Allele-specific assay reveals functional variation in the chalcone synthase promoter of Arabidopsis thaliana that is compatible with neutral evolution

Abstract

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 (pi=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.

Figure 1.

Summary of Polymorphism Location and Frequencies in the 5′ 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 The gray box indicates a region conserved across multiple Arabidopsis species (J. de Meaux, unpublished data).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 5′ 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; $, SNP780; §, SNP840; #, mutation situated in an intron; (a), sequence from Ramos-Onsins et al. (2004).

Figure 2.

Correlated Pyrosequencing Measurements with Independent Primer Pairs. Regression between pyrosequencing measurements on SNP780 obtained for ∼70 cDNA samples with two independent primer pairs (Set 719 and Set 700) (R = 0.93, P < 0.001).

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 volumetric 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 Individuals 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 pattern is reversed in light 8 h and light 24 h. Only individuals from the Kas-1 × Col-0 cross depart from this pattern. Error bars show standard error.

Figure 5.

Relative CHS cis-Regulatory Activity in F1 Individuals 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.