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Gene Networks and Chromatin and Transcriptional Regulation
of the Phaseolin Promoter in Arabidopsis
CW
ek,b
Sabarinath Sundaram,a Sunee Kertbundit,a,b Eugene V. Shakirov,a Lakshminarayan M. Iyer,c Miloslav Juríc
a,1
and Timothy C. Hall
a Institute of Developmental and Molecular Biology and Department of Biology, Texas A&M University, College Station,
Texas 77843-3155
b Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 16502 Prague 6, Czech Republic
c National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894
ORCID ID: 0000-0002-9782-7973 (T.C.H.).
The complete lack of seed storage protein expression in vegetative tissues and robust expression during embryogenesis
makes seed development an ideal system to study tissue-specific expression of genes. The promoter for the Phaseolin (phas)
gene, which encodes the major seed storage protein in bean (Phaseolus vulgaris), is activated in two sequential steps:
Phaseolus vulgaris ABI3-like factor (Pv-ALF)–dependent potentiation and abscisic acid–mediated activation. In this study,
a heterologous in vivo Pv-ALF/phas-GUS (for b-glucuronidase) expression system in transgenic Arabidopsis thaliana leaves
was used in conjunction with the powerful RNA-Seq approach to capture transcriptional landscapes of phas promoter
expression. Remarkably, expression of over 1300 genes from 11 functional categories coincided with changes in the transcriptional
status of the phas promoter. Gene network analysis of induced genes and artificial microRNA–mediated loss-of-function genetic
assays identified transcriptional regulators RINGLET 2 (RLT2) and AINTEGUMENTA-LIKE 5 (AIL5) as being essential for phas
transcription. Pv-ALF binding to the RLT2 and AIL5 promoter regions was confirmed by electrophoretic mobility shift assay. RLT2
and AIL5 knockdown lines displayed reduced expression of several endogenous seed genes, suggesting that these factors are
involved in activation of endogenous Arabidopsis seed storage gene expression. Overall, the identification of these key factors
involved in phas activation provides important insight into the two-step transcriptional regulation of seed-specific gene expression.
INTRODUCTION
Recently, much has been learned about the regulation of transcription from eukaryotic promoters, and it is now apparent that
both epigenetic and genetic processes contribute to quantitative and spatial regulation of expression. Nevertheless, the exact
molecular and cellular processes that activate specific promoters in particular tissues largely remain to be elucidated. In
mammals, deregulation of transcription may result in neoplasia,
the failure to express genes responsible for cellular differentiation or inappropriate expression of positively acting transcription
factors (TFs) and nuclear oncogenes (Cox and Goding, 1991).
Similarly, failure of the regulated gene transcription process in
plants can cause disruption of normal functions (Finnegan et al.,
1996). The plant networks responsible for the maintenance of
transcriptional regulation are only now becoming apparent, and
studies taking advantage of high-throughput methods to gain
a genomic appraisal of this situation are urgently needed to
better understand such processes. Because the seed represents such a vital stage of a plant’s life cycle and synthesis of
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Timothy C. Hall (tim@
idmb.tamu.edu).
C
Some figures in this article are displayed in color online but in black and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.112714
seed storage proteins is intrinsic to seed development, seed
development is a very attractive system to study such these
plant networks.
Expression of Phaseolin (phas), the most abundant seed
storage protein in the common bean Phaseolus vulgaris, is
regulated by both genetic and epigenetic processes. It is stringently turned off during all vegetative stages of plant development by the presence of a nucleosome that is positioned
over its TATA regions (Li et al., 1998). The bean TF Pv-ALF (for
Phaseolus vulgaris ABI3-LIKE FACTOR) is orthologous to Arabidopsis thaliana ABA INSENSITIVE3 (ABI3) and is vital for transcription from the bean phas promoter (Van Der Geest et al., 1995).
Expression of ALF, in combination with the plant growth regulator
abscisic acid (ABA), has been shown to release transcriptional
constraints on the phas promoter. Pv-ALF is a member of a B3
domain family of TFs, which are found uniquely in plants and serve
as major components required for the expression of seed-specific
genes involved in seed maturation. Amino acid sequence alignment of plant DNA binding B3 domains reveals a highly conserved
globular domain with an all-b fold (Zhou et al., 2004). The Arabidopsis genome contains over a hundred genes encoding B3-type
TFs, 14 of which form the seed-specific ABI3 subfamily (McCarty
and Chory, 2000; Finkelstein et al., 2002; Carranco et al., 2004).
Other proteins have been shown to act as key transcriptional
regulators in seed maturation processes, including ABI3, Pv-ALF,
their maize (Zea mays) ortholog Viviparous1 (VP1), and several
other Arabidopsis B3-type proteins, such as Leafy cotyledon1
(Lec1), Lec2, and Fusca3 (Fus3) (Finkelstein et al., 2002; To et al.,
2006; Suzuki and McCarty, 2008).
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The contribution of chromatin to seed-specific expression in
plants was originally illustrated by work with b-phaseolin (Li et al.,
1998). Two discrete steps in expression from the phas promoter
include (1) a potentiation step, which involves Pv-ALF–initiated
remodeling of the chromatin structure at the TATA-box region and
(2) an activation step driven by ABA that results in the synthesis of
mRNA (Li et al., 1999). The chromatin remodeling step induced by
Pv-ALF is accompanied by ordered histone modifications that are
associated with transcriptional poising and activation of the phas
promoter (Ng et al., 2006). Subsequent studies in other laboratories have complemented these findings, and it now appears that
facilitating nucleosomal repositioning may be a general feature of
B3-domain protein function (Suzuki et al., 2005; Yamamoto et al.,
2010). In addition, other factors are likely to be needed to initiate
chromatin changes mediated by Pv-ALF or other B3 TFs. Although their identity and means of recruitment are not yet known,
possible candidates are proteins involved in chromatin remodeling and signal transduction, such as kinases and phosphatases
(Chandrasekharan et al., 2003b; Ng et al., 2006).
The redundant nature of multiple seed-specific B3 TFs and
the complexity of their transcriptional networks within the seed
makes it technically challenging to identify the specific details of
transcriptional activation of seed-specific genes (Braybrook and
Harada, 2008; Suzuki and McCarty, 2008). Microarray techniques have previously been used to study the roles of B3 TFs in
gene expression at specific stages of seed development (Suzuki
et al., 2003; Le et al., 2010; Yamamoto et al., 2010). Currently,
RNA-Seq has a higher sensitivity than microarrays, permitting
detection of genes expressed at very low levels (Wang et al.,
2009). In this study, we used the RNA-Seq technique to identify
and dissect the molecular mechanisms involved in altering
the chromatin structure of the phas promoter to achieve its
transcriptional activation. Using a tightly controlled, inducible,
heterologous Pv-ALF/phas-GUS (for b-glucuronidase) system,
which includes estradiol-mediated expression of Pv-ALF and
a phas-GUS reporter construct (Ng et al., 2006), which mimics
the unique characteristics of seed-specific phaseolin expression, we characterized individual components of gene networks
that are turned on by Pv-ALF to activate phas expression in
transgenic Arabidopsis leaves. The roles of identified transcription
regulators RLT2 and AIL5 in the transcriptional activation of the
phas promoter were functionally confirmed using loss-of-function
genetic assays. Our data provide key insight as to how B3 TFs
orchestrate the initial steps of chromatin remodeling necessary for
the expression of phas and related seed maturation genes.
chromatin remodeling associated with transcriptional potentiation and activation of the seed-specific phas promoter. The
transcriptionally inhibitory chromatin at the promoter of the
phas-GUS construct in Arabidopsis leaf tissue can be experimentally remodeled in two steps: (1) potentiation, in which the chromatin
architecture is relaxed as a result of estradiol-induced expression of
Pv-ALF; and (2) transcriptional activation, triggered by the exogenous application of ABA (see Supplemental Figure 1 online).
Rosette leaves of transgenic Arabidopsis plants were exposed
to either 25 mM estradiol (4E treatment for the potentiation step)
or 200 mM ABA (4A) or both estradiol and ABA (4E4A treatment
for the activation step), as described in Methods. As a negative
control for the potentiation step, leaves were also exposed to
Murashige and Skoog (MS) medium only (4MS treatment). In
accordance with previous results (Ng et al., 2006), RT-PCR
analysis detected Pv-ALF mRNA expression in leaves during
potentiation (4E) and activation (4E4A) steps, while GUS mRNA was
observed only during the activation step (4E4A) (Figure 1A). Furthermore, GUS protein activity was detected by histochemical staining
only in the presence of both Pv-ALF and ABA (4E4A) (Figure 1B).
