Cis-regulatory elements co-opting core circadian clock regulator CCA1 underlie enhanced expression of HMA4 for metal hyperaccumulation in Arabidopsis halleri

The naturally selected extreme traits of zinc and cadmium hyperaccumulation and hypertolerance in Arabidopsis halleri depend on strongly elevated HEAVY METAL ATPase 4 (HMA4) transcript levels compared to those in the closely related Arabidopsis thaliana. This difference is regulated in cis; AhHMA4 upstream sequences alone are sufficient to confer increased expression, as previously demonstrated using reporter gene fusions stably introduced into both A. halleri and A. thaliana. However, the underlying cis-regulatory divergence specific to A. halleri remains unknown. Here, we identify cis-regulatory metal hyperaccumulation elements (MHEs) that increase AhHMA4 promoter activities by examining stably transformed reporter lines carrying partial deletions or mutations in AhHMA4 upstream sequences. MHE1 (consensus TGTAAC) functions in the distal regions of AhHMA4 promoters, and all three tandem AhHMA4 gene copies share a proximal upstream pair of MHE2 motifs (consensus AAATATCT), corresponding to the evening element. The evening element is a known target of Arabidopsis CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), a core circadian clock transcription factor that mediates light-dependent and circadian gene expression. We show that the elevated activity of the AhHMA4-1 promoter depends on MHE2 in cis and CCA1 in trans, and it can be recapitulated by introducing an intact pair of MHE2 motifs into the A. thaliana HMA4 promoter using site-directed mutagenesis. We also found that HMA4 transcript levels show diel rhythmicity in A. halleri but not in A. thaliana. In summary, we identify the causal cis-regulatory elements that co-opt a known regulator of diel and seasonal transcriptional rhythms to mediate enhanced expression of a gene critical for a naturally selected extreme trait syndrome.