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Allele-Specific Assay Reveals Functional Variation in the
Chalcone Synthase Promoter of Arabidopsis thaliana
That Is Compatible with Neutral Evolution
W
Juliette de Meaux,
1
Ulrike Goebel, Ana Pop, and Thomas Mitchell-Olds
Genetics and Evolution, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany
Promoters are thought to play a major role in adaptive evolution, yet little is known about the regulatory diversity within
species, where microevolutionary processes take place. To investigate the potential for evolutionary change in the
promoter of a gene, we examined nucleotide and functional variation of the Chalcone Synthase (CHS) cis-regulatory region
in Arabidopsis thaliana. CHS is the branch point enzyme of a biosynthetic pathway that leads to the production of secondary
metabolites influencing the interaction between the plant and its environment. We found that nucleotide diversity in the
intergenic region encompassing the CHS promoter (p ¼ 0.003) is compatible with neutral expectations. To quantify
functional variation specifically as a result of cis-regulation of CHS mRNA levels, we developed an assay using F1
individuals in which distinct promoter alleles are compared within a common trans-regulatory background. We examined
functional cis-regulatory variation in response to different stimuli representing a variety of CHS transcriptional environments
(dark, light, and insect feeding). We observed extensive functional variation, some of which appeared to be independent of
the trans-regulatory background. Comparison of functional and nucleotide diversity suggested a candidate point mutation
that may explain cis-regulatory differences in light response. Our results indicate that functional changes in promoters can
arise from a few mutations, pointing to promoter regions as a fundamental determinant of functional genetic variation.
INTRODUCTION
The cis-regulatory regions have been hypothesized to facilitate
adaptive innovations because subtle nucleotide changes may
generate novel phenotypes while preserving existing functions
(Wray et al., 2003). Promoters and other cis-regulatory regions
form a protein/DNA complex with trans-regulatory proteins
(transcription factors), thereby promoting integrative control of
expression. The functional architecture of these regions consists
of short and often redundant transcription factor binding sites
interspersed within a background sequence of apparently non-
functional regions. In some cases, binding site loss through point
mutation may be easily compensated by remaining binding sites
(Piano et al., 1999; Ludwig et al., 2000; Dermitzakis et al., 2003).
Because quasiperfect binding sites are often detected in the
sequence background of promoter regions, new binding sites
can emerge randomly through point mutation and permit a gain
of function (Stone and Wray, 2001; Dermitzakis et al., 2003).
Gene duplication has also been shown to promote gene expres-
sion variation, further supporting the idea that new functions can
evolve readily from cis-regulatory changes (Gu et al., 2004).
The evolutionary dynamics of cis- and trans-regulatory regions
remain poorly understood. Several studies suggest abundant
neutral expression variation, most of which results from variation
at the trans-regulatory level (Brem et al., 2002; Enard et al., 2002;
Von Dassow and Odell, 2002; Khaitovich et al., 2004). In addition,
a comparative study of cis-regulatory activity between two
closely related Drosophila melanogaster species reveals exten-
sive divergence at the cis-regulatory level, with most genes
having undergone more cis- than trans-regulatory changes
(Wittkopp et al., 2004). Thus, cis-regulatory evolution may be
the prime determinant of expression changes between species,
although trans-regulatory polymorphism is more important
within species (Wittkopp et al., 2004). Studies in human, fish,
Drosophila, and maize (Zea mays) document the role of specific
cis-regulatory variants in adaptive evolution (Crawford et al.,
1999; Wang et al., 1999; Schulte et al., 2000; Michalak et al.,
2001; Bamshad et al., 2002; Lerman et al., 2003; Rockman
et al., 2003). However, the fraction of naturally segregating cis-
regulatory polymorphisms that are adaptive remains unknown.
The fact that cis-regulatory function cannot be predicted from
the basic nucleotide sequence is a major hurdle to advancement
of this field. To identify functional cis-regulatory regions, phylo-
genetic footprinting is currently the most widely used approach.
This method assumes that sequence conservation in noncoding
regions indicates function, and it has proven useful in identifying
some functionally important elements in promoter sequences
(Koch et al., 2001; Boffelli et al., 2003; Cliften et al., 2003). This
approach, however, assumes that function has been conserved
across lineages and de facto ignores the adaptive innovations
that may accompany speciation. To address this issue, we must
1
To whom correspondence should be addressed. E-mail jdemeaux@
ice.mpg.de; fax 49-3641-57-1402.
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: Juliette de Meaux
(jdemeaux@ice.mpg.de).
W
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.104.027839.
The Plant Cell, Vol. 17, 676–690, March 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
focus on functional changes rather than conservation to un-
derstand the short-term evolutionary dynamics of cis-regulatory
regions.
Here, the within-species variation in a cis-regulatory region is
characterized at both nucleotide and functional levels to evaluate
the reservoir of cis-regulatory variability upon which natural
selection could act. We developed an assay of cis-regulatory
function that enables us to systematically evaluate within species
cis-regulatory variation and combine this approach with a survey
of sequence diversity in the intergenic region containing a known
promoter. This assay employs single nucleotide polymorphisms
(SNPs) in the coding region of target genes to discriminate allelic
cis-regulatory differences within F1 individuals, where allelic cis-
variants operate in a common trans-regulatory environment
(Cowles et al., 2002). We evaluated the relative proportions of
expressed parental alleles present in F1 cDNA pools using
pyrosequencing technology, similar to Wittkopp et al. (2004).
Because alleles are expressed in the same cells, this method
efficiently controls for environmental variation, allowing sensitive
detection of differences in expression.
We focused on the expression of the Chalcone Synthase gene
(CHS) because it codes for the branch point enzyme of the
flavonoid pathway. This pathway produces secondary metabo-
lites directly involved in the interaction between the organism
and its environment (Winkel-Shirley, 2001). For example, Arabi-
dopsis thaliana mutants with enhanced flavonoid biosynthesis
show higher insect resistance, in association with a physiological
cost of resistance; hence, genes controlling flavonoid production
may play a role in adaptive evolution (Johnson and Dowd, 2004).
CHS is known to be upregulated in leaves by multiple biotic and
abiotic environmental cues, such as insect feeding or light
(Reymond et al., 2000; Jenkins et al., 2001; Wade et al., 2001; H.
Vogel, personal communication). It is also developmentally reg-
ulated, as flavonoid production is elevated in flowers (Burbulis
et al., 1996). cis-regulatory variation of CHS expression was
examined in multiple conditions covering the diversity of CHS
expression environments. Concomitantly, we have assessed
molecular diversity in the intergenic region containing the well-
characterized CHS promoter (Hartmann et al., 1998; Logemann
and Hahlbrock, 2002).
Combining approaches at both nucleotide and functional
levels, we investigate the evolution of the cis-regulatory region
governing CHS expression in A. thaliana and address the
following questions: (1) is there within-species nucleotide varia-
tion in the CHS promoter? (2) Does the pattern of diversity
contain the footprint of selection? (3) Is there functional CHS cis-
regulatory variation segregating within A. thaliana? (4) What is the
putative genetic basis of naturally occurring cis-regulatory var-
iation?
RESULTS
Sequence Diversity
Approximately 1339 bp of the 59 flanking region at the CHS gene
(henceforth referred to as intergenic region) were sequenced in
28 A. thaliana accessions originating from different parts of the
world (Figure 1). This region is known to contain the CHS
promoter. Detailed serial deletion constructs have delimited the
upstream region involved in the activation of the gene, and
multiple cues have been identified that induce its expression
(Hartmann et al., 1998; Logemann and Hahlbrock, 2002).
