G-Boxes, Bigfoot Genes, and Environmental Response: Characterization of Intragenomic Conserved Noncoding Sequences in Arabidopsis

Abstract

A tetraploidy left Arabidopsis thaliana with 6358 pairs of homoeologs that, when aligned, generated 14,944 intragenomic conserved noncoding sequences (CNSs). Our previous work assembled these phylogenetic footprints into a database. We show that known transcription factor (TF) binding motifs, including the G-box, are overrepresented in these CNSs. A total of 254 genes spanning long lengths of CNS-rich chromosomes (Bigfoot) dominate this database. Therefore, we made subdatabases: one containing Bigfoot genes and the other containing genes with three to five CNSs (Smallfoot). Bigfoot genes are generally TFs that respond to signals, with their modal CNS positioned 3.1 kb 5' from the ATG. Smallfoot genes encode components of signal transduction machinery, the cytoskeleton, or involve transcription. We queried each subdatabase with each possible 7-nucleotide sequence. Among hundreds of hits, most were purified from CNSs, and almost all of those significantly enriched in CNSs had no experimental history. The 7-mers in CNSs are not 5'- to 3'-oriented in Bigfoot genes but are often oriented in Smallfoot genes. CNSs with one G-box tend to have two G-boxes. CNSs were shared with the homoeolog only and with no other gene, suggesting that binding site turnover impedes detection. Bigfoot genes may function in adaptation to environmental change.

Full text

RESEARCH ARTICLES
G-Boxes, Bigfoot Genes, and Environmental Response:
Characterization of Intragenomic Conserved Noncoding
Sequences in Arabidopsis W
Michael Freeling,
a,1
Lakshmi Rapaka,
a
Eric Lyons,
a
Brent Pedersen,
b
and Brian C. Thomas
b
a
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720
b
College of Natural Resources, University of California, Berkeley, California 94720
A tetraploidy left Arabidopsis thaliana with 6358 pairs of homoeologs that, when aligned, generated 14,944 intragenomic
conserved noncoding sequences (CNSs). Our previous work assembled these phylogenetic footprints into a database. We
show that known transcription factor (TF) binding motifs, including the G-box, are overrepresented in these CNSs. A total of 254
genes spanning long lengths of CNS-rich chromosomes (Bigfoot) dominate this database. Therefore, we made subdatabases:
one containing Bigfoot genes and the other containing genes with three to five CNSs (Smallfoot). Bigfoot genes are generally
TFs that respond to signals, with their modal CNS positioned 3.1 kb 59from the ATG. Smallfoot genes encode components of
signal transduction machinery, the cytoskeleton, or involve transcription. We queried each subdatabase with each possible
7-nucleotide sequence. Among hundreds of hits, most were purified from CNSs, and almost all of those significantly enriched in CNSs
had no experimental history. The 7-mers in CNSs are not 59-to39-oriented in Bigfoot genes but are often oriented in Smallfoot genes.
CNSs with one G-box tend to have two G-boxes. CNSs were shared with the homoeolog only and with no other gene, suggesting that
binding site turnover impedes detection. Bigfoot genes may function in adaptation to environmental change.
INTRODUCTION
Functional DNA sequence changes at a lower rate over evolu-
tionary time than sequence without function. Exon sequence
tends to be conserved, whereas functionless sequence is ran-
domized by substitution, lost by conversion, or deleted entirely.
Therefore, if two genes or chromosomal regions have diverged
from a common ancestor, be they in different species (orthologs)
or duplications within the same genome (paralogs), those few
noncoding regions that retain a high degree of sequence simi-
larity provide a measure of noncoding DNA function, where
function is inferred from conservation (Hardison, 2000, 2003). In
flowering plants, the alignment algorithm BLAST-2-sequences
(Tatusova and Madden, 1999) has been used successfully to
detect conserved noncoding sequences (CNSs) in comparisons
of maize (Zea mays) and rice (Oryza sativa) orthologous genes
(Kaplinsky et al., 2002; Inada et al., 2003) and to find intra-
genomic CNSs in Arabidopsis thaliana (Thomas et al., 2007) by
comparing the syntenic duplicates (syntenic paralogs and ho-
moeologs) retained following its most recent tetraploidy (called
the a-event). The two genomes from the a-event (Simillion et al.,
2002; Bowers et al., 2003; Maere et al., 2005) diverged to
approximately the same extent as have maize-rice and man-
mouse (Kaplinsky et al., 2002); all of these have diverged suffi-
ciently such that sequence conservations are not due to neutral
carryover. Table 1 defines important terms we use: CNS, aCNS,
gene space, and phylogenetic footprint. It should be understood
that function does not assure that noncoding sequence will be
conserved. Some sequences evolve quickly; others defy detec-
tion. CNSs are a subset of functional noncoding sequences.
Haberer et al. (2004) demonstrated that intragenomic (a)
phylogenetic footprints in proximal promoters of Arabidopsis
syntenic gene pairs could be found with an anchored alignment
tool (Brudno et al., 2004), anchoring on the 59ATG. However,
these workers did not explore sequence outside of the 500-bp
proximal promoter. Guo and Moose (2003) used LAGAN (Brudno
et al., 2003) with a Vista display (Mayor et al., 2000) to explore
CNSs within the grasses, especially between maize and rice,
concluding that transcription factor binding sites are within some
CNSs and that CNSs were sometimes shared among different
grasses.
This report explores the functions of 14,944 intragenomic
CNSs (aCNSs) retained from a set of 3179 gene space pairs
retained from the a-event in the Arabidopsis lineage (Thomas
et al., 2006, 2007). These data can be accessed by a custom
Web application called the Arabidopsis Bl2seq Viewer available
at http://synteny.cnr.berkeley.edu/AtCNS. According to Thomas
et al. (2007), these aCNSs exist in gene spaces with a modal
frequency of zero and a mean of 1.7. The median aCNS length is
25 bp, and these sites are distributed in all regions of a gene with
a general preference to be 59of a gene. The ratio of CNSs within
the gene space, going from 59to 39,59:59UTR:intron:39UTR:39is
1
To whom correspondence should be addressed. E-mail freeling@
nature.berkeley.edu; fax 510-642-4995.
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: Michael Freeling
(freeling@nature.berkeley.edu).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.107.050419
The Plant Cell, Vol. 19: 1441–1457, May 2007, www.plantcell.org ª2007 American Society of Plant Biologists