In order to identify the events and processes involved in the
transition of the phas promoter from silent to active state, we
analyzed two biological replicates via high-throughput sequencing. The RNA-Seq libraries were prepared from the four
treatments (4MS, 4E, 4A, and 4E4A) and subjected to highthroughput sequencing using the Illumina GA3 platform. The
Illumina reads were then aligned with the GUS marker gene and
the Pv-ALF transgene. As expected from RT-PCR and histochemical staining data, GUS transcripts were only detected
during the phas activation step (4E4A) (Figure 1C), whereas
Pv-ALF transcripts were detected at very high levels during both
potentiation (4E) and activation (4E4A) steps (Figure 1D), further
illustrating the high stringency of the phas-GUS reporter system.
These data further illustrate the remarkable power of the RNASeq approach to detect even the lowest abundance transcripts.
An important aspect of the heterologous system used here is
that Pv-ALF and the phas-GUS constructs are ectopically expressed in transgenic leaves, where the key endogenous Arabidopsis B3-type seed-specific TFs, such as FUS3, LEC1, LEC2,
and ABI3 itself, are not present (Gao et al., 2009). Thus, we
conclude that the RNA-Seq approach is uniquely suited to
provide a detailed analysis of kinetic changes in transcription
that occur at the phas promoter and throughout the leaf transcriptome in response to Pv-ALF expression (potentiation step)
and exogenous application of ABA (activation step).
Identification of Differentially Expressed Genes
RESULTS
Characteristics of the Inducible, Heterologous
Pv-ALF/phas-GUS System
Under natural conditions in developing seeds, events associated with potentiation and activation of the phas promoter are
inseparable. By contrast, in the estradiol-inducible heterologous
Pv-ALF/phas-GUS system, these events can be temporarily
separated (Ng et al., 2006). Therefore, this system provides
an excellent means to obtain an improved understanding of
Data obtained by Illumina sequencing were processed, filtered,
and normalized using the Illumina pipeline (http://www.illumina.
com) to generate fast-q files, which were then analyzed using
the RNA-Seq module of CLC Genomic Workbench (http://www.
clcbio.com). For biological replicate 1, 16.8 to 22.9 million reads
were obtained, and for biological replicate 2, 25.7 to 33.4 million
reads were obtained. Of these reads, over 84% were uniquely
mapped to the annotated Arabidopsis genome (TAIR10) (see
Supplemental Table 1 online). The expression level of individual
genes was quantified by counting the number of reads per
Insight to Pv-ALF–Mediated Gene Expression
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Figure 1. Expression of phas-GUS in Transgenic Arabidopsis Pv-ALF Leaves Is Dependent on Estradiol-Inducible Pv-ALF and Exogenous ABA.
(A) RT-PCR of Pv-ALF and GUS transcripts. Pv-ALF transcripts are expressed only during the potentiation (4E) and activation (4E4A) steps. GUS
transcripts are only present during the activation step. EF1a is used as a control.
(B) GUS histochemical assay in uninduced Pv-ALF plants (4MS) or plants treated with 25 mM estradiol (4E), 200 mM ABA (4A), and with both 25 mM
estradiol and 200 mM ABA (4E4A). Bar = 2 mm.
(C) and (D) RNA-Seq expression values for GUS (C) and Pv-ALF (D) transcripts under the same four experimental conditions are shown.
[See online article for color version of this figure.]
kilobase per million mapped reads (RPKM), and Quantile normalization was applied to the RPKM values by taking the mean
as the value to be normalized. A gene was considered to be
expressed if it had at least one sequence read aligned with it. We
detected expression of more than 16,000 genes in the 4MS, 4E,
4A, and 4E4A samples (see Supplemental Table 1 online). The
shapes of distributions for average RPKM values were very
similar among the four treatments of the two replicates (see
Supplemental Figure 2A online). Correlation coefficients were
calculated for RPKM values after eliminating genes with zero count
in either of the two replicates. Between the two biological replicates, correlation coefficients for each condition ranged from 0.90
to 0.94, indicating a good statistical correlation between replicates
(Figures 2A and 2B; see Supplemental Figures 2B and 2C online).
To identify for candidate genes involved in phas promoter
activation, expression data from RNA-Seq experiments were
imported into the GeneSpring GX12.5 (Agilent Technologies)
analysis tool. Statistical significance of expression values for
the two biological replicates with four conditions was analyzed
using the analysis of variance test with multiple testing corrections by the Benjamini and Hochberg method (Benjamini and
Hochberg, 1995) to correct false discovery rate, and asymptotic P
value computation was performed to determine differential expression of genes. To judge the significance of differences in
expression of most genes, we used the threshold of fold change
$2 between treatment and control samples with corrected
P value # 0.05. To detect truly differentially expressed lowexpressing TFs and chromatin-modifying genes, we used the
corrected P value # 0.2 (see below). Using this analysis, we
identified 1208 genes that were upregulated by at least twofold
(Figure 3A) and 2421 genes downregulated by at least twofold
(see Supplemental Figure 3 online) in 4E4A condition compared with the 4MS control condition. Likewise, 569 and 809
genes were up- or downregulated by at least twofold in 4E
treatment. To identify 4E-specific upregulated genes (genes
uniquely activated by Pv-ALF or potentiation specific) and 4E4Aspecific upregulated genes (ABA-dependent Pv-ALF–activated
genes), three-way Venn diagrams were constructed based on
the number of upregulated genes in any of the three conditions
(Figure 3A). This diagram showed that 275 and 116 genes were
specifically upregulated by at least twofold only in 4E4A and 4E,
respectively (see Supplemental Data Sets 1 and 2 online). In
addition, we identified 35 overlapping upregulated genes in both
4E and in 4E4A conditions and 480 overlapping upregulated
genes in both 4A and 4E4A conditions. Similarly, 183 and 878
genes were substantially downregulated during the potentiation
(4E) and ABA-mediated activation (4E4A) steps of phas-GUS
expression (see Supplemental Figure 3 and Supplemental Data
Sets 3A and 3B online). Importantly, many potentiation- and
activation step–specific Arabidopsis promoters are strongly
enriched for known or predicted Pv-ALF binding DNA elements
(see Supplemental Table 2 online).
To confirm gene expression differences identified by RNASeq, we used real-time RT-PCR analysis to validate expression
patterns of 20 genes that were upregulated either in 4E or 4E4A
treatment conditions, including low-expressed chromatin remodeling genes detected in 4E and highly expressed LATE EMBRYOGENESIS ABUNDANT (LEA) family genes expressed in 4E4A
treatment. Fold change differences in expression levels observed in
RNA-Seq and quantitative RT-PCR (qRT-PCR) data for all 20 genes
displayed similar patterns and also showed good correlations of
0.90 and 0.94 for 4E and 4E4A-specific genes, respectively (Figures
2C and 2D; see Supplemental Table 3 online).
Functional Classification and Annotation
of Upregulated Genes
Of the 1324 genes detected in both biological replicates that
were upregulated in 4E and 4E4A treatments, 1101 (;83%)
genes were annotated and functionally classified into 11 distinct
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Figure 2. Assessment of Reproducibility between the Two RNA-Seq Biological Replicates and Validation by qRT-PCR.
Correlation plots of the RPKM reads for each replicate for 4E (A) and 4E4A (B) treatments are shown. Coverage across transcripts was calculated by
counting the number of reads on aligned transcripts generated by TopHat. All correlation analysis and plots were performed using the R statistical
package. x and y axes show the number of counts generated with the program HTSeq-count. Values of Pearson’s correlation coefficient (r) are shown
on each plot. For RNA-Seq data validation by qRT-PCR, Pearson correlation coefficients for 4E (r = 0.90) (C) and 4E4A (r = 0.94) (D) were obtained by
plotting RNA-Seq data on y axis and qRT-PCR data on x axis. RNA-Seq data and qRT-PCR data are shown as fold change in gene expression
compared with the 4MS control.
[See online article for color version of this figure.]
categories that included chromatin proteins, TFs, and signaling
and seed functions (Figure 3B; see Supplemental Data Set 4
online). Activation of genes from these diverse functional categories corresponds to a robust transcriptional response that
occurs during ABA signaling and seed development. Indeed, as
reported previously in studies on ABA signaling (Cutler et al.,
2010), we observed coexpression of genes involved in seed
function, stress response (including dehydration, drought, salinity, and other environmental stresses), sugar metabolism (e.g.,
genes involved in the biosynthesis of protective sugars like
trehalose), polyamine metabolism (e.g., Arg decarboxylase
involved in putrescine biosynthesis), enzymes that detoxify reactive oxygen species, transporters, lipid metabolism, pathogen
response, and many distinct signaling pathways (Figure 3B).
This suggests a strong crosstalk between these pathways and
ABA signaling.