Fourteen nucleotide polymorphisms and seven indels were
distributed across the intergenic region (Figures 1A and 1B; see
supplemental data online for alignment). Five of seven indels
were 1 bp long. Of 21 polymorphisms, four are singletons (one
polymorphism and three indels). Fourteen haplotypes were
observed in 28 accessions, and at least one recombination
event was detected. We calculated p (per site polymorphism
rate) separately for nucleotide polymorphisms and indels and
obtained 0.0032 and 0.001, respectively (Table 1). Tajima’s D
test examines within species polymorphism and evaluates
whether its frequency distribution is compatible with the neutral-
equilibrium model (Tajima, 1989). The patterns of polymorphism
were not significantly different from neutral expectations as indi-
cated by nonsignificant Tajima’s D (D ¼ 0.6052 and D ¼
0.356, respectively; both P > 0.1). Genome-wide estimates of
diversity are available for A. thaliana that incorporate the demo-
graphic history for neutral expectations (Schmid et al., 2005). Of
195 loci analyzed by Schmid et al. (2005), 37 (18%) showed a
Tajima’s D value that was greater than the value observed at the
CHS intergenic region (S.E. Ramos-Onsins, personal communi-
cation). Thus, the pattern of polymorphism in the intergenic
region is consistent with neutral expectations derived from
either theoretical or empirical predictions. Fay and Wu’s H was
estimated using the intergenic sequence of A. thaliana croatica
as an outgroup (Table 1). This neutrality test uses an outgroup
sequence to analyze the frequency distribution of derived
mutations. Significant negative deviation from the neutral model
(i.e., an excess of high frequency derived mutations) has been
associated with a possible directional selective event (Fay and
Wu, 2000). In the CHS intergenic region, the H value was not
significantly different from zero (H ¼ 1.62; P > 0.1; Table 1).
The comparison of polymorphism to divergence ratios across
two loci (Hudson, Kreitman, and Aguade
´
[HKA] test) provides
another way of evaluating whether the evolutionary history of
a given genomic region is remarkable (Hudson et al., 1987). We
sequenced the CHS coding region in 22 A. thaliana accessions.
Polymorphism in the coding region was lower than in the inter-
genic region (p ¼ 0.0007), with five silent polymorphisms ob-
served, three of which are singletons (Figure 1B). The comparison
between promoter and coding sequence using the HKA test
detected no significant difference (x
2
¼ 1.531; P > 0.1; Table 1).
Polymorphism in the CHS promoter region was further com-
pared with two A. thaliana noncoding regions flanking different
members of a trypsin inhibitor gene family (ATTI2 and ATTI4) that
had contrasting patterns of gene expression variation (Clauss
and Mitchell-Olds, 2004). Polymorphism found in the CHS inter-
genic region and in the noncoding region upstream from ATTI2
was comparable. By contrast, nucleotide diversity found in the
noncoding region upstream from ATTI4 was an order of mag-
nitude greater (nucleotide diversity, p, reached 0.00364 and
0.02686, respectively, for ATTI2 and ATTI4, M.J. Clauss, per-
sonal communication). Within-species expression level differ-
ences were shown to segregate within A. thaliana for ATTI4 but
not ATTI2 (Clauss and Mitchell-Olds, 2004).
cis-Regulatory Evolution in A. thaliana 677
Figure 1. Summary of Polymorphism Location and Frequencies in the 59 Flanking Region of CHS in A. thaliana.
(A) Phylogenetic footprints identified by Koch et al. (2001) are outlined by black boxes along the bottom line. The gray box indicates a region conserved
across multiple Arabidopsis species (J. de Meaux, unpublished data). Polymorphisms are indicated by black and gray triangles along the middle line
according to their position on the sequence. Upper and lower triangles indicate segregating sites and indels, respectively. Gray triangles indicate
singleton polymorphisms. Two indels are longer than one nucleotide. Black bars in the top graph show the frequency of each mutation in our sample (28
ecotypes). Black arrows highlight those mutations that are found within a phylogenetic footprint.
(B) Summary of DNA variation in the 59 intergenic region upstream from CHS and in the CHS transcribed region (encompassing exon 1, intron 1, and
part of exon 2) in A. thaliana. Numbers indicate the position in the sequence relative to the first nucleotide of the CHS start codon. Positions used for the
pyrosequencing assay are indicated with bold letters. Functional cis-regulatory groups identified by our assay are indicated in the last column. *,
Undetermined positions; $, SN P780; §, SNP840; #, mutation situated in an intron; (a), sequence from Ramos-Onsins et al. (2004).
678 The Plant Cell
We also compared the polymorphism-to-divergence ratio of
the CHS promoter region with that of the ADH promoter region
studied by Miyashita (2001) using sequences from Arabidopsis
gemmifera as an outgroup. The ADH promoter region harbors
experimentally verified regulatory elements (Hoeren et al., 1998).
No significant difference was detected (Table 2). Respectively,
52 and 84% of CHS and ADH A. thaliana upstream flanking
regions aligned with those from A. gemmifera, allowing us to
compare 696 bp of the 1383-bp CHS intergenic region and 2049
bp of the 2366-bp ADH upstream region (Miyashita, 2001). Within
A. thaliana, indel events were more common in the ADH inter-
genic region than in the CHS region (38 indels in 2366 bp versus 7
indels in 1339 bp). By contrast, CHS had more indel events than
the ADH flanking region since divergence from A. gemmifera,as
indicated by the poor alignment.
Phylogenetic footprinting throughout the Brassicacae has
identified conserved fragments in the intergenic region that
were shown to promote light-induced expression (Koch et al.,
2001). Two substitutions and one indel were detected in these
conserved fragments. The two substitutions affected an H-box–
like motif and a G-box motif (positions 248 and 515, re-
spectively; see Figure 1). The G-box motif also contained a 3-bp
indel (position 520).
To evaluate whether functional constraints may have pre-
vented the occurrence of polymorphisms that affect binding
sites, we have screened the intergenic region for putative binding
sites using the PLACE database of plant enhancer elements
(Higo et al., 1999). For each haplotype, approximately half of the
1339 positions were covered by a predicted binding site. Of 21
polymorphisms, 10 (47%) affected a predicted binding site,
indicating that polymorphic positions are randomly distributed
across the region regardless of predicted binding sites. The 59
portion of the intergenic region (
;500 bp) was shown not to
affect promoter light response (Hartmann et al., 1998). Diversity
in this portion, however, did not appear to differ from the 39
portion (Figure 1).
Expression Diversity
In F1 individuals, both parental cis-regulatory regions experience
the same trans-regulatory environment; thus, the relative amount
of parental mRNAs reflects their relative activity. F1 progenies
were analyzed by comparing Col-0 with each of seven other
accessions (Cvi-0, Da1-12, Ei-2, Kas-1, Tsu-1, Lip-0, and Mrk-0).