3.0:2.0:1.2: 1.0: 1.2, giving a 59/39bias of 2.3. Thomas et al.
(2007) also showed that genes with certain functions, especially
transcription factors responding early to external stimuli, as
estimated by their gene ontology (GO) annotation, tended to
associate with different numbers of CNSs; this functional cate-
gory correlation with CNS richness validated the notion that
CNSs are functional. Finally, Thomas et al. (2007) concluded that
these CNSs are not simple sequences and not likely to either
encode or bind small RNAs. Thus, the likely general function of an
aCNS must be to bind protein or, perhaps, carbohydrates.
CNSs have been extensively studied, especially in mammals.
However, using the typical mammalian definition of CNS (e.g.,
$70% identity; $100 bp in length [Loots et al., 2000]), compa-
rably diverged plant genes would have no CNSs. Putative CNS
function includes matrix attachment regions (Avramova et al.,
1998; Glazko et al., 2003), single and multiple transcription factor
(TF) binding sites (Hardison, 2000; Loots et al., 2000; Levy et al.,
2001; Dubchak and Frazer, 2003; Guo and Moose, 2003;
Hardison, 2003; Thomas et al., 2003; Loots and Ovcharenko,
2004; Bejerano et al., 2005; Siepel et al., 2005), chromosome-
level regulatory regions (Loots et al., 2000), DNase I hypersen-
sitive sites (Gottgens et al., 2001), and enhancers of vertebrate
animal genes (such as sonic hedgehog; Goode et al., 2005).
These latter highly conserved enhancers have been shown to
mark bivalent states of chromatin associated with downregula-
tion of genes in stem cells (Bernstein et al., 2006). In plants, one
intronic CNS has been genetically analyzed and functions nor-
mally to prevent ectopic expression of the homeobox gene kn1
(Inada et al., 2003).
Given the evidence for TF binding sites in CNSs, we expect
that some of our CNSs will carry binding motifs for known plant
TFs. Because not all TF binding motifs are known, perhaps some
short sequence motifs will be enriched in CNSs compared with
nonconserved noncoding sequences and will later be found to
bind TFs. A typical animal promoter might carry as many as 6 to
15 clusters of TF binding sites, with each TF binding site being
between 5 and 12 bp, and each cluster binding four to eight dif-
ferent TF proteins (Wray, 2003). Although evidence indicates that
plant proximal 59regions carry such cis-modules (Vandepoele
et al., 2006), it is not yet clear how to apply animal results to plant
genes, especially plant genes with very long 59or 39gene spaces.
The PLACE cis-acting site database (Higo et al., 1999) lists 458
experimentally derived plant motifs (August, 2006). The median
PLACE site is 8 bp long with some degree of sequence degen-
eracy. While finding these sites in a plant genome is trivial,
proving a site is functional is a daunting task. Algorithms ana-
lyzing DNA sequence only rarely find function (Tompa et al.,
2005) without the addition of expert knowledge such as coex-
pression or phylogenetic relatedness (Prakash et al., 2004; Van
Hellemont et al., 2005). Using phylogenetic relatedness, ;1400
enhancers are deeply (>300 million years) conserved in verte-
brates (Woolfe et al., 2005).
One cis-acting binding motif is known to be vastly overrepre-
sented in the plant genome, a microsatallite, and thus was truly
expected to be enriched in CNSs: GAGA. GAGA (CTCT) motifs
are known to bind particular proteins, and these are known to
interact with chromatin remodeling complexes to alter animal
gene expression (Lehman, 2004; Meister et al., 2004; Kooiker
et al., 2005). Simple repeated sequences are also known to mark
regions of the human X chromosome that avoid silencing (McNeil
et al., 2006). To not exclude GAGA or any similar regulatory
sequence, we included simple sequences in our CNS list. In so
doing, we accepted the increased noise level expected when
vastly overrepresented sequences locate in a-syntenous gene
space by chance alone. As it turned out, few aCNSs are simple
sequence repeats (Thomas et al., 2007).
Genes retained after either a local or whole-genome duplica-
tion are a biased subset of ancestral gene content (Freeling and
Thomas, 2006). Therefore, the aCNSs may be a biased sample
of functional sites simply because the genes available for
CNS analysis are themselves biased. In the Arabidopsis lineage,
genes in the large molecular function GO categories ‘‘transcrip-
tion factor activity’’ and ‘‘protein kinase,’’ along with other genes
whose products interact in complexes, tend to be retained fol-
lowing tetraploidies at frequencies nearly double expectations
(Blanc and Wolfe, 2004; Seoighe and Gehring, 2004; Maere et al.,
2005). So far, the combined data of gene retention following
either local or whole-genome duplications in eukaryotes fits with
the predictions of the gene balance hypothesis (Freeling and
Thomas, 2006), but vertebrate tetraploidy data are more difficult
to acquire. The gene balance hypothesis (Veitia, 2002; Birchler
et al., 2003; Papp et al., 2003; Birchler et al., 2005) predicts that
Table 1. Definitions Involving CNSs and Their Identification
Plant CNS: A pairwise bl2seq ( Tatusova and Madden, 1999) hit between the nonprotein-coding sequences near usefully diverged, orthologous genes.
These sequences are at least 15-bp long with an e-value equal to or more significant than a 15/15 exact nucleotide match, and without complexity
filtration (Kaplinsky et al., 2002; Inada et al., 2003). BLAST results for Arabidopsis are displayed and may be researched in a custom viewer (http://
synteny.cnr.berkeley.edu/AtCNS). ‘‘Useful’’ levels of divergence are not so small that conservation occurs by carryover from the ancestor, without
selection, but not so great that detection is impeded, as will be further defined in the text.
Plant aCNS: As above, but the chromosomal regions are homoeologous (syntenous and paralogous) remnants of a usefully diverged tetraploidy event
(a) in the lineage (Thomas et al., 2006). Subfunctionalization is expected of homoeologous pairs, but not orthologous pairs.
Gene space: Gene space is computed after CNSs have been identified for a paired region, and each CNS has been sorted to gene. The segment of
genome between the most 59(upstream) and most 39CNS or untranslated region plus ;500 bp on each side (depending on neighboring features;
Thomas et al., 2007). Within a gene space are exons, UTRs, CNSs, known motifs, positions where specific TFs reside, and any feature that is fixed
at a chromosomal locus.
Phylogenetic footprint: The most inclusive term for conserved sequence, whether two or multiple sequences, and without stipulations as to the extent
of divergence. A CNS is a type of phylogenetic footprint.
1442 The Plant Cell

genes whose products function in subunit–subunit interactions
or participate at the top of regulatory cascades will tend to be
more susceptible to gene dosage change and therefore will be
overretained following tetraploidy. For this reason, connected
genes tend to be retained as pairs following tetraploidy. Since
CNSs can only be detected in a-gene pairs, our CNS database is
obviously biased toward connected genes.
RESULTS
Gene Retainability and CNS Richness Are Weakly
or Not Correlated
Our CNS database is necessarily confined to the analysis of
those 25% of genes that have a-pairs. To extrapolate from our
data on functional noncoding sequence to the entire genome, we
needed to access the relationship, if any, between retention
post-tetraploidy and CNS richness. When genes are sorted by
GO category and each was compared for both retention and
CNS richness, no correlation was found (data not shown; see
Supplemental Table 1 online). Because GO categories have
overlapping gene contents, we attempted the same correlation
among genes in different, nonoverlapping TF families (DATF
families in Supplemental Table 1 online; Guo et al., 2005 at http://
datf.cbi.pku.edu.cn/, July, 2005). There was no obvious corre-
lation (graph not shown; see data in Supplemental Table 1 online).
Had every gene in Arabidopsis been retained following the
tetraploidy, and thus been available for analysis, we would
have found approximately fourfold the number of aCNSs.
GAGA Motifs and PLACE TF Binding Motifs in aCNSs
GAGA
On the basis of published work (see Introduction), we expected
that CNSs would preferentially contain GAGA repeats. We ex-
pected significantly more GAGA sequences in CNS sequence
than in an equal amount of control noncoding sequence (see
Methods); this we call ‘‘enrichment.’’ For GA6 with less than two
mismatches (#10/12), CNS enrichment was 0.8, meaning that
there were actually more GA6-type sequences in control non-
coding sequence than in CNS: GA6 ¼0.6; GA5 ¼0.9; GA4 ¼
1.16; and [GA] GA[GA]AG[GA][GA]A (Kooiker et al., 2005) ¼1.0.
We expected a GAGA CNS enrichment proportion to be signif-
icantly >1. This expectation was not met.
PLACE cis-Acting Binding Motifs
Data on CNS function suggests that some CNSs might be
clusters of known TF binding sites (see Introduction). The PLACE
Angiosperm cis-acting sequence database (Higo et al., 1999)
had accumulated 431 such TF binding motifs on August 1, 2005.
Each motif and its reverse complement, in IUPAC (Prosite-type)
format, were found in both aCNS sequences and the control
noncoding sequences within the same gene spaces. By requiring
that at least 50 CNSs were hit by any motif reduced the list to 136
TF binding motifs. Enrichments ranged from 6.2-fold to 0.46-fold
with a median at 0.9-fold. Fourteen motifs gave CNS enrich-
ments more than twofold. Table 2 shows these data ranked
descending for enrichment in CNSs; the top 14 and a few
additional PLACE motifs are included here. The top-ranked eight
motifs and 10 of the top 14 carry a G-box: CACGTG (Williams
et al., 1992; Menkens et al., 1995; Gao et al., 2004). The specific
PLACE sequence CACGTGGC, the most CNS-enriched PLACE
motif (6.2-fold), is part of a Type I-AA_G-ABRE element,
CCACGTGGC, know to operate in genes responsive to both
abscisic acid (ABA) and external stress (Choi et al., 2000). TF
binding sites for jasmonic acid response (pathogenic stress;
Brown et al., 2003) and three additional known boxes (color-
coded in Figure 1 and cited at PLACE) were enriched more than
twofold in our aCNSs. Boxes DRE-CRT and ARE (thought to
confer response to auxin) are enriched to a lesser extent. We
conclude that the most CNS-enriched motif, the G-box, lies in a
noncoding gene space near particular gene pairs. The 268 CNSs
with a perfect G-box are noted in the CNS list (see Supplemental
Table 2 online). The G-box CNSs are usually positioned 59of the
ATG (229 are 59, eight are in intron, 29 are 39, and one is near a
gene we called ourselves, designated -oa) at a mean distance of
1588 bp and a range from 40 to 8958 bps upstream of the A of the
59ATG. Note that this distance is well 59of that considered to be
the normal promoter region of a gene.
Bigfoot and Smallfoot Genes
The Problem Caused by a Few Hundred Very
CNS-Rich Genes
Thomas et al. (2007) showed that some genes are particularly
CNS rich, and these genes tend to fall into GO categories that are
TF or MIR, each with a mean of approximately five CNSs/gene.
The most CNS-enriched genes (10 to 20 CNSs/gene space)
populate GO categories characterized by ‘‘response to...’’ plant
hormones, internal or exogenous stress, light, desiccation, jas-
monic acid, and so forth. This previous work engendered the
hypothesis that CNS richness characterizes, in particular, genes
that respond first to signals of all sorts: the first responder
hypothesis. However, the average paired gene in Arabidopsis
has 1.7 CNSs, and the modal gene has 0. These more CNS
average genes tend to encode enzymes or structural proteins.
The result is that a few CNS-rich genes dominate our CNS
database with their many and potentially special CNSs. To
address this problem, we created two subdatabases, one from
the longest gene spaces in Arabidopsis (Bigfoot genes) and the
other from genes with a modest number, three to five, of CNSs/
gene (called Smallfoot genes). Table 3, row 1, quantifies the
genes and sequences that define these two subdatabases, and
Table 3 compares them. Twenty-four genes were shared.
Bigfoot Genes
Figure 2 shows a screenshot of a Bigfoot gene pair in the
Arabidopsis Bl2seq Viewer (Thomas et al., 2006, 2007). Note that
it is transcribed from right to left. More often than not, the 126
pairs of Bigfoot genes exist in a region of chromosome devoid of
other genes, with CNSs populating the void (Figure 1). Compar-
isons of vertebrate genomes identified gene deserts that are
CNS Function in Arabidopsis 1443