A comparison of the genes induced in our heterologous
system with those reported in other related studies showed
a substantial overlap (see Supplemental Figure 4 online and
Supplemental Data Set 5 online). For example, in a study of
genes induced upon heterologous expression of maize VP1 in
Arabidopsis abi3 background (Suzuki et al., 2003), 194 genes
were induced upon ABA treatment and VP1 expression, of
which at least 140 genes (73%) are also induced in our heterologous system. Similarly, of 98 genes identified by Mönke et al.
(2012) using ChIP-chip and transcriptome analysis as part of the
Arabidopsis ABI3 regulon, 77 (78%) were also specifically upregulated in our system. By examining ectopic expression of
seeds-specific genes in our heterologous system, we identified
at least 50 genes (see below) that were previously reported as
being expressed only in seeds (Le et al., 2010). Notably, only
genes specifically upregulated in the 4E4A treatment overlapped
with those detected in the above studies, strongly suggesting
that the combined effect of Pv-ALF expression and ABA application recapitulates a subset of physiological responses that
naturally occur during seed development.
Analysis of Seed-Specific Genes Turned on during
Activation of the phas Promoter in Leaves
The list of genes expressed during activation of the phas promoter contains 37 genes (P value of # 0.05) that, on the basis of
domain composition, are annotated as being involved in seed
storage (Figure 4A; see Supplemental Data Set 6 online). In
Insight to Pv-ALF–Mediated Gene Expression
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Figure 3. Identification and Functional Classification of ABA-Dependent and -Independent Genes Induced in the Presence of Pv-ALF.
(A) Venn diagram of upregulated genes specific to the potentiation (4E) and activation (4E4A) steps of phas-GUS expression is shown.
(B) Functional classification of Pv-ALF–activated genes. The data consist of genes that have at least a twofold change in expression from two
independent RNA-Seq experiments. The complete list is presented in Supplemental Data Set 4 online.
addition to these, we have identified several other functional
categories of genes that have been reported to be expressed in
a seed-specific fashion (Becerra et al., 2006; Le et al., 2010;
Mönke et al., 2012). Comparison of our data with the abovementioned studies revealed that 76 Arabidopsis seed-specific
genes (P value of # 0.05) were upregulated in leaves during the
activation step of phas expression (Figure 4B). These genes
were functionally classified into 11 major categories, the largest
of which includes 18 genes that are either involved in sequestration of seed lipids or serve as seed storage proteins, such as
several cupins and oleosins (see Supplemental Figure 5 and
Supplemental Data Set 7 online). The second largest category
contains 10 seed-specific genes associated with dehydration
and stress response, such as dehydrin-like and LEA proteins.
Strikingly, only one Arabidopsis seed-specific TF was recovered
(AIL7), and this was also found in a set of previously identified 48
seed-specific TFs (Le et al., 2010). Overall, the 4E4A data set
shows that Pv-ALF and ABA trigger expression in Arabidopsis
leaves of many endogenous experimentally verified seed-specific
genes, which comprise a distinct subnetwork of the more complex natural transcriptional network in the seeds. Furthermore, our
data suggest that the general pathway for activation of seedspecific gene networks is partially conserved between bean and
Arabidopsis, such that a seed-specific TF from bean is capable
of using components of the endogenous Arabidopsis transcriptional network to induce a comparable response.
Differential Expression of a Unique Set of Core Genes
Involved in ABA Signaling
Approximately 50 genes have been reported to be part of the
core ABA signaling pathway (Cutler et al., 2010, and references
therein), of which 30 genes are differentially regulated (P values of
# 7.7E-04 to 0.09) in our heterologous system (see Supplemental
Figure 6A and Supplemental Data Set 8 online). Genes that are
upregulated include three PP2C-like phosphatases (ABI2, HAB1,
and AHG1) that function as negative regulators of the ABA signaling pathway, three kinases (OST1, CPK6, and CPK11) that
positively regulate ABA signaling, and the TF ABI5, which regulates many of the genes involved in ABA signaling. By contrast,
downregulated genes include two START-domain ABA receptors
(PYL7 and PYL9), the kinases CPK3 and SNRK2.3, which positively
regulate ABA signaling, and the farnesyltransferase b-subunit
ERA1, which attenuates ABI3 expression by modifying ABI3
(Brady et al., 2003). The presence of this assortment of differentially
regulated genes for a subset of the core ABA signaling pathway
implies that expression of Pv-ALF (or its Arabidopsis ortholog ABI3)
in conjunction with ABA hormone induces a distinct signaling response that specifically regulates a specific group of genes during
seed development. This observation may enable further dissection
of various phosphatases and kinases believed to operate at this
stage of development (Zhang et al., 2012) and may also help in
understanding how the ABA signaling pathway is fine-tuned for
seed development as opposed to other stress responses.
Analysis of Specific TFs and Chromatin Proteins Regulated
by Pv-ALF in the Presence of ABA
One of our key interests is to determine the identity of proteins
that function in the transcriptional regulation of the phas promoter and their mechanism of action. Previous DNase I footprinting (Li et al., 1999) and chromatin immunoprecipitation (Ng
et al., 2006) experiments established that changes in histone
modification status and nucleosome positioning are required to
initiate expression from the phas promoter and that chromatinassociated proteins are likely to have a major impact on phas
expression. Furthermore, in vivo footprinting analysis of the phas
promoter showed that over 20 cis-elements in the proximal part
of the promoter are bound by potential TFs (Li and Hall, 1999).
Whereas some of these factors are known or predicted, such as
the RY/Sph-element binding Pv-ALF/ABI3, the ABRE element
binding ABI5, and a CAAT-box binding protein, the precise
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Figure 4. Analysis of Seed-Specific Arabidopsis Genes Expressed during Activation of the phas Promoter in Leaves.
Relative expression profiles of identified seed storage genes (A) and other seed-specific genes (B). The color indicates the degree of fold change: red,
high; blue, low. Hierarchical clustering (heat map) was generated using the Euclidean-based method.
identity of most other factors that bind these elements still remains to be elucidated. We therefore studied the expression
patterns of TFs and chromatin proteins in our data set.
Although we detected several highly upregulated (fold change
at least 2) TF and chromatin protein coding genes in the 4E and
4E4A condition, transcriptional regulators are often known to be
expressed at very low levels (Alvarez et al., 2006; Vernimmen
et al., 2011). We identified a total of 85 upregulated TFs and
22 chromatin proteins with P values from 0.05 to 0.2 (see
Supplemental Data Sets 9 and 10 online). Overall, the identified
TFs belong to 14 distinct protein superfamilies, with at least
seven representatives from each of the NAC/NAM, MYB, AP2,
WRKY, BZIP, and C2H2 zinc finger families dominating the
landscape (see Supplemental Figure 6B and Supplemental Data
Insight to Pv-ALF–Mediated Gene Expression
Set 9 online). Genes showing the highest fold induction in 4E4A
include those encoding BZIP factor ABI5 (63x), NAC domain–
containing TFs, such as anac042 (27x), anac032 (17x), and RHL41
(20x), and the AP2 domain–containing TF DREB2 (10x). Of the highly
expressed TFs, ABI5 has a major role in regulating seed genes
regulated by ABA signaling, and it also regulates the phas promoter
by binding to the G-box element (Ng and Hall, 2008). The homeodomain TF HB-7 regulates ABA signaling by regulating PP2C and
ABA receptor expression (Valdés et al., 2012). At least 10 other
known TFs induced in our system are either induced by or are regulators of various stress responses, including ABA response. A single
member of the CAAT-box binding histone fold family, NF-YC2, is
induced in the activation step. The CCAATT-box present in the phas
promoter is responsible for uniform and high-level expression of the
phas promoter in the embryo and is thus likely bound by this CAATbox binding factor (Chandrasekharan et al., 2003a).
Of the 22 identified chromatin proteins, 12 and five were
upregulated during potentiation and activation of the phas promoter, respectively. Chromatin-associated genes induced in this
data set typically encode low abundance proteins showing up to
10-fold induction. These chromatin proteins include four nucleosome remodeling SWI2/SNF2 ATPases (CHR11, CHR18, and
CHR38) and two demethylases (LDL2 and AT5G46910) (Figure
5A; see Supplemental Data Set 10 online). Other chromatin
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proteins upregulated in the 4E and/or 4E4A treatment include
modified peptide binding adaptors containing the chromo family
of domains that binds methylated peptides and two related
proteins with a homeodomain, DDT domain, HARE-HTH domain, and WHIM motifs (RLT1 and RLT2) that are predicted to
specifically interact with the ISWI class of SWI2/SNF2 ATPases
(Aravind and Iyer, 2012). Of the structural components of chromatin, only histones H2B and H3 were upregulated upon activation. Of interest is the induction of chromatin genes involved in
DNA repair. These include PARP-1 (APP) and related enzymes
known to function in DNA repair, the RAD5 and HARP/SMARCAL-1like (Ghosal et al., 2011) SWI2/SNF2 ATPases, and the MEI1-like
multi-BRCT domain protein (Grelon et al., 2003). These data
suggest that a robust DNA repair pathway is activated under
these conditions. This observation supports the critical role of
DNA repair proteins during seed imbibition (Balestrazzi et al.,
2011) and suggests that the combined effect of ABI3/Pv-ALF
and ABA activates this pathway during seed development.