Col-0 harbored a mutation allowing us to distinguish its CHS
mRNA allele. The seven other accessions were chosen to rep-
resent nucleotide variation in the promoter region. F1 seeds
were grown in controlled conditions and examined in various
CHS transcription environments. In total, eight CHS transcription
environments were assessed (48 h dark, 8 h light, 24 h light,
organ-specific expression in flowers, 9 h insect feeding and
corresponding control, and 24 h insect feeding and correspond-
ing control). To evaluate the relative amount of parental mRNAs,
a PCR was performed on the cDNA pool using primers that
anneal to conserved regions around the SNPs differentiating
parental CHS copies. Each parental allele was then amplified
proportionately to its relative concentration in the template
solution. The relative amount of parental alleles in the PCR
solution was quantified by pyrosequencing. In a DNA sample
extracted from a heterozygous diploid individual, parental ge-
nomes are present in equal amount; thus, parental copies occur
at the same concentration. DNA extracted from a leaf of one
heterozygous F1 individual from each cross was therefore used
to calibrate the null hypothesis of no allelic difference in promoter
activity, following Cowles et al. (2002). A total of six to eight
independent F1 individuals were analyzed for each treatment
and each parental combination. An analysis of variance (ANOVA)
was performed to evaluate the effect of both experimental and
Table 1. Summary Statistics of DNA Variation in the CHS Region
59 Upstream Intergenic Region
(1339 bp, 28 Sequences)
Exon 1, Intron 1, and Exon 2
(1216 bp, 20 Sequences)
Measures of Diversity Segregating Sites Indels
a
Segregating Sites
Informative sites 13 5 (four affecting a single position) 2 (all silent)
Singletons 1 2 (one affecting one position) 4 (all silent but one)
Haplotypes 9 7 6
Haplotype diversity 0.897 0.755 0.636
Average number of nucleotide differences 4.241 1.368 0.857
p 0.0032 0.001 0.0007
u 0.0027 0.0012 0.0013
Total number of observed polymorphisms
(expected number)
14 (10.06)
b
5 (8.94)
b
D
tajima
0.6052 (P > 0.1) 0.356 (P > 0.1) 1.24 (P > 0.1)
Average number of observed differences
from A. croatica (expected number)
41.741 (45.68)
b
59.94 (56.62)
b
Number of base pairs compared 910 345.469
H (outgroup A. croatica) 1.6267 (P > 0.1) 0.726 (P > 0.1)
a
For the calculations, indels were coded as substitutions.
b
Nonsignificant HKA test (x
2
value ¼ 1.531, P ¼ 0.2160).
cis-Regulatory Evolution in A. thaliana 679
biological factors (below). Separate analyses were performed for
light treatments and for each insect feeding experiment and
corresponding control. No parent-of-origin effect was detected,
ruling out the possibility that our results reflect imprinting or
maternal effects (data not shown).
Technical Variation
Pyrosequencing measurements were repeated with two inde-
pendent primer pairs, yielding highly correlated results (R
2
¼
0.87; P < 0.0001; Figure 2). Primer pairs nonetheless significantly
affected the results, as shown by the significant effect of primer
pair upon pyrosequencing measurement in the ANOVA analysis
(Table 3). Similarly, there was a significant effect of the pyrose-
quencing technical covariate, as measured by monomorphic
peak height (see Methods). A significant effect of PCR plates was
also detected. We also investigated column and row effects by
measuring the relative allelic signal of a single sample on two
plates. We found a significant column effect that varied among
plates (data not shown). A complete sample randomization
before PCR amplification and pyrosequencing was therefore
compulsory to control for nonbiologically relevant variation.
In the allelic mixture, one or the other allele may be preferen-
tially amplified. To account for this bias, raw data were calibrated
using a K corrected standard curve (see Methods), following
Wittkopp et al. (2004). Because the treatments we used pre-
sumably caused the expression of distinct suites of genes, and
because a high PCR cycle number was required to obtain
comparable amounts of PCR products across samples (because
of low CHS expression in the dark), we wanted to make sure that
there is no treatment-dependent PCR bias in our measurement,
for which the K correction might not account. We therefore
investigated whether the cDNA gene pool could affect our
measurements. We prepared serial volumetric RNA mixtures
from homozygous genotypes placed under the different light
treatments (dark, 8 h light, and 24 h light). Three independent
retrotranscription reactions (replicates) were performed, and
pyrosequencing measurements were corrected as described in
Methods. Figure 3 shows the regressions obtained for the
different light treatments. An analysis of covariance detected
no significant interaction between light treatment and the levels
of serial mixtures (F
4,153
¼ 1.82, P > 0.1). This demonstrates that
our method accurately measures the relative amounts of paren-
tal alleles regardless of the environmental conditions in which
cDNA pools were generated. In addition, there was no significant
effect of experimental replicates (F
2,4.02
¼ 2.02, P > 0.1).
Light Treatments
CHS cis-regulatory haplotypes were found to respond differently
to the light environments as indicated by a significant effect of
light treatments upon relative expression of CHS parental alleles
within F1 heterozygotes (Table 3). Crosses were also shown to
differ, and a significant interaction between light response and
crosses was found. Homogeneous results were obtained in both
experimental trials, as indicated by a nonsignificant trial effect.
Relative to the Col-0 allele, six of seven parental alleles were less
expressed in the dark and more expressed in both 8 h and 24 h
light (Figure 4).
Results obtained for the cross between Kas-1 and Col-0 were
responsible for a large part of the genetic differences between
crosses. In this cross, Col-0 and Kas-1 CHS mRNA levels in dark-
maintained leaves did not differ significantly from the heterozy-
gous DNA control. Although fewer repetitions were available for
the cross between Ler-0 and Col-0, a similar trend toward equal
allelic expression was observed. For the other light treatments,
all parental combinations gave similar results.
We further tested whether the differential promoter activity
between Col-0 and the other accessions was background de-
pendent. We crossed Ei-2, Lip-0, and Cvi-0 with both Lz-0 and
Ag-0, two accessions with promoter and coding sequences
similar to Col-0, and evaluated relative CHS mRNA allelic
abundance in three light treatments (six crosses in total; Table
4). In comparison with Ei-2, Lip-0, or Cvi-0, mRNA alleles from
Lz-0 and Ag-0 were more abundant in the dark and less
abundant after 8 h or 24 h of light. Lz-0 and Ag-0 cis-regulatory
alleles thus behaved similarly to the Col-0 cis-regulatory allele
(significant treatment effect, F
2,90
¼ 17.55, P < 0.001; Table 4). No
Table 2. Polymorphism to Divergence Ratios in CHS and
ADH Intergenic Regions
Characteristics of Variation
Promoter
CHS
Promoter
ADH
a
Number of sequences 27 14
Number of polymorphic sites in
A. thaliana
14 42
Expected number of polymorphic sites 17.88 38.12
Number of base pairs 1335 2366
Average number of differences from
A. gemmifera
52.00 203.00
Expected number of differences 48.20 207.00
Number of base pairs compared 696 2049
HKA x
2
0.669
P value 0.413
a
Data from Miyashita (2001).
Figure 2. Correlated Pyrosequencing Measurements with Independe nt
Primer Pairs.
Regression between pyrosequencing measurements on SNP780 ob-
tained for
;70 cDNA samples with two independent primer pairs (Set
719 and Set 700) (R ¼ 0.93, P < 0.001).
680 The Plant Cell
significant cross effect was detected, indicating that these
results were independent of genetic background. We also took
advantage of an additional SNP (SNP840) distinguishing Lip-0
from Ei-2, Ag-0, and Lz-0 CHS coding sequences and looked at
relative expression levels in response to light changes. The cross
between Lip-0 and Ei-2 was significantly different, whereas
crosses between Lip-0 and either Lz-0 or Ag-0 were comparable
to results obtained for the cross between Lip-0 and Col-0. The
relative expression levels of Lip-0 and Ei-2 were independent of
light treatment (F
2,15
¼ 0.026, P ¼ 0.97), in contrast with the other
two crosses (Lip-0 with either Lz-0 or Ag-0; F
3,31
¼ 10.86, P <
0.001). The Lip-0 allele was significantly more expressed than the
Ei-2 allele in all three light conditions (F
3,16
¼ 48.62, P < 0.001;
minimum P < 0.001 for comparison between DNA measurement
and light treatments).
Thus, the light treatments allowed us to distinguish at least
two functionally distinct allelic promoter groups: group 1 (compris-
ing Col-0, Ag-0, and Lz-0) and group 2 (comprising Lip-0, Ei-0,
Mrk-0, Cvi-0, Tsu-1, and Da1-12). The Kas-1 and Ler-0 prom-
oter alleles appear to form a third functional group (group 3) that
does not respond to dark differently from the Col-0 allele, al-
though differences were detected in the insect feeding trial
(Figure 1B; see below). The cross between Ei-2 and Lip-0 indi-
cates that additional cis-regulatory diversity exists within these
broadly defined groups.