Table 2. DATF Families and Counts in July, 2005, Showing Nonrandom Distribution of Both CNS Richness and Frequency of Genes That Are
Bigfoot; Bigfoot Genes Are Possible among Retained Pairs Only
TF Families
Total
Genes
Genes
Retained
Retention
Frequency
Average No.
CNS/Gene
Average Length
Total CNS/Gene
Fraction
Bigfoot
Bigfoot
Frequency
TF:LUG 1 0 0.00
TF:NZZ 1 0 0.00
TF:SAP 1 0 0.00
TF:CCAAT-DR1 2 2 1.00 2.00 75.00 0 of 2
TF:ULT 2 2 1.00 3.00 66.00 0 of 2
TF:S1Fa-like 2 0 0.00
TF:VOZ 2 0 0.00
TF:GIF 3 2 0.67 0.00 0.00 0 of 2
TF:HRT-like 3 2 0.67 0.00
TF:MBF1 3 2 0.67 0.00 0.00 0 of 2
TF:PBF-2-like (Whirly) 3 2 0.67 1.00 21.00 0 of 2
TF:LFY 3 0 0.00
TF:PcG 4 2 0.50 0.00 0.00 0 of 2
TF:C2C2-YABBY 5 0 0.00
TF:FHA 5 0 0.00
TF:EIL 6 2 0.33 0.00 0.00
TF:LIM 6 4 0.67 0.50 10.50
TF:BES1 6 2 0.33 5.00 140.00 0 of 2
TF:ALFIN 7 6 0.86 1.00 19.33 0 of 6
TF:CAMTA 7 6 0.86 1.00 32.67 0 of 6
TF:ARID 7 4 0.57 3.00 132.50 0 0f 2
TF:E2F/DP 8 2 0.25 0.00 0.00
TF:CPP 8 4 0.50 8.00 239.50 0 of 4
TF:PLATZ 9 4 0.44 5.00 140.00 0 of 4
TF:HMG 10 4 0.40 0.00 0.00 0 of 4
TF:CCAAT-HAP2 10 6 0.60 1.67 38.67 2 of 6 0.33
TF:TAZ 10 6 0.60 3.33 117.67 0 of 6 0.00
TF:GRF 10 4 0.40 6.00 286.50 0 of 4
TF:SRS 10 6 0.60 11.00 514.67 2 of 6 0.33
TF:TUB 11 2 0.18 2.00 37.00 0 of 2
TF:CCAAT-HAP3 11 2 0.18 7.00 131.00 0 of 2
TF:PHD 12 2 0.17 3.00 68.00 0 of 2
TF:JUMONJI 13 2 0.15 1.00 28.00 0 of 2
TF:CCAAT-HAP5 13 4 0.31 2.00 73.00 0 of4
TF:GARP-ARR-B 13 6 0.46 2.67 100.33 2 of 6 0.33
TF:ABI3/VP1 13 6 0.46 13.67 528.33 4 of 6 0.67
TF:Nin-like 14 8 0.57 3.00 82.25 0 f 8 0.00
TF:ZIM 15 10 0.67 1.40 28.00 0 of 10 0.00
TF:ZF-HD 15 4 0.27 13.50 450.00 4 of 4 1.00
TF:GeBP 16 4 0.25 0.00 0.00 0 of 4
TF:SBP 17 6 0.35 2.67 101.67 2 0f 6 0.33
TF:ARF 23 2 0.09 6.00 211.00 2 of 2 1.00
TF:HSF 24 4 0.17 2.50 79.50 0 of 4
TF:TCP 24 8 0.33 5.13 179.00 2 of 8 0.25
TF:Trihelix 29 12 0.41 2.33 68.67 2 of 12 0.17
TF:C2C2-GATA 29 16 0.55 5.38 210.75 4 of 16 0.25
TF:AUX/IAA 29 16 0.55 5.63 211.75 2 of 16 0.13
TF:C2C2-co-like 31 16 0.52 3.00 118.25 0 of 16 0.00
TF:GRAS 33 15 0.45 3.27 85.67 6 of 15 0.40
TF:C3H 35 16 0.46 2.06 48.13 2 of 16 0.13
TF:C2C2-DOF 36 16 0.44 6.19 213.88 6 of 16 0.38
TF:B3 39 4 0.10 6.50 116.00 0 of 4
TF:AS2 42 8 0.19 4.25 118.75 2 of 8 0.25
TF:GARP-G2-like 43 16 0.37 7.00 239.13 2 of 16 0.13
TF:WRKY 73 24 0.33 3.75 109.00 4 of 24 0.17
TF:bZIP 75 30 0.40 3.87 122.40 2 of 16 0.13
(Continued)
1444 The Plant Cell

adjacent to TF and developmental genes and are full of deeply
conserved noncoding sequences (Ovcharenko et al., 2005);
Bigfoot gene space seems to be a plant convergence on the
gene desert phenomenon. The 252 Bigfoot genes are noted in
Supplemental Table 1 online. Using GOStat (see Methods), there
were 140 to 142 genes each in four overrepresented (P ¼0.00)
GO categories: 0005488, binding; 0003677, DNA binding;
0003700, TF activity; and 0003676, nucleic acid binding. Other
significantly overrepresented GO categories (P < 3 310
8
) were
several with the phrase ‘‘regulation of...’’ and GO:0008755
hormone-mediated signaling. Bigfoot genes are predominantly
(;66%) TF genes. Other very broad categories of Bigfoot gene
pairs are 16 unknown protein pairs, six protein kinase pairs, 18
enzyme pairs, one RNA binding pair and, interestingly, two DVL
gene pairs (A12N061 and A08N202). DVL genes (genes that were
not in The Arabidopsis Information Resource [TAIR]; May, 2005),
are a group of genes encoding small polypeptides that, when
overexpressed, confer developmental phenotypes (Wen et al.,
2004). The GOstat results for Bigfoot genes ( Table 4) expand
upon the ‘‘first responder’’ list of GO terms that are most CNS
rich of all a-pairs in Arabidopsis ( Thomas et al., 2007). It was not a
surprise that particularly long gene spaces (Bigfoot) tended to be
CNS rich.
We investigated whether or not Bigfoot genes occur at random
in the phylogenetic trees of gene families. To answer this ques-
tion, we looked more closely at the 56 TF families recognized by
DATF (Table 2). Twenty-four of 44 a-paired homeobox (HB)
genes are Bigfoot (55%), but 0 of 20 MADS box a-paired genes
are Bigfoot. The range of percentage of Bigfoot among all TF
families is greater yet (even though some families have so few
Table 2. (continued).
TF Families
Total
Genes
Genes
Retained
Retention
Frequency
Average No.
CNS/Gene
Average Length
Total CNS/Gene
Fraction
Bigfoot
Bigfoot
Frequency
TF:HB 94 44 0.47 9.64 333.95 24 of 44 0.55
TF:MADS 108 20 0.19 4.38 138.57 0 of 20 0.00
TF:NAC 116 40 0.34 5.05 185.00 8 of 40 0.20
TF:C2H2 131 44 0.34 5.82 225.91 8 of 44 0.18
TF:AP2/EREBP 146 75 0.51 5.16 158.67 28 of 75 0.37
TF:bHLH 168 68 0.40 5.75 188.32 14 of 68 0.21
TF:MYB 207 100 0.48 6.00 213.90 28 of 100 0.28
Totals 1852 724 0.39
Figure 1. The Fasta Motifs from the PLACE Database That Are Most Enriched in CNSs Compared with Adjacent Noncoding NonCNS Gene Space.
The color codes are explained within the figure. Citations for each motif are at PLACE (http://www.dna.affrc.go.jp/PLACE/).
CNS Function in Arabidopsis 1445