Arabidopsis Gene Networks Involved in the Establishment of
the Potentiation and Activation Steps of phas Expression
Although overexpression of several chromatin proteins and TFs
correlates with phas promoter potentiation and activation, the
Figure 5. Expression Profiles of Chromatin Genes Identified by RNA-Seq in Leaves and Molecular Interactions of Candidate Genes with Pv-ALF/ABI3.
(A) Heat map of chromatin genes activated in 4E and 4E4A treatments. The color indicates the degree of fold change: red, very high; yellow, high; blue,
low. Hierarchical clustering heat map was performed using Euclidean distance method.
(B) Map of molecular interactions between Pv-ALF/ABI3 and Pv-ALF–responding chromatin genes. Purple nodes denote regulatory edge. Green
triangles define TFs. Orange circles on At3g13682 indicate small molecule modifications.
(C) Coexpression analysis of TFs regulated by Pv-ALF/ABI3.
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challenge remains to identify exactly which regulators specifically target the phas promoter. We addressed this using two
topical bioinformatic approaches, namely, VirtualPlant software
(Katari et al., 2010) for chromatin related proteins and ATTED II
(Obayashi et al., 2011) for TF interactions with ABI3/Pv-ALF.
ABI3 was included in the input gene list in place of bean Pv-ALF,
since the most parsimonious hypothesis is that exogenous Pv-ALF
exerts its function through the Arabidopsis ABI3-dependent gene
network. Results from the VirtualPlant network analysis revealed
that ABI3/Pv-ALF bears a regulatory edge complementary
only with 4E-specific PARP-1/APP, RLT1, and RLT2 genes
(Figure 5B), suggesting that Pv-ALF initially regulates transcription of these genes during the potentiation step.
While VirtualPlant analysis indicated that ABI3/Pv-ALF interacts
with several TFs, ATTED-II analysis revealed that the strongest
coexpression connection emerged only for AINTEGUMENTALIKE5 (AIL5; At5g57390), a poorly characterized TF (Tsuwamoto
et al., 2010), and for an extensin-like gene At2g27380 (Figure
5C; see Supplemental Figure 7 online). Importantly, the AIL5 gene
was expressed upon addition of ABA and was also exceptionally
highly expressed under 4E4A treatment, but not in other conditions. Furthermore, promoter analysis using the Known CIS
Analyzer AGRIS software indicates that the AIL5 promoter harbors many Pv-ALF binding motifs (three ABRE-like, six G-box,
and eight RAV1-A motifs). Taken together, these observations
indicate that Pv-ALF may specifically upregulate expression of
RLT2/RLT1/PARP-1(APP) and AIL5, which in turn may directly
or indirectly regulate phas expression. Hence, these computational results provide a rationale for genetic tests aimed at understanding the functional role of these candidate genes in the
two-step transcriptional activation of the phas promoter.
RLT2 and AIL5 Are Required for Efficient Activation
of phas-GUS Expression in Transgenic Arabidopsis Leaves
To dissect the putative network of genes involved in the activation of phas-GUS expression, we first focused on the two
most likely candidates, RLT2 and AIL5, computationally predicted to be the key factors is the potentiation and activation
steps of phas expression, respectively. To functionally characterize the role of these proteins in phas-GUS transcription, the
Pv-ALF plants used to perform RNA-Seq experiments in this
study were supertransformed with artificial miRNA (amiRNA)
constructs targeting either RLT2 or AIL5 genes. This approach
uses expression of an endogenous microRNA precursor engineered to yield a 21-nucleotide double-stranded amiRNAs
complementary to and directed against either RLT2 or AIL5
(Alvarez et al., 2006; Schwab et al., 2006). Expression of RLT2
and AIL5 amiRNAs was driven by the constitutive cauliflower
mosaic virus 35S promoter. Three knockdown lines for each
gene were selected and used to investigate the function of the
corresponding gene during the Pv-ALF–mediated phas-GUS
expression in leaves. All experiments were performed with either
untreated leaves (4MS condition) or leaves treated with estradiol
and ABA as described above (4E4A condition).
In the uninduced 4MS condition, expression of RLT2 mRNA in
the RTL2-13, RTL2-2, and RTL2-11 amiRNA lines decreased by
37 to 50% compared with the untransformed Pv-ALF control
line (Figure 6A, black bars). The relatively low reduction of RLT2
expression by the gene-specific amiRNA could be partially due
to difficulties in reducing the steady state level of certain housekeeping transcripts subject to negative feedback regulation
(Ossowski et al., 2008). Nevertheless, no reduction in RLT2 expression was observed in the vector-only control line. As expected from
the RNA-Seq data, upon induction (4E4A treatment), RLT2 mRNA
levels increased over fourfold in the control Pv-ALF line (Figure 6A,
white bars). By contrast, no induction of RLT2 gene expression
was observed in the three amiRNA lines, and RLT2 mRNA levels in
4E4A condition stayed near the 4MS levels. Remarkably, while
GUS mRNA can only be detected in the 4E4A-induced condition
(white bars), its expression in all three RLT2 amiRNA lines was
reduced by up to 98% in comparison to the Pv-ALF control line
(Figure 6B). Thus, this drastic decrease in phas-GUS expression
in the leaves of RLT2 amiRNA lines largely correlates with the
lack of RLT2 induction in 4E4A condition. Importantly, knockdown of RLT2 expression did not affect the expression of the
Pv-ALF transgene (Figure 6C), suggesting that RLT2 functions
downstream of Pv-ALF to regulate the potentiation step of expression from the phas promoter. Taken together, these data
point to a specific mechanism, in which the threshold RLT2 level
in 4MS condition is not sufficient to activate phas-GUS expression in leaves, and suggest that Pv-ALF–dependent induction of RLT2 is necessary to trigger phas-GUS expression. A
similar threshold mechanism for the basal level of p53 and toll
like receptor 9 expression has recently been described for the
activation of apoptosis and immune responses in human cells
(Assaf et al., 2009; Kracikova et al., 2013).
As with the RLT2 amiRNA lines, real-time RT-PCR analysis
revealed that, in the uninduced 4MS condition, AIL5 mRNA
levels in the three independent AIL5 knockdown lines were reduced on average by 56%, compared with the untransformed
Pv-ALF control line (Figure 6D, black bars). Upon induction
(4E4A treatment), AIL5 mRNA levels increased 2.7-fold over
4MS levels in the control Pv-ALF line (Figure 6D, white bars). By
contrast, the level of AIL5 gene expression in AIL5-1, AIL5-2,
and AIL5-5 amiRNA lines, while slightly increased, did not
exceed the 4MS levels in uninduced control Pv-ALF leaves.
Importantly, GUS mRNA levels in 4E4A condition were 62 to
78% lower in the AIL5 amiRNA lines than in the control Pv-ALF
plants (Figure 6E), indicating that AIL5 is required for efficient
transcriptional activation of the phas-GUS transgene. Similar to
the situation in the RLT2 amiRNA lines, Pv-ALF expression was
not altered in AIL5 amiRNA lines (Figure 6F).
Interestingly, downregulation of the RLT2 and AIL5 genes also
reduced the induction of several endogenous Arabidopsis seed
genes in the 4E4A condition in transgenic leaves. Specifically,
RLT2 downregulation resulted in a decrease of expression by
56 to 90% for LEA (At3g17520), 65 to 91% for cupin (At4g36700),
and 86 to 90% for oleosin (At5g40420) compared with the
control Pv-ALF line (see Supplemental Figure 8 online). Similarly,
downregulation of AIL5 also resulted in decreased mRNA expression of cupin, oleosin, and LEA genes by up to 83, 85, and
69%, respectively (see Supplemental Figure 8 online). Taken
together, these data not only provide functional evidence implicating RLT2 and AIL5 genes as key regulators of a gene
network used by Pv-ALF for the transcriptional activation of
Insight to Pv-ALF–Mediated Gene Expression
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Figure 6. RLT2 and AIL5 Regulate phas Expression in Leaves.