The mean observed percentage of Col-0 mRNA allele reached
a maximum of 60% in samples collected after 48 h in the dark
and a minimum of 40% in samples collected after either 8 or 24 h
of intense illumination (Figure 4). Thus, the mRNA from group 1
(Col-0, Ag-0, and Lz-0) accessions is up to 50% (1.5-to-1 ratio)
Table 3. ANOVA of Allele-Specific CHS Transcript Levels
Source SS-Type III
a
df Mean Square F-Ratio P
Relative allele expression in response to light treatments (model R
2
¼ 0.78)
Trial
b
0.17249 1 0.17249 4.43 0.0819
Primers 0.01745 1 0.01745 15.71 0.0001
Treatment
c
0.42930 4 0.10733 33.71 0.0019
Cross 0.07455 6 0.01242 16.17 0.0011
Technical covariate 0.09176 1 0.09176 86.62 <0.0001
Treatment 3 cross 0.13544 24 0.00564 5.08 <0.0001
Trial 3 treatment
d
0.01341 4 0.00335 3.02 0.0177
Trial 3 cross 0.00454 6 0.00076 0.68 0.6659
Plate within trial 0.23077 6 0.03846 34.63 <0.0001
Error 0.44093 483 0.00093
Relative allele expression after 9 h of insect feeding (model R
2
¼ 0.57)
Trial
b
0.01562 1 0.01562 0.44 0.5214
Primer 0.01191 1 0.11907 6.09 0.0143
Treatment
c
0.07948 2 0.03974 5.24 0.1547
Cross 0.15651 6 0.02609 2.78 0.1149
Technical covariate 0.02207 1 0.02207 11.28 0.0009
Treatment 3 cross 0.14858 12 0.01238 6.33 <0.0001
Trial 3 treatment 0.01603 2 0.00801 4.1 0.0177
Trial 3 cross 0.06096 6 0.01016 5.19 <0.0001
Plate within trial 0.14189 6 0.23648 12.09 <0.0001
Error 0.51643 264 0.00196
Relative allele expression after 24 h of insect feeding (model R
2
¼ 0.62)
Trial
b
0.01836 1 0.01836 0.61 0.4697
Primer 0.01983 1 0.01983 9.17 0.0027
Treatment
c
0.09018 2 0.04509 79.31 <0.0001
Cross
e
0.04108 5 0.00822 95.13 <0.0001
Technical covariate 0.12713 1 0.12713 58.77 <0.0001
Treatment 3 cross 0.11060 10 0.01106 5.11 <0.0001
Trial 3 treatment 0.00059 2 0.00030 0.14 0.8727
Trial 3 cross 0.00045 5 0.00009 0.04 0.9990
Plate within trial 0.23549 6 0.03925 18.14 <0.0001
Error 0.51049 236 0.00216
a
SS, sum of squares.
b
Trial effect corresponds to independent repetition of the experimental gene induction treatments. This effect was treated as a random source of
variation.
c
The null expectation of no cis-regulatory allelic differences modeled by amplifications from heterozygous DNA samples is included in the treatments.
d
Trial 3 treatment effect and cross effect are not significant if data from the Kas-1 3 Col-0 cross are deleted from the analysis.
e
Because the Kas-1 3 Col-0 cross differed markedly from the other crosses, it was deleted from the analysis of the 24 h insect feeding trial.
cis-Regulatory Evolution in A. thaliana 681
more abundant in the dark than the mRNA from group 2
accessions. By contrast, in light, mRNA from group 2 is
;50%
more abundant than mRNA from group 1. Given that CHS is
induced by light and repressed in the dark (Jenkins et al., 2001),
this indicates that the expression of group 2 CHS alleles is tightly
regulated by light environment, whereas the expression of group
1 alleles is more loosely regulated.
In samples collected from flowers, group 1 cis-regulatory
alleles showed weaker activity than the other alleles to which
they were compared. Results for organ-specific expression in
flowers were thus similar to those for leaf tissue under 8 h or 24 h
of light induction. Because flower development is also regulated
by light, organ-specific expression in flowers is not independent
of the light environment, and the specific effect of the develop-
mentally regulated CHS transcription environment upon relative
cis-regulatory activity is difficult to assess. However, our results
indicate that the flower-specific transcriptional environment has
the same effect as the (very stressful) 8 h or 24 h strong light
environments.
Insect Treatments
Leaves were collected after either 9 h or 24 h of insect feeding,
together with control leaves from individuals upon which no larva
had been placed. We analyzed results obtained after 9 h and 24 h
separately, in comparison with their corresponding controls. For
9 h of insect feeding, no significant treatment effect was found,
but a significant interaction between crosses and treatments was
observed (Table 3). The SLICE analysis (see Methods) detected
significant heterogeneity among crosses for each of two treat-
ments (9 h insect feeding; F
6,264
¼ 11.09, P < 0.0001; 9 h insect
control; F
6,264
¼ 9.42, P < 0.0001). Three of seven crosses
showed significant heterogeneity across treatments (DNA, 9 h
insect feeding and 9 h control). Both control and insect-damaged
leaves showed an excess of Col-0 mRNA allele relative to the
mRNA of the Cvi-0, Da1-12, and Ei-2 genotypes (least squares
Figure 3. Standard Curves for Calibration of the Raw Data.
Regression obtained between the proportion of the Col-0 allele in serial
volumetric mixtures and the pyrosequencing measurement. Serial vol-
umetric mixtures were performed using cDNA pools resulting from
different light treatments as well as genomic DNA. The regression
coefficients (R) were 0.99, 0.90, 0.94, and 0.97 for DNA, dark, light 8 h,
and light 24 h, respectively. ANOVA indicated no interaction between
allelic proportion and treatment effect (P > 0.1).
Figure 4. Relative CHS cis-Regulatory Activity in F1 Individu als from Seven Parental Combinations in Response to Light Environment.
F1 individuals obtained from crosses between Col-0 and seven other ecotypes were analyzed. For each cross, relative levels of the Col-0 CHS mRNA
allele are indicated for each of the light treatments (dark, light 8 h, and light 24 h). The bold black line indicates the expected value for equal promoter
activity of both parental haplotypes in the cross. In the dark, the Col-0 parental allele is generally more expressed than its counterpart. The patternis
reversed in light 8 h and light 24 h. Only individuals from the Kas-1 3 Col-0 cross depart from this pattern. Error bars show standard error.
682 The Plant Cell
mean comparisons; maximum P ¼ 0.0097). No effect of insect
feeding on the differential response of parental alleles could be
detected (P ¼ 0.07; Scheffe
´
’s multiple mean comparison test
between 9 h insect and control). Although no significant trial
effect was detected, the interaction between trial and treatment
and trial and cross was significant. This is probably related to our
experimental conditions. For insect feeding trials, plants were
placed in the shade under reduced light intensity, but temper-
ature and light levels were not strictly controlled.
For 24 h insect feeding, the offspring of Kas-1 3 Col-0 differed
markedly (Figure 5). We thus conducted the ANOVA without
this parental genotype combination. A significant interaction
between treatments and crosses was observed, although the
treatment effect alone was not significant (Table 3). Similar to
the 9 h insect treatment, no consistent effect of insect feeding
upon differential response of parental alleles was detected.