a-paired genes that they cannot be studied at this time). Break-
ing the 24 HB Bigfoot genes into subfamilies, the HD-ZIPI and
HD-ZIPII subfamilies are almost all Bigfoot (90%), while those
genes in the sister HD-ZIPIII and -IV subfamilies are more like the
average TF. The Bigfoot notation measures a CNS property that
is positively correlated with CNS richness but is a unique feature
of gene space. As with CNS richness, Bigfoot genes characterize
some gene lineages but not others.
Smallfoot Genes
We then subjected a list of 1197 Smallfoot genes to the same
analyses described for Bigfoot genes. Table 5 shows the GOStat
results: 45 terms were enriched at P < 0.001, and 20 of them
involve signal transduction; the six highest ranked terms identify
protein kinase genes, including GO:004713, protein Tyr kinase
activity (rank 1, P ¼9E-12), and GO:004674. GO:0045449,
regulation of transcription (rank 8, P ¼1.9E-9), and GO:003700,
TF activity (rank 24, P ¼E-7), are also represented. Lower-
ranked terms include cytoskeleton and RNA helicase. Note that
no term involving signal transduction was significantly enriched
in Bigfoot genes (Table 4), so Bigfoot and Smallfoot genes
function quite differently.
Enriched and Purified Motifs in Bigfoot aCNSs
Bigfoot Genes
As presented previously, most Bigfoot genes encode TFs, most
are CNS rich, and all use an exceptional amount of chromosomes
for conserved function (Figure 2, Table 2). Even so, Bigfoot CNSs
comprise only 4.3% of the average Bigfoot gene space. The
Bigfoot subdatabase contains 2538 aCNS sequences (see Sup-
plemental Table 2 online) whose total sequence lengths sum to
91,970 bps of nonoverlapping, aligned (59-39)aCNS sequence.
These CNSs are distributed around Bigfoot genes with a greater
overall 59:39bias (3.1:1) than the similar statistic for the average
gene (1.8:1). The frequency and distribution of these CNSs will, of
course, influence the frequency and distribution of motifs located
within CNSs; that’s why these CNS distribution data are reviewed
here.
Random 7-bp Motif Enrichment and Purification
Supplemental Table 3 online lists all 16,384 (4
7
) possible 7-bp
DNA sequence motifs and data on their properties, hit numbers,
and hit locations in the aligned Bigfoot subdatabase. Data on
each motif in the database include number and frequency of
nonoverlapping hits to aCNS (for both complement and reverse
complement), hits to gene space control sequences, the ratio of
aCNS hits to control hits (called ‘‘fold enrichment’’ if aCNSs are
preferred and ‘‘fold purification’’ if noncoding nonCNS gene
space is preferred), and a x
2
evaluation of significance (see
Methods) for those 7-mers with hits totaling 10 or more; nominal
P values are listed and evaluated in a special column based on
Bonferroni corrections for multiple tests (see Methods). Figure
3A plots the number of occurrences of each of the 16,384 motifs
plus its reverse complement in aCNSs (yaxis) versus an equal
amount of gene space control sequences (xaxis; see Methods).
Table 3. Comparison of Features of Bigfoot and Smallfoot Genes, Where Bigfoot Genes Have >4 bp of Syntenous, CNS-Rich Space 39159of the
CDS, Smallfoot Genes Have Three to Five CNSs, and 24 Genes Are Both Bigfoot and Smallfoot
Feature Bigfoot Genes Smallfoot Genes
Number of genes (CNSs; total bp CNS; total
bp control noncoding)
252 genes (2897; 459,548 bp; 26,141,400 bp) 1197 genes (7532; 252,934 bp; 5,426,926 bp)
GO categories Narrow spectrum, 78% TFs; many respond to
environmental or hormonal signals ( Table 4)
Broad spectrum. Signal transduction, then
transcription, metabolism, and cytoskeleton
(Table 5)
Does the direction of transcription influence
enrichment/purification of a random 7-mer?
None significantly preferred complement
versus reverse complement, and only one
pair had a nominal P < 0.001 (see
Supplemental Table 3 online)
Many 7-mers were polar. Of the eight
significantly enriched, two were significantly
polar and four were polar at P nominal
<0.001. The next most significantly enriched
were 13% polar (see Supplemental
Table 4 online)
How important is purifying selection in
determining the overall sequence
of gene space?
Very. The difference between slope 0.45 and
the expected 1.0 of the hits CNS versus hits
control of the trend line of Figure 3A implies
massive purification of CNSs.
Very. The difference between the slope 0.51
and 1.0 on the similar plot as discussed for
Bigfoot genes attests to importance of
purification (data not shown).
Which 7-mer sequences tend to be purified
from CNSs (removed)?
Most purified are long runs of A or T, and
purified are almost any sequence with more
than three As or Ts in a row. In general,
[AþT] is purified. More research is needed.
Similar to Bigfoot genes. A general definition of
CNS should include sequences purified and
sequences under positive selection.
Which 7-mers are enriched? Are they
TF binding sites?
The most significant carry CACGTG (G-box
binds bZIP or HLH TFs), plus a few known
TF binding sites, but most are unknown
(see Supplemental Table 3 online).
One of the eight significant 7-mers could bind
a MYC TF (see Supplemental Table 4
online). As with Bigfoot genes, significant
and ‘‘worth interest’’ 7-mers are mostly
unknown (e.g., 59-CTTCTTC).
1446 The Plant Cell

Given normal distributions, and no difference between aCNS
sequence and nearby noncoding sequence, a trend line in Figure
3A should emerge with a slope of 1.0; this slope, the null
hypothesis, reflects a 1:1 motif hit ratio between CNS and control
noncoding sequence. Unexpectedly, the slope of the trend line in
Figure 3A is 0.46. Many of the 7-bp motifs that are most
overrepresented in noncoding space (enclosed in the largest
oval in Figure 3A) have been purified from aCNS sequences, so
much so that the slope of the trend line is far below 1.0. Those
motifs most overrepresented in noncoding sequence in general
(high AþT sequence and especially in runs; Figures 3B and 3C)
tend not to be in CNSs. There are 150 significantly purified
7-mers and 282 additional 7-mers that have low enough P values
to be worth interest (see Methods). Supplemental Tables 3 and
4 online have these 7-mer rows color-coded in pink and blue,
respectively.
We found 14 motifs, seven pairs, that are significantly enriched
(overrepresented, the opposite of purified) in Bigfoot aCNSs;
those furthest from and above the trend line are circled in Figure
3. Most of these motifs have a core ACGT. Each point on Figure
3, each representing the sum of complement and reverse com-
plement hits, was evaluated for significance of difference from
an expected (via null hypothesis) 1:1 aCNS:control ratio (see
Methods). All data points enclosed within circles or ovals in
Figure 3A are significantly unexpected (see Supplemental Table
3 online).
Among those 7-mers enriched in aCNSs, 14 (including reverse
complements) are enriched significantly. These 14 are listed in
the legend of Figure 3. Ten of these 14 motifs carry a complete
G-box, or are consistent with overlapping a complete G box, and
all have the core ACGT. The additional significantly enriched
7-mer motifs were experimentally unprecedented: a CATG core
and a CCAC/GTGG core. The fold enrichment for these 14 most
significant 7-bp sequences varies from 3.21 (TCACATG and
reverse complement: 61 hits) to 9.89 (CACGTGC and reverse
complement: 40 hits). Among all 8-mers with hits in Bigfoot gene
aCNSs >9, GCACGTGC was most enriched at 23-fold (n¼10).
This maximum-enriched 8-mer is clearly related to the most
enriched PLACE motif (stress and ABA responsiveness) dis-
cussed previously: CACGTGGC. Among all 8 bp, this PLACE
motif also ranks among the most enriched in the Bigfoot aCNSs
at 9.7-fold (n¼22).
There are 28 7-mers, those that hit Bigfoot CNSs in the ‘‘worth
interest’’ category of significance; these usually share core motifs
CATG, ACGT (G-box core), or CCAC/GTGG that are known to be
associated with significantly enriched 7-mers and sometimes
group into additional orderly patterns.
GAGA motifs are not significantly enriched. The GAGAGAG/
AGAGAGA 7-mers (and reverse complements) were 2.1-fold en-
riched with an insignificant P value, one below our ‘‘worth
interest’’ category. The most significantly enriched GAGA-like
sequence is GGAGAAG and its reverse complement at 2.75-fold
enrichment (nominal P ¼0.003), with 30 hits in the Bigfoot aCNS;
this 7-mer is also not ‘‘worth interest.’’
Sequences That Are Missing (Purified) from the aCNSs
of Bigfoot Genes
We use the term ‘‘purified’’ to be the opposite of ‘‘enriched’’ to
connote the selective process that presumably removed unfit
Figure 2. Partial Viewer Screen Shot of Frozen Gene Space of an HD-ZIP TF Homoeologous Gene Pair, an Exemplary Bigfoot Gene Space.
Orientation is /; transcription is right to left. The space is enclosed by CNS1 at the 59end and CNS12 at the 39end. CNS10 is colored blue because it
is oriented backward (/þ) from exon orientation. The solid colored exons of the models (GenBank from TAIR, The Institute for Genomic Research
[TIGR] version 5) have no untranslated region annotations, suggesting that cDNA evidence was lacking. CNS15 is colored red, denoting that it was
invalidated during the proofing stages of CNS database construction (Thomas et al., 2007).
CNS Function in Arabidopsis 1447