Expression levels of RLT2 (A), AIL5 (D), GUS ([B] and [E]), and Pv-ALF ([C] and [F]) were analyzed in three independent supertransformed lines carrying
either amiRNA-RLT2 ([A] to [C]) or amiRNA-AIL5 constructs ([D] to [F]), as well as in untransformed control Pv-ALF plants and plants supertransformed
with vector only (VC) are shown. qRT-PCR reactions were performed using RNA samples isolated from uninduced 4MS (black bars) or induced 4E4A
(white bars) samples. Expression values plotted are average of three biological replicates. Error bars represent SD. Double asterisks represent significant
differences between expression in an uninduced Pv-ALF line and an uninduced amiRNA lines (P value < 0.05, Student’s t test). Single asterisks
represent significant differences between the induced Pv-ALF line and induced amiRNA lines (P value < 0.001, Student’s t test). Expression levels of
GUS and Pv-ALF in uninduced 4MS samples are below the detection limit.
phas-GUS in leaves, but also indicate that RLT2 and AIL5 can
activate expression of several endogenous Arabidopsis seedspecific genes in the leaves of Pv-ALF plants.
Downregulation of RLT2 and AIL5 Affects Endogenous Seed
Gene Expression in Seeds
The apparent role of RLT2 and AIL5 in the activation of seedspecific gene expression in leaves indicates that these genes
may also be involved in the transcriptional regulation of endogenous seed genes. To address this possibility, we examined expression of various genes involved in seed maturation processes
in the mature seeds of Pv-ALF and amiRNA lines not subjected to
any treatment. Similar to the situation in leaves, RLT2 expression
in the seeds of RLT2 amiRNA lines was reduced by 34 to 49%
(Figure 7A), whereas AIL5 expression in the seeds of AIL5
amiRNA lines was reduced by 47 to 60% compared with the
seeds of control Pv-ALF plants (Figure 7B).
As expected in the absence of estradiol, Pv-ALF transcripts
were not detected in the mature seeds of Pv-ALF plants and all
amiRNA lines. However, GUS expression was detected in all
lines analyzed, even in the absence of induced Pv-ALF and
exogenous ABA (Figure 7C). This is likely due to the expression
of endogenous ABI3 protein (Figure 7D) and naturally elevated
levels of intracellular ABA during the seed maturation processes
in Arabidopsis. Nevertheless, GUS transcript levels in the seeds
of amiRNA-RLT2 and amiRNA-AIL5 lines were reduced by 35 to
40% in comparison with the seeds from the control Pv-ALF line
(Figure 7C). Furthermore, accumulation of maturation phase–
specific mRNA transcripts that are necessary for seed development,
such as cupin, oleosin, CRC, and LEA genes (Parcy et al., 1994;
Crowe et al., 2000), was also decreased in seeds of all amiRNA
knockdown lines (Figures 7E to 7H). By contrast, expression
levels of endogenous Arabidopsis seed-specific B3 TFs ABI3
and FUS3, or proteins known to be major regulators of seed
development, such as ABI4 and ABI5 (Brocard-Gifford et al.,
2003), were not altered in RLT2 and AIL5 knockdown lines
(Figure 7D; see Supplemental Figure 9 online). These data
strongly suggest that RLT2 and AIL5 are involved in the regulatory processes that activate seed storage gene expression
in Arabidopsis seeds. Interestingly, despite this reduction in
expression of some seed storage genes, all individual RLT2
and AIL5 knockdown lines investigated here did not show abnormal changes in seed development. This is likely due to the
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The Plant Cell
(RRL) and used in qualitative electrophoretic mobility shift assays
(EMSAs) (Shakirov et al., 2009). PCR-amplified 32 P-labeled
365-bp DNA fragments corresponding to the 2340 to +25 regions
of putative target promoters were used as probes. Although
the DNA sequence specificity of B3-domain TFs is not well
defined, Arabidopsis ABI3 protein is known to bind to ABRE
motifs and RY/G-box elements (Ezcurra et al., 2000). As expected from previous studies (Carranco et al., 2004), a retarded
band was observed when Pv-ALF was present in the binding
mixture containing the phas promoter DNA (Figure 8, lane 4).
Similar shifted bands of various intensities were also detected
when the binding mixture contained the RLT2 or AIL5 promoters
(Figure 8, lanes 8 and 12). The shifted complexes were only
observed in the presence of Pv-ALF protein, as incubation of the
target DNA probes with the reticulocyte extract alone did not
yield a similarly shifted band (Figure 8, lanes 3, 7, and 11).
Furthermore, incubation of RRL-expressed Pv-ALF protein
with the 2340 to +25 promoter region of At1g26680 gene (a
negative control sequence harboring no known or predicted
Pv-ALF binding motifs) did not yield a retarded band (Figure 8, lanes
1 and 2). Competition EMSAs were next used to further confirm
sequence specificity of Pv-ALF binding to RLT2 and AIL5 promoters. As anticipated for a specific competitor, the addition of
100-fold excess of cold (unlabeled) phas, RLT2, and AIL5 PCR
products resulted in complete disappearance of the corresponding
shifted bands, which in all cases were replaced by a faster migrating
diffuse smear, likely representing dynamic dissociation of the
Figure 7. Seeds of RLT2 and AIL5 amiRNA Knockdown Plants Show
Reduced Expression of Endogenous Arabidopsis Seed Maturation Genes.
Expression of RLT2 (A), AIL5 (B), GUS (C), ABI3 (D), Cupin (E), Oleosin
(F), CRC (G), and LEA (F) was analyzed by qRT-PCR using RNA isolated
from fully developed seeds of three independent supertransformed lines
carrying either amiRNA-RLT2 or amiRNA-AIL5 constructs, as well as
from untransformed control Pv-ALF plants. Seeds were not treated with
estradiol or exogenous ABA prior to analysis. Expression values plotted are
average of three biological replicates. Error bars represent SD. Asterisks
represent significant differences (P value < 0.023, Student’s t test).
[See online article for color version of this figure.]
fact that plants harbor multiple redundant pathways governing
expression of seed-specific genes, which can compensate for the
shortage of certain seed storage proteins with the increased accumulation of other similar proteins during seed filling. This scenario
has recently been demonstrated in soybean (Glycine max),
where RNA interference–mediated suppression of two major
seed storage proteins, glycinin and conglycinin, resulted in
rebalancing of soybean’s proteome, maintaining wild-type levels
of seed protein and storage triglycerides (Schmidt et al., 2011).
Pv-ALF Binds to the Putative RLT2 and AIL5 Promoter
Regions in Vitro
Having shown that RLT2 and AIL5 are required for phas-GUS
expression, we next asked if Pv-ALF can directly bind putative
RLT2 and AIL5 promoters in vitro. Recombinant Pv-ALF protein
was expressed in the rabbit reticulocyte lysate expression system
Figure 8. Qualitative Analysis of Pv-ALF Interaction with RLT2 and AIL5
Promoter Sequences in Vitro.
Equal amounts of Pv-ALF protein were incubated with the indicated
promoter sequences and the resulting protein-DNA complexes separated by native PAGE. Unprogrammed RRL reactions lacking
ectopic DNA template for Pv-ALF protein were used as negative controls
for each individual DNA probe (lanes 1, 3, 7, and 11). Specific Pv-ALF–
DNA complexes (lanes 4, 8, and 12) are indicated by a black arrow.
Asterisks designate a nonspecific band present in the negative RRL-only
control reactions (lanes 3, 7, and 11). The promoter region of the
At1g26680 gene (-ve probe), which harbors no known or predicted
Pv-ALF binding motifs, was used as a negative control for Pv-ALF
binding (lanes 1 and 2). Competition assays were performed with a 100-fold
excess of unlabeled PCR products corresponding to either the specific
phas (lane 5), RLT2 (lane 9), and AIL5 (lane 13) promoter regions or to
nonspecific promoter region of the At1g26680 gene (lanes 6, 10, and 14).
32P-labeled
Insight to Pv-ALF–Mediated Gene Expression
Pv-ALF-32P–labeled DNA complex in the presence of specific
competitors (Figure 8, lanes 5, 9, and 13). By contrast, the addition of 100-fold excess of cold nonspecific At1g26680 probe
did not result in any change in the profile of Pv-ALF–shifted band
(Figure 8, lanes 6, 10, and 14), further confirming the specificity
of Pv-ALF interaction with the phas, RLT2, and AIL5 promoters
in vitro. Taken together, these results provide biochemical evidence for the ability of Pv-ALF to interact with RLT2 and AIL5
promoters in vitro and clearly suggest that Pv-ALF may directly
stimulate expression of RLT2 and AIL5 genes in vivo.