Nonetheless, Kas-1 and Col-0 showed significantly different
responses in control relative to insect-damaged leaves. Indeed,
for insect-damaged leaves, both parental alleles were expressed
at the same level, but for control leaves, the Kas-1 parental allele
was more expressed than the Col-0 allele (
;0.7 and 0.3, hence,
more than twofold expression difference). A comparable trend
was observed for the Ler-0 genotype, although missing data
prevented its incorporation in the ANOVA analysis. However,
when the Col-0 3 Kas-1 cross was excluded from the analysis,
the SLICE analysis detected a significant heterogeneity among
crosses within treatment (24 h insect feeding; F
5,236
¼ 7. 52, P <
0. 0001; 24 h insect control; F
5,236
¼ 4. 08, P ¼ 0. 0014).
Molecular Basis of Functional Change
At the nucleotide level, the comparison of the Col-0 and Tsu-1
sequences reveals that group 1 promoter alleles differ from other
alleles by at least one mutation. This mutation is located in the
intergenic region on position 674 (Figure 1B). This mutation
consists of an adenine residue replacing the ancestral guanine
residue. It affects a cis-element involved in the light repression of
Asparagine Synthase expression in pea (Pisum sativum) (Ngai
et al., 1997). This element occurs within a fragment covering
position 688 to 641 that is conserved in the Arabidopsis
genus (J. de Meaux, unpublished data). The putative functional
consequences of this mutation are discussed below.
At alignment position 739, the sequence TTGGCA has
changed to TTGACA in 9 out of 28 accessions. TTGACA, which
contains the W-box core TGAC to which WRKY transcription
factors bind, occurs at elevated frequencies in defense-related
genes (Maleck et al., 2000). This potential gain-of-function poly-
morphism is situated in a region where the AT content alternates
in phase with the helical DNA repeat. This feature is a possible
nucleosome signal (Thastrom et al., 1999). As a result of DNA
wrapping in a nucleosome, the TTGACA site is likely to be
spatially close to a region
;80 bp away, which is highly
conserved across A. thaliana species, and could participate in
the interaction among transcription factors controlling CHS
expression. However, the two accessions (Cvi-0 and Kas-1)
carrying this newly evolved motif did not show correlated cis-
regulatory characteristics in our functional assay.
Finally, the Ler-0 and Kas-1 functional group (group 3) could
not be correlated with the specific occurrence of a single
mutation in the intergenic region. No difference from the Col-0
haplotype could be identified that was simultaneously shared by
Ler-0 and Kas-1 and absent from the other accessions used in
the functional study. This indicates that either several mutations
compensate for each other to produce a similar functional
phenotype or that the molecular basis of the group 3 functional
alleles lies outside the region we have sequenced.
DISCUSSION
Functional genetically based variation within species is a pre-
requisite for adaptive evolution. However, within species surveys
of cis-regulatory diversity are scarce, and little is known about
the adaptive importance and fate of this diversity. We combined
an analysis of nucleotide variation in the CHS promoter region
with a robust assay of CHS cis-regulatory variation to evaluate
the reservoir of diversity in this region which may contribute to
evolution in A. thaliana. Although selection has not left any
detectable signature in the CHS promoter region, we demon-
strate that functional cis-regulatory polymorphisms segregate
within A. thaliana. In addition, our study indicates that functional
Table 4. ANOVA of Allele-Specific CHS Transcript Levels in Crosses Not Involving the Col-0 Genotype
Source (Model R
2
¼ 0.79) SS
a
df MS
b
F-Ratio P
Treatment
c
0.085 2 0.042 17.504 <0.001
Cross
d
0.011 5 0.002 0.887 0.493
Technical covariate 0.011 1 0.011 4.594 0.035
Cross 3 treatment 0.032 10 0.003 1.323 0.23
Plate 0.108 1 0.108 44.319 <0.001
Plate 3 genotype 0.031 5 0.006 2.546 0.033
Plate 3 treatment 0.036 2 0.018 7.320 0.001
Error 0.218 90 0.002
a
SS, sum of squares type III.
b
MS, mean square.
c
Three treatments were compared (dark, 8 h light, and 24 h light).
d
Six crosses were analyzed.
cis-Regulatory Evolution in A. thaliana 683
cis-regulatory variation can arise from a small number of muta-
tions.
Neutral Pattern of Div ersity in the Promoter Region
We surveyed nucleotide variation in the intergenic region up-
stream from the CHS coding region. The 39 part of this region has
been shown experimentally to drive gene expression in response
to light and pathogen elicitor molecules (Hartmann et al., 1998;
Logemann and Hahlbrock, 2002). The pattern of polymorphism
segregating in this region is compatible with theoretical and
empirically based neutral expectations, as indicated by the
various summary statistics available to test for deviations from
the neutral equilibrium model (Table 1). Neutral evolution is also
indicated by the fact that the ratio of divergence to polymorphism
in the CHS promoter region is statistically undistinguishable from
that of the CHS transcribed region, as well as the ADH 59
upstream region (Miyashita, 2001; Ramos-Onsins et al., 2004).
Nonetheless, it remains possible that selection has acted locally
in some populations. Our sampling does not allow us to test this
hypothesis. The CHS promoter region differs from the ADH
promoter region in the extent to which A. gemmifera and A.
thaliana sequences can be aligned. The alignable region be-
tween A. thaliana and A. gemmifera comprises
;50% of the
CHS upstream region, whereas it covers >80% of the ADH
region. Similar results are found when comparing the A. thaliana
CHS promoter region to Arabidopsis croatica. Intraspecific data
from additional loci are needed to evaluate how much the CHS
intergenic region typifies the level of functional constraints in cis-
regulatory regions and, thus, the level of polymorphism and
divergence that can be expected in functional noncoding re-
gions. As a first estimation, our comparison with the ATTI gene
family indicates that the levels of diversity in the CHS intergenic
region are comparable with those found in regions showing less
expression variation (Clauss and Mitchell-Olds, 2004).
Functional Variation
To assess functional cis-regulatory variation, we paired distinct
parental promoter alleles within F1 heterozygotes and assessed
the relative expression levels of parental mRNA alleles that are
distinguishable by at least one nucleotide polymorphism. This
approach has been used previously to study both within- and
among-species cis-regulatory polymorphisms (Cowles et al.,
2002; Yan et al., 2002; Wittkopp et al., 2004). Here, we adapted
the method to assess cis-regulatory variation in a broad range
of inducing or repressing transcriptional environments. By con-
trolling for multiple sources of experimental bias (PCR, techni-
cal covariates, and experimental error) and ruling out possible
bias unrelated to our biological purpose (e.g., no cDNA pool–
dependent bias and no maternal effect), we have developed
a robust assay for identifying significant functional differences
among alleles. This assay efficiently controls for changes in
expression as a result of trans-regulatory and environmental
differences. Indeed, the standard deviation for pyrosequencing
measurements performed on cDNA samples is comparable in
magnitude to that for measurements on DNA samples from
heterozygous individuals. Most of the variation in our assay is
actually due to PCR rather than to environmental differences
affecting expression in different F1 individuals. This contrasts
with methods evaluating variation in total gene expression, such
as cDNA microarrays or real-time PCR, where plant-to-plant
variation in total expression levels complicates intraspecific
evaluation of expression variation (Townsend, 2004).
Figure 5. Relative CHS cis-Regulatory Activity in F1 Individu als from Seven Parental Combinations in Response to 24 h of Insect Feeding.
F1 individuals obtained from crosses between Col-0 and seven other ecotypes were analyzed. For each cross, relative levels of the Col-0 CHS mRNA
allele in a 24 h insect-damaged plant and corresponding control are indicated. The bold black line indicates the expected value for equal promoter
activity of both parental haplotypes, as measured on DNA from heterozygous individuals. Error bars show standard error.