sequence from functional regions of gene space. A total of 150
7-bp sequences are significantly purified from BF aCNSs, and an
additional 282 7-mers are purified at ‘‘worth interest’’ levels of
significance. Many of the significantly purified 7-mers are highly
abundant in noncoding space, which accounts for the 0.46 slope
of the trend line in Figure 3A. Figures 3B and 3C plot all purified
7-mers that hit a Bigfoot CNS at least 10 times in the order of their
purification significance, with the most significant on the left
(arrows in Figure 3C at P nominal < 0.00001, P nominal < 0.001,
and P nominal < 0.05). The xaxes are percentage of GC of the
7-mer (Figure 3B) and 7-mers with runs of 4 nucleotides (Figure
3C). It can be seen from Figures 3B and 3C that significant
purification involves runs of A and T and generally high percent-
age of AT, the very sequences that characterize the bulk of
noncoding space.
Distribution of Significantly Enriched or Purified
7-bp Sequences within Bigfoot Gene Space and
Strand Preference
The 59:39ratio of the 2538 CNSs around Bigfoot genes, the genes
used for this 7-mer analysis, is 3.1: 1, and the ‘‘worth interest’’
7-mers 3.8:1 59:39. Those 14 most significantly enriched 7-mers
exhibit a 59:39ratio of 7.3:1. The mean 59aCNS hit position for the
Table 4. 256 Bigfoot Genes Are Enriched for Particular GO Categories (Ranked by Significance)
Rank GO Gross Function GO Description Count of 256 Total Count GOStat P Value
1 GO:0006355 Transcription Regulation of transcription, DNA dependent 54 1114 0
2 GO:0006350 Transcription Transcription 96 1965 0
3 GO:0045449 Transcription Regulation of transcription 96 1845 0
4 GO:0006139 Transcription Nucleoside, -tide, base NA metabolism 96 3144 0
5 GO:0019222 Regulation of... Regulation of metabolism 97 1915 0
6 GO:0003677 Transcription DNA binding 104 2792 0
7 GO:0003700 Transcription TF activity 104 2060 0
8 GO:0050791 Regulation of... Regulation of physiological processes 97 2118 0
9 GO:0019219 Regulation of... Nucleic acid regulation of 96 1859 0
10 GO:0050794 Regulation of... Regulation of cellular processes 96 2079 0
11 GO:0031323 Regulation of... Regulation of physiological process 96 1896 0
12 GO:0050789 Regulation of... Regulation of biological process 97 2331 0
13 GO:0003676 Transcription NA binding 106 3956 0
14 GO:0051244 Regulation of... Regulation of cellular physiological process 96 2075 0
15 GO:0006351 Transcription Transcription, DNA-dependent 54 1158 4.52E-86
16 GO:0005634 Nucleus Nucleus 78 2536 2.24E-75
17 GO:0044238 Metabolism Primary metbolism 113 8953 1.42E-28
18 GO:0044237 Metabolism Cellular metabolism 116 9761 3.61E-26
19 GO:0008152 Metabolism Metabolism 121 11086 4.75E-23
20 GO:0043231 Organelle Intracellular membrane-bound organelle 114 10654 3.07E-20
21 GO:0043227 Organelle Membrane-bound organelle 114 10655 3.07E-20
22 GO:0050875 Organelle Cellular physiological process 125 12540 1.67E-19
23 GO:0043229 Organelle Intracellular organelle 115 11089 4.68E-19
24 GO:0043226 Organelle Organelle 115 11090 4.68E-19
25 GO:0009987 Cell Cellular process 126 13047 3.02E-18
26 GO:0005622 Cell Intracellular 119 11949 3.92E-18
27 GO:0042221 Response to... Response to chemical stimulus 25 1142 5.34E-14
28 GO:0009719 Response to... Response to endogenous stimulus 24 947 1.34E-09
29 GO:0009723 Response to... Response to ethylene stimulus 11 138 1.38E-09
30 GO:0009725 Response to... Response to hormone stimulus 20 655 1.95E-09
31 GO:0009628 Response to... Response to abiotic stimulus 25 1627 9.37E-08
32 GO:0005623 Cell Cell 130 18237 1.01E-06
33 GO:0009873 Response to... Ethylene-mediated signaling pathway 6 51 1.88E-06
34 GO:0009753 Response to... Response to jasmonic acid stimulus 8 124 2.02E-06
35 GO:0009861 Response to... Response to wounding stress 8 154 1.02E-05
36 GO:0009651 Response to... Response to salt stress 7 112 1.33E-05
37 GO:0000160 Signal transduction Two-component signal transduction
(phosphorylation)
6 74 1.56E-05
38 GO:0009751 Response to... Response to salyclic acid stimulus 7 121 2.11E-05
39 GO:0006970 Response to... Response to osmotic stress 7 134 3.95E-05
40 GO:0009611 Response to... Response to wounding 8 210 8.55E-05
41 GO:0009737 Response to... Response to ABA stimulus 7 177 0.00023
42 GO:0009733 Response to... Response to auxin stimulus 8 261 0.00040
43 GO:0009814 Response to... Response to pathogen 8 264 0.00040
1448 The Plant Cell

significantly enriched 7-mers is 3.15 kb upstream of the 59ATG;
the mean 39position is 1.9 kb downstream from the stop codon
upstream. Data for the 28 ‘‘worth interest’’ 7-mers are similar.
These average locations within Bigfoot gene space are well
outside the transcription unit and outside the 0 to 500-bp
proximal promoter that is generally studied experimentally.
None of the 7-mers to Bigfoot CNSs showed preference for
orientation with regard to the direction of transcription; there
were neither significant nor ‘‘worth interest’’ motifs (see Supple-
mental Table 3 online). This is in contrast with similar data for
Smallfoot genes, as will be shown.
Smallfoot Gene Data
We prepared an aligned subdatabase of aCNSs with genes
labeled as Smallfoot genes as described and subjected this gene
list to the same analyses as was the Bigfoot gene list (Supple-
mental Table 5 online is the 7-mer to Smallfoot genes data sheet).
Table 5. 1197 Smallfoot Genes Are Enriched for Particular GO Categories (Ranked by Significance)
Rank GO Gross Function GO Description Count of 1197 Total Count GOStat P Value
1 GO:0004713 Signal transduction Protein Tyr kinase activity 46 650 9.09E-12
2 GO:0005524 Signal transduction ATP binding 84 1630 8.22E-11
3 GO:0004674 Signal transduction Protein Ser-Thr kinase activity 56 927 1.39E-10
4 GO:0030554 Signal transduction A nucleotide binding 84 1663 1.80E-10
5 GO:0017076 Signal transduction Purine nucleotide binding 91 1909 1.06E-09
6 GO:0006468 Signal transduction Protein–amino acid phosphorylation 60 1071 1.06E-09
7 GO:0045449 Transcription Regulation of transcription 88 1845 1.77E-09
8 GO:0019219 Transcription Nucleic acid metbolism 88 1859 2.62E-09
9 GO:0000166 Signal transduction Nucleotide binding 99 2186 2.93E-09
10 GO:0051244 Regulation of... Regulation of physiological process 95 2075 3.27E-09
11 GO:0050794 Regulation of... Regulation of cellular process 95 2079 3.27E-09
12 GO:0016773 Signal transduction P-transferase activated alcohol acceptor 64 1210 3.27E-09
13 GO:0016310 Signal transduction Phosphorylation 62 1166 4.67E-09
14 GO:0004672 Signal transduction Protein kinase activity 57 1038 4.95E-09
15 GO:0031323 Regulation of... Regulation of cellular metabolism 88 1896 5.34E-09
16 GO:0050791 Regulation of ... Regulation of physiological process 95 2118 8.65E-09
17 GO:0019222 Regulation of ... Regulation of metabolism 88 1915 9.13E-09
18 GO:0006796 Signal transduction Phosphate metabolism 63 1229 1.84E-08
19 GO:0006793 Signal transduction Phosphorus metabolism 63 1230 1.84E-08
20 GO:0006350 Transcription Transcription 88 1965 4.08E-08
21 GO:0043283 Enzyme Biopolymer metabolism 113 2742 5.32E-08
22 GO:0006464 Signal transduction Protein modification 72 1513 6.06E-08
23 GO:0050789 Regulation of... Regulation of biological processes 99 2331 9.50E-08
24 GO:0003700 Transcription TF activity 90 2060 1.01E-07
25 GO:0043412 Signal transduction Biopolymer modification 73 1637 1.39E-06
26 GO:0016772 Signal transduction Transferring P-containing groups 81 1896 2.27E-06
27 GO:0006139 Signal transduction Nucleobase, -side, -tide, nucleic
acid metabolism
120 3144 2.41E-06
28 GO:0005634 Nucleus Nucleus 101 2536 2.92E-06
29 GO:0016301 Signal transduction Kinase activity 72 1657 5.52E-06
30 GO:0007169 Signal transduction Transmembrane receptor protein
Tyr kinase signaling pathway
16 137 6.17E-06
31 GO:0007167 Signal transduction Enzyme-linked receptor protein signaling pathway 16 137 6.17E-06
32 GO:0003677 Transcription DNA binding 106 2792 1.92E-05
33 GO:0005515 Regulation of... Protein binding 82 2027 2.35E-05
34 GO:0044238 Metabolism Primary metabolism 275 8953 4.34E-05
35 GO:0007010 Cytoskeleton Cytoskeletal organization and biogenesis 17 191 0.00010
36 GO:0015630 Cytoskeleton Microtubule cytoskeleton 13 125 0.00024
37 GO:0030163 Enzyme Protein catabolism 19 294 0.00027
38 GO:0007166 Signal transduction Cell surface receptor-linked signal
transduction
16 187 0.00028
39 GO:0007018 Cytoskeleton Microtubule-based movement 9 63 0.00041
40 GO:0003676 Transcription Nucleic acid binding 134 3956 0.00043
41 GO:0046910 Enzyme Pectinesterase inhibitor activity 9 64 0.00045
42 GO:0007017 Cytoskeleton Microtubule-based process 11 101 0.00064
43 GO:0003724 Enzyme RNA helicase activity 5 17 0.00075
44 GO:0005875 Cytoskeleton Microtubule-associated complex 9 69 0.00076
45 GO:0043285 Enzyme Biopolymer catabolism 19 315 0.00100
CNS Function in Arabidopsis 1449