DISCUSSION
Inducible, Heterologous Pv-ALF/phas-GUS System for the
Discovery of Seed-Specific Transcriptional Networks
in Arabidopsis
The development of the heterologous phas-GUS expression system, which includes estradiol-mediated expression
of Pv-ALF and the phas-GUS reporter construct, provides a
powerful strategy for dissecting the molecular events involved in
expression of seed-specific genes (Ng et al., 2006). Transcriptional activation of the phas promoter is achieved through a series of changes in its chromatin architecture from repressed to
potentiated to activated form. Importantly, the heterologous
expression system described here has been shown to work not
only in bean and Arabidopsis, but also in tobacco (Nicotiana
tabacum) (Li et al., 1999). These findings indicate that, although some aspects of phas activation in different dicot
lineages are expected to be species specific, the major players
and pathways involved in the two-step expression of seedspecific genes are evolutionarily conserved among Fabales,
Brassicales, and Solanales.
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When constructing the likely Arabidopsis gene network responsible for the activation of phas-GUS expression, our
approach relied on several key hypotheses, which were
proposed in previous reports. First, because histone disposition
and chromatin modifications are essential for phas-GUS expression (Li et al., 1999; Ng et al., 2006), our method centered on
the analysis of chromatin genes (i.e., genes already known to be
involved in chromatin biology). Second, we assumed that the
mode of action of Pv-ALF in transcription of seed-specific genes
is similar to that of Arabidopsis ABI3. Arabidopsis ABI3 and bean
Pv-ALF are indeed orthologous and share over 46% amino
acid identity and have the highest similarity among all B3-type
Arabidopsis proteins (Bobb et al., 1995). Interestingly, the more
distant maize VP1 (a monocot member of this orthologous
group) can complement ABI3 defective Arabidopsis plants
(Suzuki et al., 2001), further supporting the functional similarity
between Pv-ALF and ABI3 action. In addition, Pv-ALF and ABI3
exhibit similar developmentally regulated expression and DNA
binding patterns (Gao et al., 2009). Finally, while we cannot
completely rule out the possibility that some of the genes
uniquely present in 4E or 4E4A are irrelevant due to nonspecific
Pv-ALF binding to their promoters, a majority of proteins identified in this analysis do have a presumed connection to ABI3.
Epigenetic Regulation by Pv-ALF–Mediated
phas Expression
To dissect epigenetic mechanisms involved in Pv-ALF-mediated
phas expression, we used a curated list previously derived
comprehensive list of chromatin proteins (Iyer et al., 2008) and
compared their transcription profiles across all treatments. Our
RNA-Seq data demonstrated a remarkable enrichment of seedspecific proteins and many families of chromatin modifying and
remodeling enzymes. These results support previous observations
Figure 9. Model Depicting Sequential Changes in Chromatin Modifications over the phas Promoter during Potentiation and Activation.
In the repressed state during vegetative growth, the promoter is repressed by nucleosomes bearing dimethylated H4-K20. Pv-ALF–mediated potentiation (Step 1) is predicted to recruit RLT2, a component of ISWI chromatin-remodeling complex that also contains the CHR11-like SWI2/SNF2
ATPase. During this stage, ordered histone modifications occur by demethylation of histone H3-K4, acetylation of H3-K14 and H4-K5, and histone
methylation (Ng et al., 2006). As illustrated in Step 1, this results in remodeling of the chromatin architecture over the TATA region of the phas promoter
but does not lead to transcriptional activation in the absence of ABA. During the ABA-dependent activation illustrated in Step 2, Pv-ALF induces AIL5,
which activates the expression of the phas promoter.
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The Plant Cell
that dynamic changes in chromatin architecture are likely to be
required for the highly regulated expression of seed-specific
genes. This conclusion is also consistent with earlier reports
that many genes involved in embryogenesis are repressed during
vegetative growth or in vegetative tissues by several independent
mechanisms of chromatin remodeling, which are dependent on
Pickle (Li et al., 2005), Brahma (Tang et al., 2008), trihelix repressors
(Gao et al., 2009), and polycomb group proteins (Makarevich et al.,
2006). In addition, our results provide new insight and candidates
for the developmentally programmed mechanisms required for
changing the chromatin landscape in order to initiate the expression of the phas promoter.
Molecular Mechanism of Seed-Specific
ABI3/Pv-ALF–Mediated phas Chromatin Remodeling
Results from amiRNA knockdown and EMSA experiments establish the requirement for RLT2 and AIL5 in Pv-ALF–dependent
expression of the phas promoter. Based on these findings and
the identification of potential regulators of histone modifications,
we envision a specific model of Pv-ALF–dependent phas activation (Figure 9). Upon estradiol treatment, the binding of Pv-ALF to
the phas promoter first triggers ordered histone modifications by
as yet uncharacterized factors. Pv-ALF also induces expression
of the homeobox chromatin protein RLT2 and the ISWI protein
CHR11 (At3g06400). RLT2 is characterized by a homeodomain
fused to a DDT domain, a HARE-HTH domain, and three WHIM
motifs (Aravind and Iyer, 2012). The WHIM motifs have been
computationally and experimentally shown to be absolutely essential for the interaction with the ISWI family of SWI2/SNF2
ATPases (Aravind and Iyer, 2012). CHR11 is the sole ISWI that is
induced in our system, and its high expression during the potentiation (4E) step of phas expression supports its cofunctionality with RLT2 in the complex. A recent yeast two-hybrid study
in Arabidopsis also confirms this interaction (Li et al., 2012).
Thus, several lines of evidence suggest that RLT2 may promote
the potentiated state of phas chromatin through its interaction
with CHR11. ISWI-like ATPases like CHR11 use ATP hydrolysis
to regulate nucleosome spacing by either optimizing it to facilitate repression or by randomizing it to facilitate transcriptional
activation (Corona and Tamkun, 2004). Thus, it is likely that the
rotationally and translationally positioned repressive nucleosome
over the phas promoter, previously observed by our group (Li
et al., 1998), might be randomized to facilitate its activation. We
propose that the combined action of RLT2 and CHR11 proteins
modifies phas chromatin, thereby providing a platform for the
second step of phas expression (transcriptional activation).
During the ABA-dependent activation step, Pv-ALF may bind
several cis-elements present in the AIL5 promoter. AIL5 (also
known as Embryomaker) contains two AP2 domains and has
a documented role in the transition from vegetative to embryonic
phase of development (Nole-Wilson et al., 2005; Tsuwamoto
et al., 2010). Other Aintegumenta-like proteins implicated in
embryo development include Baby Boom (promotes differentiation of Arabidopsis embryonic stem cells) (Boutilier et al., 2002)
and Eg-AP2-1 protein in oil palm (Elaeis guineensis) (Morcillo
et al., 2007). Most importantly, downregulation of AIL5 and RLT2
results in reduced expression of the GUS marker gene, as well
as of endogenous seed-specific genes encoding cupin, oleosin,
and LEA (this article). Thus, our data show that RLT2 and AIL5
act as important regulators of phas transcriptional activation.
In conclusion, we provide evidence that our heterologous
Pv-ALF/phas-GUS expression system recapitulates a specific
phase of seed development when the ABI3-like TFs interact with
the ABA signaling pathway. In addition to providing a comprehensive transcriptional profile of the system during potentiation
and activation, our analysis identifies a specific ABA-dependent
signaling cascade and several transcriptional regulators that are
active during this phase of seed development. In particular, the
expression of a small yet diverse set of chromatin modifying and
remodeling proteins supports previous observations that dynamic
changes in chromatin architecture are likely to be required for the
highly regulated expression of seed-specific genes. This conclusion is also consistent with earlier reports that many genes
involved in embryogenesis are repressed during vegetative
growth or in vegetative tissues by several independent mechanisms of chromatin remodeling (Li et al., 2005; Makarevich et al.,
2006; Tang et al., 2008; Gao et al., 2009).
In combination with network analysis, our results provide insight and identify candidates for the developmentally programmed
mechanisms required for changing the chromatin landscape to
initiate expression from the phas promoter. Additionally, our
analysis points toward several other distinct proteins and
pathways that are potentially regulated in a manner similar to the
phas promoter, such as a multitude of seed-specific proteins
and DNA repair pathways.