684 The Plant Cell
CHS expression is repressed in the dark and induced in light
(Jenkins et al., 2001). Its expression is also regulated by the
circadian clock, is induced by herbivory or elicitors (Reymond
et al., 2000; Logemann and Hahlbrock, 2002), and is upregulated
in flowers (Burbulis et al., 1996). We evaluated CHS cis-regula-
tory variation in all these transcription environments in A. thaliana
and thus identified at least three functional groups of light-
sensitive promoter alleles. Group 1 cis-regulatory alleles are
loosely light regulated, whereas group 2 cis-regulatory alleles are
tightly light regulated, being relatively more repressed in the dark
and more induced in light. The third group of cis-regulatory
alleles is found in the Kas-1 and Ler-0 genotypes, where CHS
mRNA alleles are relatively more induced in the light but less
repressed in the dark, as indicated by the higher level of Kas-1
mRNA allele measured in the early morning on control plants of
the insect trial. These cis-regulatory differences are apparently
independent of the genetic background in which they were
observed because similar light responses were detected in
multiple crosses. Our results demonstrate that extensive cis-
regulatory variation exists within species and that the analysis
of expression in a single environment underestimates cis-
regulatory polymorphism.
The functional cis-regulatory differences we observe are small
in magnitude and reach a maximum ratio of 1.5 to 1, as in the
case of Col-0 versus Cvi-0 in the dark. Nevertheless, they are
likely to substantially influence flavonoid production. CHS is a key
enzyme in the flavonoid pathway, and its activity is thought to be
regulated primarily at the transcriptional level (Mol et al., 1996;
Noh and Spalding, 1998). In addition, it has been shown to form
enzymatic complexes by interacting directly with other enzymes
of the pathway (Burbulis and Winkel-Shirley, 1999). Thus, a 50%
difference is likely to modify significantly the metabolite flux
through the flavonoid pathway (Borevitz et al., 2000; Liu et al.,
2002).
Rather than transcriptional differences, our results could also
be explained by allele-specific differences in mRNA stability. A
genome-wide study of mRNA stability in A. thaliana indicates that
unstable mRNAs are rare (1%), whereas allelic variation of ex-
pression levels seems more common, as indicated by studies
in humans and maize (Bray et al., 2003; Guo et al., 2004). The
CHS mRNA is not unstable (Gutierrez et al., 2002), and the SNP
polymorphism does not seem to affect the minimum free energy
of mRNA folding, which means that global pairing patterns are
similar for all three mRNA variants (Hofacker, 2003). Together,
this suggests that stability differences among these mRNA
variants are unlikely.
The Molecular Basis of Functional Variation
Because the intergenic region was shown to influence CHS
expression (Hartmann et al., 1998; Logemann and Hahlbrock,
2002), it is relevant to ask which promoter polymorphisms
may explain the observed functional variation. Functional cis-
regulatory changes can result from allele-specific differences in
chromatin decondensation. However, the functional polymor-
phism uncovered in this study does not correlate with molec-
ular changes in regions known to be associated with nucleosome
positioning (Lomvardas and Thanos, 2002). Alternatively, allele-
specific variation in methylation susceptibility has been shown
to affect expression of the SUPERMAN gene in A. thaliana
(Jacobsen andMeyerowitz,1997). The genetic basis of that differ-
ence remains nonetheless unclear, and this phenomenon does
not seem to be widespread, as it has not been observed at other
loci. Our results instead suggest that variation in transcription-
factor binding specificity is responsible for the functional cis-
regulatory variation of CHS expression observed in A. thaliana.
We did not detect functional constraints indicative of purifying
selection against new polymorphisms within putative transcrip-
tion factor binding sites. Most of these putative binding sites are
neither overrepresented nor relevant for CHS expression and,
hence, may be nonfunctional (Sandelin and Wasserman, 2004).
Nonetheless, for 3 out of 10 polymorphic putative binding sites,
a prediction regarding potential functional relevance can be
made using information from our functional assay, combined
with a careful scrutiny of the binding site sequence context (see
below).
We identified one point mutation at alignment position 674
that strictly correlates with the functional group of promoter
alleles that are loosely light regulated. An experimental confir-
mation of the specific role of this mutation is not currently
feasible. The changes in activity are likely to be too small to be
reliably detected with a transient expression system using a re-
porter gene. Further allele-based methods are needed for testing
the genetic basis of small changes in promoter activity. Despite
this limitation, several lines of evidence strongly suggest that
this mutation has functional consequences. This mutation occurs
in a nucleotide island that is conserved among Arabidopsis
species (J. de Meaux, unpublished data), within a nucleotide box
that was first observed in the promoter of the pea Asparagine
Synthetase, for which transcription is negatively regulated by
light (Ngai et al., 1997). The tightly light-regulated promoter
alleles, which harbor the wild-type box, are more upregulated in
light than the loosely light-regulated alleles, which harbor the
mutated box. Because the wild-type box characterized in pea
participates in light-mediated repression of gene expression, this
suggests that the mutated box causes stronger repression than
the wild-type box. It is interesting to note that the G-to-A change,
which presumably alters transcription-factor binding, is likely to
change the structural configuration of DNA in the box (Brukner
et al., 1995) and occurs in a position of less stringent nucleotide
conservation (Ngai et al., 1997). This mutation suggests that
functional cis-regulatory variation can result from relatively few
changes; thus, the potential for adaptive evolution can readily be
generated in promoter regions (Wray et al., 2003).
Three mutations occur in phylogenetic footprints; however, it
was not possible to relate them to any functional allelic group.
Two of these mutations are singletons carried by the Lz-0 or
Ler-0 accessions and occur within a G-box. Although highly con-
served in the Brassicacae (Koch et al., 2001), this box was shown
to be nonfunctional with respect to light regulation in A. thaliana
(Hartmann et al., 1998). Consistent with this previous study,
we could not detect a cis-regulatory difference correlating with
these changes. A third mutation is carried by several Indo-Asian
accessions (Kas-1, Sorbo, Shakhdara, and Hodja) and affects
a highly conserved H-like box (Koch et al., 2001). Because Kas-1
and Ler-0 seemed to be similarly affected in their response to the
cis-Regulatory Evolution in A. thaliana 685
biological clock and do not share this mutation, it is unclear
whether this second mutation significantly affects expression in
A. thaliana. Our result has consequenc es for the interpretation of
phylogenetic footprints. Indeed, if conservation indicates func-
tional constraints at a macroevolutionary scale, we cannot
exclude the possibility that such constraints have been recently
relaxed at a microevolutionary scale, resulting in neutral variation
within phylogenetic footprints.
We found a newly evolved WRKY transcription factor binding
site (W-box) at relatively high frequency. These boxes are usually
found to be overrepresented in defense-regulated promoters
(Maleck et al., 2000). However, a careful analysis of the sequence
context of this box indicates that, if functional, this new W-box is
expected to interact with the nearby functional region because
of possible nucleosome position. It is interesting to note that
sequences carrying the newly evolved W-box never have any
of the three polymorphic positions, spaced by 9 and 7 bp,
respectively, which immediately precede the highly conserved
region starting at alignment position 688 (see above). All three
of these mutations affect a TGA trinucleotide, and TGA is the
most flexible trinucleotide according to the bendability scale of
Brukner et al. (1995). This W-box was not associated with
functional cis-regulatory variation in our assay. Nonetheless, it
illustrates how our understanding of promoter function may
benefit from investigation of the sequence context surrounding
putative binding sites. In the future, the characterization of
naturally segregating cis-regulatory diversity should contribute
to the elucidation of promoter function because it generates
molecular hypotheses for functional variation in promoter re-
gions.