Tables 4 and 5 show that the gene GO annotations of Bigfoot and
Smallfoot genes are very different. In general, the 7-mers
enriched were very different as well, as summarized in Table 3
and Supplemental Table 4 online. Those eight sequences (com-
plement only) enriched significantly and 53 sequences enriched
at a ‘‘worth interest’’ significance level were, with one possible
exception (a MYC gene binding site; see Supplemental Table 4
online), without experimental precedent and not the same as
were hit in the Bigfoot database. The most significantly enriched
7-mer is 59CTTCTTC, and there are several more reasonably
similar sequences ranking among the 61 most significant. There
are several sequences that are 59CACG-like, all of unknown
function. In general, 7-mers that significantly hit Smallfoot CNSs
are approximately five times more numerous but about half as
enriched as are the comparable Bigfoot 7-mers (Table 3). If we
had twice as many 7-mer hits, as would be the case with a twice-
as-large database, there would be a manifold increase in the
number of significantly enriched 7-mers.
Figure 3. The Nature of Random 7-Mer Hits to Bigfoot Gene aCNSs.
(A) Plot is the number of each 7-mer motif in CNSs ( yaxis) versus number of hits in control noncoding DNA ( xaxis) where the expected hit ratio is 1:1
(primary data; see Supplemental Table 3 online). Each point is a particular 7-mer. The slope of the correlation line, 0.46 and not 1.0, and the volume of
the points that define it (large oval) imply that many overrepresented 7-mers are removed (purified) from aCNSs (rightmost oval is the most purified).
Some 7-mers are most enriched in aCNSs (circle at left). The 14 most significantly enriched 7-mers, in descending order of their significance, are as
follows (all P < E-5): 59-ACACGT, CACGTGT, CACGTGA, TCACGTG, CACGTGC, GCACGTG, CACGTGG, CCACGTG, ACGTGGC, GCCACGT,
CATGTGA, TCACATG (the MYCATERD1 box at PLACE: dehydration stress [Tran et al., 2004]), GGACCAC, and GTGGTCC (not in PLACE).
(B) and (C) These graphs illustrate the nucleotide content of purified 7-mers. The xaxis is all purified 7-mers ranked from most significantly purified
(lowest P value) on left to least significant on right, with arrows denoting the boundaries of the three nominal P value groups that are below P ¼0.05
(95% confidence is all to the left of the rightmost arrow).
(B) Plots percentage of GC of 7-mer ( yaxis) versus significance of purification.
(C) Plots ‘‘yes or no’’ to the question, ‘‘Is there a run of four nucleotides in this 7-mer?’’ versus significance of purification, where a vertical line denotes
‘‘yes.’’ The three arrows denote, from left to right, nominal P values for significance of purification: P ¼10
5
,10
3
, and 0.05. Note that 7-mer purification
is elevated with high percentage of AT and runs of the same nucleotide.
1450 The Plant Cell

For 7-mers to Bigfoot genes, it didn’t matter whether the
complement or reverse complement was used. Smallfoot gene
CNSs hit significantly by 7-mers are often biased (Table 3; see
Supplemental Tables 3 and 4 online) to one strand or the other.
This directionality fits the compact nature of Smallfoot genes and
tends to validate the 7-mer motif’s functionality in relation to
transcription or translation of Smallfoot genes. Twenty-five
percent of significantly enriched and 5% of ‘‘worth interest’’-
enriched 7-mers are biased significantly (Bonferroni-corrected;
see Methods). These percentages rise to 50 and 13%, respectively,
if strand bias is judged at the P nominal < 0.001 level. Even purified
sequences to Smallfoot genes show strand bias at approximately
one-third the values of enriched 7-mers. Under each of these
conditions, every 7-mer to Bigfoot gene CNS showed zero stand
bias; Bigfoot CNSs provided an exceptionally useful control.
G-Boxes in CNSs Are Not Significantly Associated
with Conserved Neighbor 7-Mers or Motifs, Except
Other G-Boxes
The G-box, CACGTG, has been studied in detail for two de-
cades. Some of these studies have found associated motifs that,
with the G-box, comprise a cis-acting array. However, the
G-boxes have been in core promoters (not as far upstream as
our CNS G-boxes). Studies on ribulose-1-5-bisphosphate car-
boxylase small subunit genes, for example, have discovered a
conserved modular array consisting of a G-box and an adjacent
I-box (Arguello-Astorga and Herrera-Estralla, 1998), and a GCC
motif (jasmonic acid, GCCGCC) was found near a G-box in
pmt1a gene of tobacco (Nicotiana tabacum; Xu and Timko,
2004), and both were necessary for jasmonic acid induction.
Weaker associations between G-boxes and adjacent sequences
have been reported (Giuliano et al., 1988). The Arabidopsis
G-box and an I-box–like motif (ATAATCCA) were associated with
photosynthesis GO terms in Arabidopsis (Vanderpoele et al.,
2006). None of these G-box–linked motifs or any PLACE motif
was found significantly overrepresented in the same CNS as a
G-box. The single exception is another G-box (see Supplemental
Table 2 online). The frequency of finding an exact G-box in a CNS
is 0.02 (297/14,944). Of the 136 G-box CNSs that were long
enough to extend 20 bp on both sides, nine had at least one other
exact G-box in this 46-bp sequence, for a frequency of 0.07. A
crude expectation for two or more G-boxes in any one CNS by
chance would be (0.02)
2
¼0.0004. The observed 0.07 exceeds
null hypothesis expectations by 175-fold. We also used the
DIALIGN Web application (Morgenstern et al., 2006) on all 267
G-box CNSs and their reverse complements, anchored on the
G-boxes and strict global positioning around all G-boxes, to
search for statistically significant nucleotides or motifs con-
served near G-boxes; no motifs were found other than the G-box
itself. We compiled a table of all CNSs and all significantly
enriched Bigfoot 7-mers and looked for any co-occurrence of
7-mer pairs to CNSs; we found nothing significant.
Evaluation of Shared CNSs
Some sorts of negative data are important. With trivial excep-
tions, there were zero Arabidopsis aCNSs, not even our most
significant CNSs, detectable by bl2seq in other Arabidopsis
gene space except the duplicate from the a-tetraploidy. Sup-
plemental Table 5 online contains these data. We examined 23
Bigfoot gene pairs with an out-group gene from the same
lineage, a gene categorized by Bowers et al. (2003) to have
diverged from a-duplicate genes as part of the b-tetraploidy.
(The b-tetraploidy, more ancient than the a-tetraploidy, occurred
after the monocot-dicot split [Bowers et al., 2003], but it remains
unclear whether or not the b-tetraploidy is in poplar [Tuskan et al.,
2006].) Not one CNS was conserved between either a-gene and
any b-out-group gene. These three-gene comparisons were at
the tips of each of the 23 gene trees. The b-tetraploidy event has
been estimated to have happened ;100 million years ago
(Bowers et al., 2003; Maere et al., 2005). Therefore, unlike the
case in vertebrates, where some enhancer CNSs are conserved
for >350 million years (see Introduction), the maximum level of
CNS conservation in higher plants is relatively shallow. That does
not mean that the function specified by these CNSs is not
conserved; it means that we cannot detect such conservation at
the sequence level.
Supplemental Table 5 online also conveys an exception to the
above result: occasionally CNSs are duplicated, usually in tan-
dem, within the gene space of a single gene.
DISCUSSION
Limitations of Intragenomic Footprints
We can only analyze 25% of the gene content of Arabidopsis,
which is the gene content that was retained in pairs following
a-tetraploidy. We found that those genes retained, and studied,
are not expected to have more or fewer CNSs than the remaining
75% of the minimized genome would have had they all retained
post-tetraploidy or if there were a brassicoid genome diverged to
the same extent as are a-pairs. To our knowledge, no Arabidop-
sis relative useful for pairwise (CNS) research has been se-
quenced to date.
Without an out-group branching off the Arabidopsis lineage
before the a-tetraploidy, we cannot measure subfunctionalized
or neofunctionalized (gain-of-function) sequences, although post-
tetraploidy duplicate genes certainly diverge (Gu et al., 2002a,
2002b; Makova and Li, 2003; Raes and Van de Peer, 2003; Gu
et al., 2004; Haberer et al., 2004; Li et al., 2005; Rastogi and
Liberles, 2005; Roth et al., 2006). However, sometimes diver-
gence is slower than expected (Koonin, 2005; Chapman et al.,
2006), or divergence may vary by gene functional category (Ha
et al., 2007). What we measure are sequences shared by
homoeologs for the entirety of the time since tetraploidy. These
7470 noncoding sequence pairs in our aCNS database are likely
to specify the same function today as they provided in the
ancestral gene before tetraploidy. Cotton (Gossypium hirsutum),
a candidate out-group, is simply too ancient to facilitate Arabi-
dopsis–cotton CNS discovery (data not shown), and poplar is
even more distant. Papaya (Carica papaya; in the family Carica-
cae, in the same order as Arabidopsis) could constitute a useful
out-group when the sequence is finished (Lai et al., 2006). Lack of
a properly positioned out-group or usefully diverged genomic
CNS Function in Arabidopsis 1451