METHODS
Plant Material and Treatments
Arabidopsis thaliana ecotype Columbia plants homozygous for both the
estradiol-inducible XVE-HA-PvALF effector and the -1470phas-GUS reporter (Ng et al., 2006) (hereinafter referred to as the Pv-ALF line) were
grown at 24°C/16°C day/night (16/8-h photoperiod and 70 to 80% humidity) in MetroMix 200 potting soil. Rosette leaves from 10 3- to 4-weekold plants were collected randomly and transferred to MS liquid medium
(Murashige and Skoog, 1962) for four independent treatments with gentle
agitation at room temperature in the continuous light. The treatments were
as follows: uninduced control without the addition of estradiol or ABA
(4MS), 25 mM estradiol (Sigma-Aldrich) treatment (4E), 200 mM ABA
(Sigma-Aldrich) treatment (4A), and both 25 mM estradiol and 200 mM
ABA (4E4A). For the 4E4A treatment only, samples were treated in sequential order. First, leaves were incubated for 4 h in MS liquid medium
containing 25 mM b-estradiol, then rinsed with running distilled water to
remove estradiol, and then incubated for another 4 h with MS liquid medium
containing 200 mM ABA. After treatment, leaves were frozen in liquid N2 and
stored at 280°C until further analysis. Histochemical staining for GUS
activity was performed on the treated leaves using 5-bromo-4-chloro-3indolyl-b-D-glucuronic acid (Sigma-Aldrich) as substrate (Jefferson et al.,
1987).
RNA Isolation and Library Preparation for RNA-Seq
Transcriptome Analysis
Total RNA was extracted from leaves using the RNeasy mini kit (Qiagen)
according to the manufacturer’s protocol. RNA integrity was confirmed
using the Agilent Bioanalyzer RNA NanoChip (Agilent Technologies).
Insight to Pv-ALF–Mediated Gene Expression
Samples were prepared for whole transcriptome analysis using mRNASeq kit according to manufacturer’s protocol (Illumina). Briefly, mRNA was
purified from total RNA (10 mg) using oligo(dT) magnetic beads (Illumina).
Following purification, the mRNA was fragmented using divalent cations
under elevated temperature, and cleaved RNA fragments were used for
first-strand cDNA synthesis using reverse transcriptase (Invitrogen) and
random primers. Second-strand cDNA synthesis was then performed
using RNaseH and DNA polymerase I (Invitrogen). The resulting cDNA
fragments were subjected to end repair and ligation of adapters. The
ligated products were loaded on a 2% agarose gel and a gel slice corresponding to ;250 bp was excised, purified, and enriched by PCR for
use in creating the final cDNA library. Following a check of quantity and
quality on the Agilent 1000 Chip (Agilent Technologies), the cDNA library
was used for cluster generation. Single-end sequencing (72-bp reads)
was performed on the Genome Analyzer II following the manufacturer’s
protocol (Illumina) at Bioinformatics Core Facility, Texas A&M University.
Bioinformatic Analysis
Illumina GA pipeline 1.3 software was used for image deconvolution
and quality value calculations. The raw reads were cleaned by removing
adaptor sequences, empty reads, and low-quality sequences. TAIR10
annotations and sequence files for individual annotated genome features
(genes, cDNAs, 39 untranslated regions, 59 untranslated regions, introns,
exons, and intergenic regions) were downloaded from The Arabidopsis
Information Resource ftp://ftp.Arabidopsis.org/home/tair/Sequences.
CLC Genomics workbench version 4.0 was used to align the obtained
Illumina reads with the Arabidopsis reference genome, allowing up to two
mismatches. The RNA-Seq analysis tool (ERANGE software) of CLC
Genomic Workbench (http://www.clcbio.com) was used to measure gene
expression values in RPKM, a normalized measure of exon read density
that allows transcript levels to be compared both within and between
samples (Mortazavi et al., 2008). These values were used to calculate
gene expression levels and to discover novel exons based on the annotated reference genome. Quantile normalization (Bolstad et al., 2003)
was performed for all Illumina runs across the four treatments of two
biological replicates. Genes with more than 13 median coverage with
the RPKM value of more than 1.25 was considered for further analysis.
Correlation plots of the RPKM reads for each replicate were used to
assess the reproducibility of RNA-Seq experiments. Coverage across
transcripts was calculated by counting the number of reads on aligned
transcripts generated by TopHat (Trapnell et al., 2009). All correlation
analysis and plots were performed in R.
Data analysis and subsequent statistical tests for the two biological
replicates were performed using Genespring GX version 12.5 software
(Agilent Technologies). Filtered expression data were imported into the
GeneSpring Experimental Tool. To identify genes activated by Pv-ALF
and ABA, entities were filtered based on a fold change of at least 2
between the conditions. One parameter with four experimental conditions
(4MS, 4E, 4A, and 4E4A), each with two biological replicates, was used for
the statistical analysis. The inbuilt statistical package of the GeneSpring
software automatically applied the one-way analysis of variance with
multiple testing corrections by the Benjamini and Hochberg method
(Benjamini and Hochberg, 1995) to correct false discovery rate, and
asymptotic P value computation was performed to determine the change in
expression of a particular gene. Transcripts with absolute fold change values
of at least 2 with the corrected P value of < 0.05 (or <0.2 where specified) were
included in our analysis as differentially expressed genes. Hierarchical
clustering of the transcriptome data was performed using the Ward criterion
and the Euclidean method (Soukas et al., 2000).
Identification of putative Pv-ALF binding sites in the promoter regions (1000
bp; both DNA strands) of genes specifically upregulated in 4E and 4E4A was
performed using the Motif Sampler bioinformatics tool (http://www.Arabidopsis.
org/tools/bulk/motiffinder/index.jsp) at the TAIR10 genome database.
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Annotation and Classification
For functional classification of genes, the following procedure was
adopted. Domain architectures for the genes were first predicted by
profile searches using HMMER software with a database of Pfam profiles
in combination with locally compiled profiles not reported in Pfam (Iyer
et al., 2008). Annotations from The Arabidopsis Information Resource
(TAIR10 database, http://www.Arabidopsis.org/tools/bulk/go/index.jsp)
(Berardini et al., 2004), Gene Ontology terms, and orthology with other
model eukaryotes were used to manually refine our annotations. Identified
transcriptional regulators were divided into chromatin proteins and TFs
(Iyer et al., 2008), which can be differentiated based on their domain
composition. Chromatin proteins are composed of domains involved in
the posttranslational modification of histones and nuclear proteins and
recognition of distinct histone or DNA modifications. In addition, chromatin proteins also include multiple types of ATP-dependent proteins or
protein complexes that alter chromatin structure either on a local or
chromosomal scale. TFs use DNA binding domains to regulate transcription, typically at promoter elements.
Network Analysis
Coexpression network analysis was performed using the Arabidopsis
trans-factor and cis-element prediction database ATTED-II (Obayashi
et al., 2009, 2011) to identify genes coexpressed with input genes. The
extent of gene coexpression network view was calculated based on the
Pearson’s correlation coefficient and mutual rank (MR; i.e., calculated as
the geometric mean of the correlation rank of gene A and gene B and of
gene B to gene A). Three types of edges (lines) are used to draw the
networks: bold edges (MR < 5), normal edges (5 # MR > 30), and thin
edges (MR # 30). The VirtualPlant software was used to visualize the
known molecular interactions between the selected genes. This program
uses a more extensive gene interactions database, which is based on
known protein–protein interactions, enzymatic pathways, and transcriptional regulation pathways (Gutiérrez et al., 2005; Katari et al., 2010).
Transformation of Arabidopsis Pv-ALF Line with amiRNA Constructs
To generate AIL5 and RLT2 knockdown Arabidopsis plants, gene-specific
amiRNAs were engineered by replacing the original miR319a sequence in
the pRS300 plasmid with an artificial sequence (21-mer) specific to either
AIL5 (59-TGGAATGATTGTTATACCCAT-39) or RLT2 (59-TTGATATTCACGAATAGGCGT-39), as previously described (Alvarez et al., 2006). The
list of primers used for amiRNA cloning is provided in Supplemental Table
4 online. The constructs were cloned into the pCBK05 vector (Riha et al.,
2002) behind the constitutive 35S promoter using the SpeI restriction
site. The resulting AIL5 and RLT2 amiRNA constructs were introduced
into Agrobacterium tumefaciens strain GV3101 by electroporation and
supertransformed into the homozygous Pv-ALF line by the floral dip
method (Clough and Bent, 1998). Arabidopsis supertransformants were
selected on MS agar containing 20 mg/L phosphinotricine (Duchefa). Three
knockdown lines against each gene were selected and used to investigate the
function of the corresponding gene during the Pv-ALF–mediated phas-GUS
expression. All experiments were performed after treating the leaves
with estradiol and ABA as described above.