METHODS
Sequencing
All Arabidopsis thaliana seeds were obtained from the Nottingham
Arabidopsis Stock Centre. Young leaves from each accession were
ground in liquid nitrogen, and DNA was subsequently purified following
standard cetyl-trimethyl-ammonium bromide protocol. CHS is single
copy in the A. thaliana genome (Koch et al., 2000). To amplify the CHS
intergenic region, we designed a forward primer in the closest adjacent
putative open reading frame 59 upstream from CHS (59-TCT-
CCGGTCTGCATTGTGC-39) and a reverse primer in the first CHS exon
(59-GTAGTCAGGATACTCCGC-39). The adjacent open reading frame
is of unknown function and is annotated Mac12.12 in the A. thaliana
genome (http://www.arabidopsis.org). PCR was conducted in a solu-
tion (2 mM MgCl
2
, 1 unit of Taq polymerase [Qiagen, Valencia, CA], and
0.2 mM deoxynucleotide triphosphate) buffered using the manufac-
turer’s recommendations and containing 2 pmol of each primer for a total
volume of 25 mL. PCR products were obtained with 35 cycles as follows:
948C, 30 s/528C, 30 s/688C, 3 min. Two independent PCRs were
performed for each accession, and their mixed PCR products were
directly sequenced on both strands with an ABI3700 capillary sequencer
(Applied Biosystems, Foster City, CA) using primers placed approxi-
mately every 500 bp. In case of ambiguous sequence results, the whole
procedure was repeated. Sequences were assembled with Seqman 5.0
(DNASTAR, Madison, WI), and each variable site was checked by
examining sequence chromatograms. The orthologous region was also
sequenced from Arabidopsis croatica and Arabidopsis gemmifera,two
species closely related to A. thaliana, using primers 59-AGGACAATCGTT-
GATCCAG-39 and 59-GTAGTCAGGATACTCCGC-39. The PCR was con-
ducted as described above, with the exception of the use of a 548C
annealing temperature for PCR cycling. Two independent PCRs were
performed and products were cloned using the TOPO TA cloning kit
(Invitrogen Life Technologies, Paisley, UK). Six clones per PCR were
sequenced as above. The CHS exons 1 and 2 were sequenced in 20 A.
thaliana accessions as well as in A. croatica following Ramos-Onsins et al.
(2004).
Expression Analysis
Crosses
A first set of eight crosses was performed between accession Col-0
and each of the following accessions: Lip-0, Mrk-0, Kas-1, Cvi-0, Ler-0,
Tsu-0, Da(1)-12, and Ei-2. These nine accessions were chosen to repre-
sent the sequence diversity found in the intergenic region. Reciprocal
crosses were performed to control for maternal effects. Seeds from each
reciprocal cross were sown in a sterilized potting soil/vermiculite mix
and were vernalized in the dark at 48C for 7 d followed by 7 d in Voetsch
reach-in chambers (12 h day, 208 C day temperature, 168C night temper-
ature, 70% humidity) for germination. Germinated seedlings were then
transplanted into single pots and assigned to random positions in the
reach-in chambers in the same conditions as described above. For each
reciprocal cross, 20 seedlings per cross (40 seedlings in total for each
parental combination) were grown for 5 weeks. A second set of seven
crosses was performed between Ag-0, Lz-0, Cvi-0, Ei-2, and Lip-0
(Ag-0 3 Cvi-0, Ag-0 3 Ei-2, Ag-0 3 Lip-0, Lz-0 3 Cvi-0, Lz-0 3 Ei-2,
Lz-0 3 Lip-0, and Lip-0 3 Ei-2), which were analyzed only under different
light conditions (see below).
Light Treatments
CHS gene expression is repressed in the dark and strongly induced in
light (Jenkins et al., 2001). Plants were placed in the dark for 48 h at 208C
followed by 24 h of strong white light at 158C (700 mmol/cm
2
), and
samples were collected at the end of the dark phase, after 8 h light, and at
the end of the light phase. We thus analyzed three independent light
environments: dark, 8 h strong light, and 24 h strong light. Soil temper-
ature in the pots reached 258C as a consequence of illumination. One
newly expanded leaf of
;1 cm in length was harvested from each plant.
Leaves were immediately put into liquid nitrogen either after 48 h in the
dark or after 8 h or 24 h in strong light. To control for biological clock
effects between trials on CHS expression levels, the experiment was
always started at the same time of the day (12:00
PM). For each light
treatment, leaves from four distinct plants per cross (two from each
reciprocal cross) were harvested. Two independent trials separated by
a 5-month interval were conducted, for a total of eight plants per cross
and per treatment. For the above mentioned second set of crosses, eight
F1 individuals per cross were analyzed for their response to light in a single
trial.
Organ-Specific Expression in Flowers
CHS is upregulated in flowers where flavonoids are produced abundantly
(Burbulis et al., 1996). Plants where a leaf had been harvested after 48 h in
the dark were further grown for several weeks under standard light
conditions in a reach-in chamber until they flowered. A single flower per
plant was harvested for RNA extraction. Flowers from four distinct plants
per cross (two from each reciprocal cross) were harvested. Two in-
dependent trials separated by a 5-month interval were conducted, for
a total of eight plants per cross.
686 The Plant Cell
Insect Feeding Trials
CHS expression is induced upon feeding by Plutella xylostella larvae (H.
Vogel, personal communication). For insect induction, 4th instar larvae
were starved overnight. In the early morning, two larvae were placed on
each plant assigned to the insect treatment. After 9 and 24 h, herbivore-
damaged leaves, as well as leaves from insect-free control plants, were
harvested. For each insect treatment and each associated control, leaves
from four distinct plants per cross (two from each reciprocal cross) were
harvested. Two independent trials separated by a 5-month interval were
conducted, for a total of eight plants per cross and per treatment. Insect
feeding trials resulted in four independent treatments: 9 h insect feeding,
9 h control, 24 h insect feeding, and 24 h control.
RNA Extraction, cDNA Synthesis, and Quantitative Pyrosequencing
Immediately after collection, 1 to 2 cm
2
of sampled leaf material was
placed into liquid nitrogen and stored at 808C. RNA was extracted using
500 mL of Trizol (Invitrogen Life Technologies) and following the manu-
facturer’s protocol. Precipitated RNA was resuspended in RNA storage
solution (Ambion, Austin, TX) after 20 min of air drying. Total RNA
concentrations were evaluated by spectrophotometry, and
;4 mgof
total RNA was used for each cDNA synthesis. cDNA was synthesized
using Superscript III RT (Invitrogen Life Technologies) following the
manufacturer’s protocol with the following modifications: 100 units of
Superscript III-RT and 20 units of RNaseOUT (Invitrogen Life Technolo-
gies) for a 20-mL reaction. Also, incubation time for retrotranscription was
extended to 2 h. To ensure that RNA samples were free of DNA
contamination, a PCR was also performed on several RNA samples
before the RT, following the protocol described below. No amplification
product was observed.
Pyrosequencing is a method based on the stochiometric photochem-
ical reaction triggered by the nucleotide by nucleotide extension of
a sequencing primer. The photochemical reaction allows quantification of
nucleotides at polymorphic positions relative to neighboring monomor-
phic positions, hence estimating allele-specific relative expression levels
(Ahmadian et al., 2000; Neve et al., 2002).
To control for possible position effects in the thermocycler, cDNA
samples together with DNA extracted from heterozygous plants were
randomly distributed across 96-well plates before PCR. A 1-mL volume of
the cDNA solution was used for PCR with 5 pmol of each primer, 5 pmol of
ROTI-Mix (Carl Roth, Karlsruhe, Germany) mixed nucleotides, 25 pmol
MgCl
2
, 1 unit of Taq polymerase (Qiagen, Valencia, CA), and the PCR
buffer provided by the manufacturer. Two sets of primers were used
to assess SNP 780, which differentiates Col-0, Lz-0, and Ag-0 from all
other accessions: Set 719 (59Biotin-CAAGGTTGCTTCGCCGGC-39/
59-GGTAACGGCTGTGATCTC-39) and Set 700 (59Biotin-AGCGTCTC-
ATGATGTACC-39/59-TTTCTCTCCGACAGATGTG-39). Primer set 700
was also used to assess SNP840 differentiating Lip-0 from the other
accessions.