sequences fundamentally limits this and all current work using
the a-tetraploidy of Arabidopsis.
Results That Validate the Functionality of CNSs
Table 6 summarizes our major results. Three results imply CNS
functionality independent of evolutionary conservation. (1) CNS
richness varies with gene GO category (Thomas et al., 2007).
Genes annotated GO ‘‘respond to. . .’’ are most CNS-rich, then
TF genes in general, then signal transduction genes, then met-
abolic genes, and then, with zero CNSs, genes encoding ribo-
somal subunits and components of the mitochondria. Our GO
term analyses of Bigfoot (Table 4) and Smallfoot (Table 5) genes
substantiates this result. (2) The most significantly CNS-enriched
motifs in all CNSs and in the Bigfoot CNS list are G-box motifs
(Figures 2 and 3A). G-boxes are proven to bind known families of
TFs (Menkens et al., 1995; Toledo-Ortiz et al., 2003), lending
credibility to our database. (3) The significantly and worth interest
CNS-enriched motifs in the Smallfoot but not (0%) Bigfoot CNSs
are often aligned in relation to the direction of transcription.
Twenty-five percent of the significantly enriched and 6% of those
worth interest are polar, and these values increase to 50 and
13%, respectively, if we reduce the significance level to P
nominal <0.001. Bigfoot 7-mers show zero polarity. The CNS
enrichment significance of complement versus reverse comple-
ment is given in Supplemental Tables 3 and 4 online. CNS
polarity, based on the direction of transcription, implies a func-
tion physically linked to the transcription process, which fits
Smallfoot gene spaces with their CNSs being close to or within
the transcriptional units. We could think of no alternative to
function that could account for these three results, but experi-
mental verification of aCNS function remains outstanding. Pre-
sumably, this function is to bind or affect the movement (e.g.,
boundary elements) of regulatory factors. Small RNAs binding
gene transcripts are unlikely candidates for binding aCNS se-
quence (Thomas et al., 2007), although such binding is possible
for 1.3% of the CNSs. We are left with the hypothesis that CNSs
generally bind proteins or other macromolecules. CNSs of the
sort that are aligned near Smallfoot gene exons behave like
typical TF binding sites. On the other hand, CNSs far upstream of
Bigfoot exons, even though they may carry a G-box, are more
like enhancers.
Many CNSs May Be Explained by Selection for or against
Particular Sequences, but Almost All of These Sequence
Motifs Were Unknown Previously
As seen from the slope of the regression line in Figure 3A (0.46 and
not the expected 1.0) and in the description of the similar line
comparing Bigfoot and Smallfoot genes in Table 3 (0.51 and not
the expected 1.0), it is clear that CNS regions of noncoding gene
space have been significantly purified of many particular 7-mer
sequences. CNSs are defined largely by what sequences they do
not contain. Apparently, whenCNSs are interrupted by many sorts
of sequences, function is impeded. Thus, a CNS must tend to be a
module of function rather than a chancecloseness of independent
binding sequences. In other words, a CNS generallydoes not have
spacer DNA thatcould be any sequence. We know thatruns of five
or six of any nucleotide and runs of four of A or T are particularly
removed from CNS space. AAAAAAA in Smallfoot CNSs is
purified at 0.13 in CNS versus in noncoding control space (P ¼0).
With exceptions, Bigfoot and Smallfoot CNSs have had very
similar sequences removed but are characterized by very differ-
ent enriched 7-mers (see Supplemental Tables 3 and 4 online).
Almost all of our significant or worth interest 7-mers have no
experimental history. For example, there are nine 7-mers of
the 50 most enriched in Smallfoot genes marked ‘‘High GC’’ (e.g.,
59-CGTGGCC, 59-CGGCGCC, or 59-GAGCCGT of Supplemen-
tal Table 4 online). The core CCGTCC may be similar to a few of
these; this is called Box A (Logeman et al., 1995) in the PLACE
database (Higo et al., 1999). Our results indicate that the linear
sequence of conserved noncoding DNA sometimes explains
function. We need much more CNS data (10-fold) in plants if we
are to generate enough hits to fully evaluate linear DNA sequence
as an explanation for CNS function. The sorghum sequence as a
rice comparison should be perfectly suited to this aim (Inada
et al., 2003). It remains possible that a conserved CNS sequence
is but one of several or many that maintains identical functional
conformations.
Table 6. Four Major Conclusions as Clues to CNS Functions
1. Some CNS-enriched motifs, like the G-box and jasmonic acid ( jerr or GCC box in Figure 1 and MYCATERD1 box in Figure 3A), have a rich
experimental history, but the vast majority of CNS-enriched motifs have yet to be studied. The G-box in very 59CNSs may be functionally distinct
from the well-described G-box in proximal promoters of light-sensitive genes. Adding the Bigfoot and Smallfoot genes together, CNSs are
significantly enriched in 22 7-mer sequences, and another 81 7-mers are enriched (nominal P < 0.001) to an extent ‘‘worth interest.’’ Similar counts
for purified 7-mers is 388 and 564; CNSs may be more what sequences they are not than what they are.
2. Those genes with the most CNSs tend to be ‘‘first responder’’ genes, these being TF genes 64% of the time ( Thomas et al., 2007), induced by
environment or hormonal signals (Table 4). Those genes that take up an extraordinary length of chromosome, Bigfoot genes, tend to be CNS rich
and are especially represented among ‘‘first responder’’ genes, these being TF genes of particular subfamilies or classes (i.e., HD-ZIPI). Genes with
two to five CNSs tend to be involved in signal transduction and not transcription (Table 5). The modal gene is not regulatory and has 0 CNSs
(Thomas et al., 2007).
3. Unlike the situation in vertebrates, we found no evidence for highly conserved (100 to 400 million years) noncoding sequences within the gene trees
of Arabidopsis. Even so, the most conserved sequences in plants seem to resemble the most conserved noncoding sequences in animals in terms
of regulatory/developmental function and location in gene voids.
4. There is no one functional explanation for CNS richness. While Bigfoot genes, G-box CNS genes, ‘‘first responder’’ genes, and TF genes all tend to
be CNS rich, analyses of the detailed gene contents of these categories finds more uniqueness than overlap.
1452 The Plant Cell

Genes with Zero CNSs
It is curious why the modal gene carries zero CNSs and is also
expected to contain many TF binding sites. We think there is an
explanation: the expected TF binding sites are either conserved
as motifs but not as recognizable linear sequence or that there
has been enough binding site turnover among redundant se-
quences in a cluster to obscure the conserved function, as has
been demonstrated in Drosophila (Ludwig et al., 1998, 2005;
Moses et al., 2004, 2006) and mammals (Frith et al., 2006). When
Arabidopsis a-gene pairs are anchored on the 59-A of their ATG
and compared using a modified local alignment program, such
as DIALAIGN (Brudno et al., 2003; used by Haberer et al., 2004),
convincing footprints were detected in the 500-bp upstream
region. These less significant intragenomic footprints include
potentially the TF binding sites that are missing in our modal pair
of homoeologs. Motif-finding algorithms coupled with phyloge-
netic footprinting between poplar and Arabidopsis have been
successful in finding convincing arrays of TF binding sites in the
promoters of dicot MADS box TF genes (De Bodt et al., 2006).
Additionally, Vandepoele et al. (2006) found motifs in Arabidopsis
genes using three input data sets: genome sequence of Arabi-
dopsis, genome-wide expression data for Arabidopsis, and the
complete genome of the related out-group dicot, poplar. Con-
fining themselves to 500 to 1000 bp upstream of exon 1, these
workers used alignments to find Arabidopsis-poplar orthologs
and used a nonalignment-based MotifSampler (Thijs et al., 2001,
2002) to find overrepresented, conserved (functional) short
proximal promoter cis-acting motifs among coregulated genes.
There is adequate evidence that a TF binding site in the proximal
promoters of Arabidopsis genes can sometimes be identified if
the correct tools are used. Presumably, future research, perhaps
using binding site reconstruction tools such as MONKEY (Moses
et al., 2004, 2006), will find that CNSs, and especially the larger
CNSs, contain more than one binding motif, or that anchoring on
CNSs might permit finding conserved motifs adjacent to CNSs
and far from exons. The fact remains that we did not find any
motif associations within CNSs with the exception that G-boxes
tend to occur multiple times in CNSs.
We suggest that plant researchers interested in cis-regulatory
modules include all noncoding gene space as we define it (Table
1) and not just a bit of 59sequence.
CNS Function and Gene Expression
The most significant short sequence enriched in CNSs is the
G-box; this is a clue as to the role(s) of CNSs in gene expression.
However, because the historically documented G-box is the
most significantly CNS-enriched TF binding motif does not mean
that the G-box–containing CNSs should exhibit historical prop-
erties. It is possible that the G-box has many functions and those
CNS-associated have not been studied. The placement of the
average G-box is in a CNS 1.5 kb upstream of exon 1, and the GO
annotation of the G-box–containing gene spaces did not include
response to light signals. This far 59location is not far by Bigfoot
gene standards with their mean CNS 3.1 kb 59. Perhaps the
G-boxes within CNSs are as yet unstudied enhancers. On the
other hand, research on G-box–like motifs has linked them to
several different functions. The ACGT core embedded in a num-
ber of related sequences is known to bind TGA-type bZIP TFs
(Izawa et al., 1993), although this core is not always necessary for
binding (de Pater et al., 1994). The phenotypic consequences of
such binding can involve both positive and negative regulation of
transcription in response to developmental cues, ABA, auxin,
pathogenic stress, salicylic acid, and ethylene (Tucker et al.,
2002). However, the GOStat results on our genes with G-box
CNSs did not yield any significant term in the ‘‘response to. . .’’
genre except ‘‘response to ethylene.’’ Further research on these
distant CNS G-boxes and their genes is needed.
All three of the original studies of CNSs in plants (Kaplinsky
et al., 2002; Guo and Moose, 2003; Inada et al., 2003) noted
that,compared with man-mouse, there were far fewer CNSs in
plants and they were far shorter, so much so that the definition
used by mammalian workers to define a CNS would find no CNSs
in plants at all. This observation was predicated on the compa-
rability of data from man-mouse and maize-rice, both with exons
diverged to ;85% nucleotide sequence identity over long high-
scoring pairs from BLAST-2-sequences. Our work on aCNSs in
Arabidopsis is consistent with the observation that plants have
vastly fewer and shorter CNSs than mammals. Most compelling
is the difference in distribution of CNSs along the grass or
Arabidopsis chromosome versus the man-mouse chromosome.
In plants, CNSs are almost always clustered near exons so that
sorting them to gene was feasible; mammalian intergenic regions
are often covered with CNSs, and raising bl2seq mismatch
penalties does not help. Sorting them to gene without experi-
mental data would generate ambiguities. Inada et al. (2003)
suggested that CNSs largely keep stem cell/meristem genes
from being ectopically expressed, where they might cause
disease/cancer, and that many more genes use this regulatory
mode in mammals compared with plants due to several differ-
ences in biology. This hypothesis was based on one case study
and some arguments. Our results support this useful but un-
tested hypothesis.
CNS Richness, Bigfoot Genes, and Evolvability
When faced with stress, such as must occur with environmental
change, animals move and plants endure on penalty of extinc-
tion. Major trends in animal form and development might be seen
primarily as adaptations involving organismal and cellular move-
ment, with trends involving synapses becoming paramount over
evolutionary time in the metazoan lineage. Perhaps the compa-
rable major (most complex, perhaps) trends during higher plant
evolution might be in those gene lineages that, when expressed
as functional modules, modify abilities to endure. Over the last
500 million years of plant evolution, the maximums of adaptation
to environmental extremes have probably gone up and not down
over time; maximums of plant morphological complexity have
certainly gone up (Freeling and Thomas, 2006). Table 4 details
the many ‘‘response to. . .’’ GO categories that are significantly
overrepresented in the Bigfoot database. We suggest that Big-
foot genes and other CNS-rich genes include those special
endurance genes, genes we have lumped together in the general
category ‘‘first responder’’ to environmental signals. As an illus-
tration, the CNS-rich TF family HB, the even more CNS-rich
CNS Function in Arabidopsis 1453

