. Furthermore, Dof2 dis-
played positive correlation with PIP1 and RD26, the
core regulatory factors in the coexpression network.
More importantly, expression of Dof2 increased in group
A. From correlation analysis of the TF-Gene-Network,
the correlation coefficient of the Tg TF-Gene-Network is
larger than that of the WT TF-Gene-Network. These
results show the regulatory ability of Dof2 and Dof3 to
greatly decrease and the genes regulated by these tran-
scription factors are not closely related the Tg in the
transcription regulation network of WT “Figure 6”.
4. DISCUSSION
The Tg Arabidopsis plants containing PgTIP1 demon-
strated promising traits for agricultural application. In
this study, we aimed to understand the gene expression
changes due to transformation with PgTIP1. Whole ge-
nome expression analysis using gene chips was em-
ployed to identify differences in transcription profiles
between Tg and WT plants. A random variance model
corrected t-test was used to assess the detection value of
samples and to effectively reduce the residual caused by
small sample sizes by sufficiently increasing the degrees
of freedom [5]. We identified 5796 genes categorized
according to the time factor, 391 differentially expressed
genes categorized according to the Tg factor, and 388
differentially expressed genes characterized according to
both Tg and time factors. Thus, transformation of ex-
ogenous PgTIP1 into Arabidopsis induces expression
changes of endogenous Arabidopsis genes.
Data from gene coexpression network analysis re-
vealed 2 genes, PIP1 and RD26, localized to the network
cores. PIP1 is a plasma membrane aquaporin. PIP1
members increase the water permeability of cells ex-
pressing these aquaporins [14,15]. RD26 encodes a NAC
transcription factor. Seedlings of RD26-overexpressed
plants have large leaf blades and short petioles, while
RD26-overexpressed plants have small leaf blades and
long petioles [16]. PIP1 and RD26 are up-regulated to
coordinate PgTIP1 expression.
Tfscan illuminates 2 important transcription factors,
Dof2 and Dof3, regulating gene networks. Dof proteins
are DNA-binding proteins with one finger domain tran-
scription factor. Dof-domain proteins play critical roles
as transcriptional regulators in plant growth and devel-
opment [17]. The TF-Gene-Network of Tg reveals that
PIP1 and RD26 are regulated by Dof2. This suggests
that synergism of PgTIP1 expression and Dof2 enhance
Figure 5. TF-Gene-Network of transgenic (Tg); red nodes represents up-regulated genes and blue nodes
represent down- regulated genes in Tg type. The blue boundary circle shows that PIP1 and RD26 are
regulated by Dof2.

H. Y. Chen et al. / Advances in Bioscience and Biotechnology 1 (2010) 384-390 389
Figure 6. TF-Gene-Network of wild type (WT); red nodes represent up-regulated genes and blue nodes
represent down- regulated genes in the WT. The blue boundary circle shows that genes related to water
channel and growth are independent from Dof2.
Tg seed growth in Arabidopsis.
In conclusion, from coexpression networks, we de-
duced that the genes PIP1 and RD26 are key expression
genes activated following transformation of PgTIP1.
Genes PIP1 and RD26 are activated to influence growth
of seeds concurrently with PgTIP1 expression. Tran-
scription factor Dof2 plays an important role in regula-
tion of PIP1 and RD26 and their relative genes, which
revealed a way to study and control Arabidopsis thaliana
seed growth. Studying Tg plants with altered expression
of key genes using the proposed model is necessary to
better understand functions of key genes in Tg Arabi-
dopsis plants containing PgTIP1.
5. ACKNOWLEDGEMENTS
We thank Dr. Aiping Zang for maintaining the PgTIP1 transgenic
Arabidopsis plants. This work was supported by grants from the Na-
tional High Technology Research and Development Program of China
(863 Program) (Grant No. 2006AA10Z112), the National Transgenic
Program (Grant No. 2009ZX08004-008B), the Knowledge Innovation
Program of the Chinese Academy of Sciences (Grant No. KJCX2-
YW-L08), the China Manned Space Flight Technology Project, and the
National Natural Science Foundation of China (Grant Nos. 30770197,
90917009, 31070237).
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