Real-Time Quantitative RT-PCR
Total RNA from leaf tissue was isolated and purified using RNAeasy mini
kit (Qiagen) following the manufacturer’s instructions. Total RNA from
mature seed tissue was isolated as described previously (Oñate-Sánchez
and Vicente-Carbajosa, 2008) and purified using an RNAeasy mini elute
Qiagen RNA kit. In both cases, DNA was digested using Qiagen RNaseFree DNase set. DNA-free RNA was used to perform first-strand cDNA
synthesis with oligo(dT) primers using the SuperScript III One-Step
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The Plant Cell
RT-PCR system (Invitrogen). Sequence-specific primers for real-time PCR
were designed using the Primer-Blast online tool (Rozen and Skaletsky,
2000). Real-time qRT-PCR was conducted with the Power SYBR Green
PCR Master Mix 7500 (Applied Biosystems) using the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for
30 s, and 60°C for 1 min, in a total reaction volume of 15 mL. Three
biological replicates were performed for each gene. The relative quantities
of PCR products were calculated using the equation 2 (2DDCT) at a
threshold value of 0.02 (Livak and Schmittgen, 2001). Data were normalized to a control housekeeping gene, EF1a, which is stable under
treatment conditions. Data were further calculated relative to the control
Pv-ALF line or uninduced condition, which were designated as one.
Reactions were performed in ABI PRISM 7000 (Applied Biosystems).
Student’s t test was used to determine statistical significance between the
uninduced and induced treatments, and the Pearson correlation coefficient (r) was used to determine statistical correlation between the
RNA-Seq data and qRT-PCR data (see Supplemental Tables 5 and 6
online for the list of primers used for gene expression analysis).
In Vitro Translation and EMSA Assays
The bean Pv-ALF coding region was subcloned into the T7 expression
vector pET28a (Novagen) for in vitro protein expression using the TnTcoupled RRL kit (Promega). Expression of Pv-ALF was verified using
a separate control reaction performed in the presence of [35S]Met and
was visualized following electrophoresis on SDS-PAGE and exposure to
phosphor imager screens. The qualitative EMSA assays were conducted
as described (Shakirov et al., 2009) with slight modifications. Briefly, each
reaction (15 mL total volume) contained 4 mL of RRL-translated Pv-ALF
protein (or 4 mL of Pv-ALF–free RRL lysate for negative controls), 0.5 pmol
of 32P-labeled PCR product corresponding to the 2340 to +25 promoter
regions of either phas, RLT2, AIL5, or At1g26680 genes, 3 mL of 5 3 DNA
binding buffer (100 mM Tris-HCl, pH 8.0, 250 mM NaCl, 50 mM MgCl,
5 mM EDTA, 5 mM DTT, and 25% glycerol), and 1 mL each of nonspecific
RNA and single-stranded DNA oligonucleotides, as described (Shakirov
et al., 2009). Reactions were incubated at room temperature for 15 min.
For competition assays, 1003 of cold (unlabeled) competitor DNA was
mixed with the radioactively labeled probe prior to the addition of the
protein. The complexes were separated on 5% polyacrylamide gel
(acrylamide:bisacrylamide 29:1) for 2 h at 150 V in 0.83 TBE (Tris Borate
EDTA) at room temperature, dried, and exposed to phosphor imager
screens. Screens were scanned by a Pharos FX Plus molecular imager
and visualized with Quantity One v.4.6.5 software (Bio-Rad).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL
database or the Arabidopsis Genome Initiative database under the following accession numbers: Pv-ALF, U28645; ABI3, At3g24650; RLT1,
At1g28420; RLT2, AT5g44180; AIL5, At5g57390; ABI2, At5g57050; HAB1,
At1g72770; AHG1, At5g51760; OST1, At4g33950; CPK6, At2g17290;
CPK11, A1g35670; PYL7, At4g01026; PYL9, At1g01360; CPK3,
At4g23650; SNRK2.3, At5g66880; ERA1, At5g40280; ABI5, At2g36270;
Anac042, At2g43000; Anac032, At1g77450; RHL41, At5g05410; DREB2,
At5g05410; CHR11, At3g06400; CHR18, At1g4830; CHR38, At3g42670;
LDL2, At3g13682; JMJ, At5g46910; PARP1, At5g22470; RAD5, At5g22750;
LEA, At3g17520; Cupin, At4g36700; Oleosin4, At5g40420; CRC, At4g28520;
and FUS3, At3g26790.
Supplemental Figure 2. Assessment of Reproducibility between the
Two RNA-Seq Biological Replicates.
Supplemental Figure 3. Analysis of Overlapping Genes That Are Downregulated during the Pv-ALF–Mediated phas Activation.
Supplemental Figure 4. Comparison of the Number of Genes Identified
in This Study with Genes Detected in Related Studies.
Supplemental Figure 5. Functional Classification of Seed-Specific Arabidopsis Genes Expressed during Activation of the phas Promoter in Leaves.
Supplemental Figure 6. Expression Profiles of Genes Involved in the
ABA Signaling Pathway and Transcription Factors Preferentially Expressed during Pv-ALF–Mediated phas Activation.
Supplemental Figure 7. Coexpression Analysis of Transcription Factors
Regulated by Pv-ALF Expression.
Supplemental Figure 8. RLT2 and AIL5 Knockdown in a Pv-ALF Background Reduces Expression of Seed-Specific Genes in 4E4A-Treated Leaves.
Supplemental Figure 9. Seeds of RLT2 and AIL5 amiRNA Knockdown Plants Do Not Show Reduction in the Expression of Endogenous Arabidopsis ABI3-Related Genes.
Supplemental Table 1. RNA-Seq Sequence Summary.
Supplemental Table 2. Occurrence of Specific Binding Sites Enriched
in the Promoters of ABA-Independent and ABA-Dependent Genes
Upregulated upon Pv-ALF Induction.
Supplemental Table 3. Validation of RNA-Seq Data by qRT-PCR.
Supplemental Table 4. List of Primers Used for amiRNA Cloning.
Supplemental Table 5. List of Primers Used for RT-PCR Analysis.
Supplemental Table 6. List of Primers Used for qRT-PCR.
Supplemental Data Set 1. List of ABA-Independent Genes Activated
by Pv-ALF Expression.
Supplemental Data Set 2. List of Genes Specifically Upregulated in
Leaves upon Pv-ALF Induction and Addition of ABA (4E4A) Only.
Supplemental Data Set 3. List of ABA-Independent Genes Downregulated by Pv-ALF Expression.
Supplemental Data Set 4. Classification and Annotation of Genes Upregulated during Potentiation (4E) and Activation (4E4A) of the phas Promoter.
Supplemental Data Set 5. Comparison of Genes Identified in This
Study with genes Detected in Related Studies.
Supplemental Data Set 6. List of Genes Encoding Seed-Related
Proteins Expressed in the Pv-ALF/phas-gus Heterologous System
during phas Activation.
Supplemental Data Set 7. List of Arabidopsis Seed-Specific Genes
Expressed in Leaves of Pv-ALF Plants during Activation of the phas
Promoter by Pv-ALF and ABA.
Supplemental Data Set 8. List of ABA Signaling Pathway Genes
Differentially Regulated during Pv-ALF–Mediated phas Activation.
Supplemental Data Set 9. List of Transcription Factors That Are
Overexpressed in the 4E and 4E4A Treatments.
Supplemental Data Set 10. List of Chromatin Proteins That Are Overexpressed in the 4E and 4E4A Treatments.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Dynamic Two-Step System Used to Study Potentiation and Activation of the phas Promoter in Transgenic Arabidopsis Leaves.
ACKNOWLEDGMENTS
This work was supported by National Science Foundation Grants MCB
0843692 and MCB 1260947 to T.C.H., by MEYS-Czech Grant ME10038
Insight to Pv-ALF–Mediated Gene Expression
to M.J., and by intramural funds of the National Institutes of HealthNational Library of Medicine to L.M.I. We thank Charles Johnson and EunGyu No (TX AgriLife Bioinformatics Service) for their help with Illumina GAII;
Patricia Klien, Rodolfo Aramayo, and Phil Beremand for help with data
analysis; Ginger Stuessy for plant culture; James Hardin and David Reed
for computational services; and Gene Technologies Laboratory staff for
sequencing.
AUTHOR CONTRIBUTIONS
T.C.H., S.S., and S.K. designed the project. S.S. and S.K. carried out
research work. E.V.S performed gel shift assay. S.S. and L.M.I. analyzed
the RNA-Seq data and interpreted the data. M.J. performed the comparative analysis using TopHAT. All authors contributed to writing the article.
Received April 15, 2013; revised May 23, 2013; accepted June 15, 2013;
published July 19, 2013.
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Gene Networks and Chromatin and Transcriptional Regulation of the Phaseolin Promoter in
Arabidopsis
Sabarinath Sundaram, Sunee Kertbundit, Eugene V. Shakirov, Lakshminarayan M. Iyer, Miloslav
Jurícek and Timothy C. Hall
Plant Cell; originally published online July 19, 2013;
DOI 10.1105/tpc.113.112714
This information is current as of June 16, 2017
Supplemental Data
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