subfamily, HD-ZIP (homeodomain-basic leucine zipper), and
especially the Bigfoot HD-ZIPI class are known to be induced
by environmental stress (Henriksson et al., 2005). The relation-
ship between CNSs and signal response invites further research.
Recent research from the Z.J. Chen laboratory (Ha et al., 2007)
found that a-duplicates that were induced by environmental
stress had more divergent expression patterns than a-duplicates
expressed programmatically in the course of development.
Most Arabidopsis genes are positioned close to one another
on the chromosome and have zero aCNSs. Bigfoot genes are not
only exceptional in size, but, as first responders, function high in
the regulatory cascade. If CNS richness implies a high level of
gene regulation, then Bigfoot genes are, at the same time, high-
level regulatory genes and also the most highly regulated (Inada
et al., 2003; Thomas et al., 2007). This enigma suggests a
systems sort of regulatory model. During the course of higher
plant evolution, mutations (subfunctionalizations and neofunc-
tionalizations) in particular genes, important genes, may help
explain adaptations to changing environments. Bigfoot genes
present exceptionally large targets for regulatory mutation. Since
Bigfoot genes are exceptionally enriched in ‘‘response to...’’
genes (Table 4), the report that ‘‘response to...’’ homoeologs may
diverge in expression particularly rapidly (Ha et al., 2007) sup-
ports these conclusions. All else being equal, it seems wise to
look at alleles, derivatives and cooptions involving these large,
CNS-rich, first responder genes to better understand the genetic
basis of plant endurance.
METHODS
a-Pairs, Gene Spaces, and the aCNS Database
We used the Arabidopsis thaliana intragenic CNS database as by Thomas
et al. (2007) and its supplemental data. When pairs, gene spaces, or CNSs
gain information due to this study, that information is added to our data
tables as Supplemental Tables 1 and 2 online.
Gene Categories and Statistics
Genes were categorized by GO term (per Thomas et al., 2007), as MIR
genes, and those genes that were also TFs were subcategorized into
DATF’s 56 families (Database of Arabidopsis Transcription Factors, July,
2005; http://datf.cbi.pku.edu.cn/) (Guo et al., 2005). Except for MIR
genes, genes encoding RNA were not counted in this study. When a gene
in a category list had an uncalled, unannotated, or vaguely annotated
homoeologous partner, we did not add the poorly annotated gene to the
GO annotation lists. However, we did update the DATF lists, thereby
increasing the number of genes above that on the original list (documen-
ted in Supplemental Table 1 online, columns B and C). Genes we called
ourselves in the process of validating a-gene pairs were not used when
GO terms were needed. Our analysis did not find new MIR genes except
as additional duplicates in known gene spaces; MIR genes were from
‘‘RNA Families Database of Alignments and CMs’’ or Rfam (Thomas et al.,
2007). Gene lists derived from the annotation keyword Bigfoot and
Smallfoot and gene lists from motif enrichment analyses, like ‘‘contains a
G-box CNS’’ (lists from Supplemental Table 1 online) were evaluated for
GO category representation using GOStat (http://gostat.wehi.edu.au)
(Beissbarth and Speed, 2004), using TAIR GO annotations, with a P value
cutoff #0.001.
Analysis of CNS Sequence for Over- and Underrepresentation of
Particular DNA cis-Acting Motifs or 7-Mer Sequences
We used the x
2
test to evaluate the significance of a PLACE (cis-acting
binding sequence) motif or random 7-mer hit to CNSs versus hits to
control noncoding gene space. We limited data to having 10 hits minimum
to facilitate this test. The results are the nominal P values displayed in
Supplemental Tables 3 and 4 online. In instances where many, usually
thousands, of motifs/7-mers were assessed in the same experiment,
nominal P values were multiplied by this repeat number, generating a
Bonferroni-corrected P value. Values are considered significant if their
corrected P value is <0.05 and color-coded pink in our spreadsheets.
Based on observations to be presented, motifs/sequences giving values
with P values just below Bonferroni significance (nominal P < 0.001) are
identified as ‘‘worth interest,’’ but are not called ‘‘significant’’; these are
color-coded blue.
The 7-mers were chosen because they were the longest random
sequences that gave us adequate numbers of hits to CNSs >9 and
because Guo and Moose (2003) used them previously.
CNS sequences were from Thomas et al. (2007); we include these in
Supplemental Table 2 online. The control nongenic sequences were
prepared by extracting the total gene space from our manual annotations
and then by partitioning sequence fragments into categories representing
aCNSs, noncoding nonCNS controls, and exons. We also prepared CNS
and control noncoding sequence fragments from a subdatabase com-
posed only of gene spaces from 126 pairs of genes labeled as Bigfoot
pairs and 1197 pairs called Smallfoot genes. CNS and control fragments
were from the gene pairs actually used to prepare the database. There
were always far more noncoding nonCNS control sequences than CNS
sequences. Therefore, we normalized the hits to our control sequences
so we could compare our aCNS hits to control hits, assuming a 1:1
correlation would be expected, or that there would be no difference in
7-mer content between aCNS and control sequence. When there were
$10 CNS hits, significance of difference from the 1:1 expectation was
estimated by x
2
and corrected for multiple tests (see above). Most ratios
were not significantly different than 1.0. Those ratios >1.0 were marked as
‘‘O’’ in Supplemental Tables 3 and 4 online, and those #1 were marked as
‘‘U’’ whether or not the difference from 1.0 was significant or ‘‘worth
interest.’’
Evaluation of Our Results and Conclusions with TIGR Assembly
Version 5 in Light of Versio n 6
After we froze our aCNS database, TAIR released a newer version of
their annotations, TIGR assembly 6. Using the lists of changes available
from the TAIR website (ftp://ftp.arabidopsis.org/Genes/TAIR6_genome_
release/), we estimated the effect these changes might have on our data.
Of the 6358 genes we analyzed in pairs, 551 genes were revised. Of these,
228 changed gene models, 195 changed protein structures only, and 353
new splice variants were added. No genes used in our analysis were
deleted or had splice variants removed. No ‘‘new’’ exons are represented
as aCNSs in our database, presumably because this would require
misannotation of both homoeologs, not just one. The aCNS database
prepared using version 5 gene annotation did not require correction in
light of version 6 annotation (Thomas et al., 2007).
Websites Cited
Websites used in our study include the following: Public Arab-
idopsis Synteny Viewer 1.0 (http://synteny.cnr.berkeley.edu/AtCNS/),
Arabidopsis Small RNA Project (http://asrp.cgrb.oregonstate.edu/),
BLASTView (ensemble) set on Arabidopsis TIGR assembly (http://atensembl.
arabidopsis.info/Multi/blastview?species¼arabidopsis_thaliana), Data-
base of Arabidopsis Transcription Factors (http://datf.cbi.pku.edu.cn/),
1454 The Plant Cell

GOstat application (http://gostat.wehi.edu.au), MultAlin: Multiple se-
quence alignment by Florence Corpet (http://prodes.toulouse.inra.fr/
multalin/multalin.html), NCBI Blast: National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/BLAST/), PLACE: Plant cis-
acting sequence database(http://www.dna.affrc.go.jp/PLACE/), R project
for statistical computing (http://www.r-project.org), and TAIR Arabidopsis
assembly 6 download (ftp://ftp.arabidopsis.org/Genes/TAIR6_genome_
release/).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Table 1. Merged Gene and Pairs List, with Data
Sorted by Gene or Pair.
Supplemental Table 2. Master aCNS List with Data Sorted by aCNS.
Supplemental Table 3. 7-Mer to Bigfoot Data Sheet, with Data
Sorted by Random 7-Mer Sequence.
Supplemental Table 4. 7-Mer to Smallfoot Data Sheet.
Supplemental Table 5. Results on Shared aCNSs.
ACKNOWLEDGMENTS
We thank Damon Lisch for discussions, our College of Natural Re-
sources for partial subsidy of the Statistics and Bioinformatics Consulting
Service, and especially the National Science Foundation (DBI-034937
to M.F.).
Received January 18, 2007; revised March 10, 2007; accepted April 19,
2007; published May 11, 2007.
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