SNP quantity was assessed using the PyrosequencerAB device
(Biotage, Uppsala, Sweden) and following the manufacturer’s protocol
for vacuum sample preparation and pyrosequencing reactions. Sequenc-
ing primers used for SNP780 and SNP840 were 59-GAGGA-
CACGTGCTCCA-39 and 59-TGTGTCAGGGTCCG-39, respectively. For
both SNPs, relative SNP concentration was measured as the ratio of
a polymorphic peak relative to a monomorphic peak. Polymorphic peaks
corresponding to more than one position (i.e., a monomorphic and
a polymorphic position or two monomorphic positions) were not used in
the analysis. Genotypes Lip-0 and Col-0 differ in the CHS coding
sequence at two nucleotide positions (SNP780 and SNP840), permitting
experimental replication with two independent SNPs. Both SNPs were
assessed on PCR fragments obtained with the primer set 700, and
expression levels were significantly correlated (R
2
¼ 0.84, P ¼ 0.003). To
calibrate the pyrosequencing measurements, serial volumetric mixtures
of homozygote DNA or RNA were performed (0.2:0.8, 0.3:0.7... up to
0.8:0.2). Volumetric proportions were corrected for concentration and
technical bias using the K correction proposed by Wittkopp et al. (2004),
in which K is the ratio of the technical bias (measured as the deviation of
the heterozygote DNA measurement from 50%) over the volumetric bias
(measured as the deviation of the 50/50 volumetric mixtures from 50%).
The technical bias accounts for possible preferential amplification of
a parental allele, and the volumetric bias accounts for inaccuracy in the
estimation of total DNA (or RNA) levels before the mixture. True propor-
tions were obtained from transforming volumetric proportions (VP) with
the following function: K * VP/(1 VP þ K * VP).
Data Analysis
Population Genetics
Sequences were aligned with Megalign 5.03 (DNASTAR). The DnaSP 3.84
program (Rozas and Rozas, 1999) was used for both intraspecific and
interspecific analyses of nucleotide polymorphism (Table 1). Nucleotide
diversity was calculated as p, the average number of nucleotide differ-
ences among pairs of sequences, and as u, the proportion of segregating
sites (Watterson, 1975; Tajima, 1983; Nei, 1987). To compute the indel
diversity statistics, each indel was coded as a substitution, the rest of
the sequences being equal. Patterns of nucleotide polymorphism were
summarized by the following test statistics: Tajima’s D, based in the
differences between two estimators of intraspecific diversity, and Fay and
Wu’s H, which makes use of an outgroup sequence to analyze the
frequency of derived polymorphisms (Tajima, 1989; Fay and Wu, 2000).
The HKA test is based on the prediction that, for a particular region of the
genome, the rate of divergence between species is proportional to the
levels of polymorphism within spec ies (Hudson et al., 1987). This test
compares the ratio of intraspecific polymorphism to interspecific di-
vergence in two loci. HKA tests were p erformed for silent positions using
silent segregating sites and the silent divergence value (Nei, 1987) to
compare the intergenic region and the CHS coding region. For compar-
ison of multiple intergenic regions, all positions were considered to be
silent. These three neutrality tests (D, H, and HKA) focus on different
characteristics of nucleotide polymorphism and thus effectively summa-
rize the evolutionary history of the CHS intergenic region.
Promoter Analysis
Sequences were searched for known transcription factor binding sites
using version 16.0 of the PLACE database (Higo et al., 1999). This
database was chosen because it is publicly available and is regularly
updated (403 binding site entries on April 13, 2004). We wrote a Perl script
that reads a sequence alignment, automatically queries PLACE with each
sequence in the alignment (gaps removed), and outputs those binding
sites that are affected by a polymorphic site. The script is available from
the authors upon request.
Statistical Analysis of Expression Data
A significant correlation was detected between the pyrosequencing
measurement and the signal-to-noise difference estimated by comparing
the height of a monomorphic peak to basal signal. Following the
manufacturer’s advice, we discarded data where monomorphic peaks
showed a signal below 10 units. We subsequently incorporated the
signal-to-noise difference as a technical covariate in the pyrosequencing
measurement. We performed separate analyses for light induction
treatment (including organ-specific expression in flowers) as well as for
each insect treatment. In all analyses, measurements obtained for DNA
cis-Regulatory Evolution in A. thaliana 687
samples of heterozygous individuals were used to calibrate the null
hypothesis in which both promoter alleles have equal activity.
An ANOVA was performed with the GLM procedure implemented in
SAS using the following statistical model:
X
ijklm
¼ m þ G
i
þ I
j
þ T
k
þ P
kl
þ N
m
þ GI
ij
þ IT
jk
þ GT
ik
þ C þ e
ijklm
;
where m is the grand mean, G
i
is the effect of the i
th
cross, I
j
indicates the j
th
induction treatment, T
k
represents the k
th
trial, P
kl
is the effect of the l
th
plate in the k
th
trial, N
m
indicates the m
th
primer set, C is a technical
covariate, and GI
ij
,IT
jk
, and GT
ik
represent interactions between cross 3
treatment, treatment 3 trial, and cross 3 trial, respectively. Because of
missing data in some cells of our data set, it was not possible to include
the effect of the maternal genotype in this model. This effect was thus
analyzed separately using a similar model that did not incorporate the
interactions between factors.
In each well, X
ijklm
is the ratio of a polymorphic peak over a mono-
morphic peak obtained by pyrosequencing. In the GLM model, trial, PCR
plate within trial, and interactions involving the trial factor were treated as
random effects. To dissect significant interaction effects, we used the
SLICE procedure in SAS to test for heterogeneity among treatments
within a genotype or among genotypes within treatment. For hetero-
zygous DNA, no heterogeneity among crosses was detected. When
expression levels were significantly heterogeneous, we performed a sep-
arate ANOVA for each cross or each treatment followed by a least
squares mean comparison test (Tukey’s test) to identify statistically
differentiated treatments or genotypes. For the additional crosses, given
that we had only one trial and two plates, all effects were handled as fixed
effects in the ANOVA.
For the analysis of the effect of cDNA pool upon pyrosequencing
measurement, the following linear model was tested:
X
ijk
¼ m þ A
i
þ I
j
þ R
k
þ AR
ik
þ C þ e
ijk
;
where m is the grand mean, A
i
is a continuous covariate corresponding
to the i
th
proportion of Col-0 allele, I
j
indicates the j
th
induction treatment,
R
k
represents the k
th
retrotranscription batch, C is a technical covariate,
and AR
ik
represents interaction between proportion 3 treatment. R
k
and
AR
ik
are random effects.
Sequence data from this article have been deposited with the EMBL/
GenBank data libraries under accession numbers AJ867819 to
AJ867845. Accession numbers for the A. gemmifera and A. croatica
sequences are AJ868239 and AJ868240, respectively.
ACKNOWLEDGMENTS
We thank J. Bishop, M.J. Clauss, A. Lawton-Rauh, S.E. Ramos-Onsins, P.
Wittkopp, J. Zavala, and two anonymous reviewers for hel pful discussions
and/or comments on the manuscript. This work was supported by the
Max Planck Gesellschaft and by the Bundesministerium fuer Bildung
und Forschung/Jena Centre for Bioinform atics Initiative 0312704F.
Received September 21, 2004; accepted January 6, 2005.
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