Putative cis-regulatory elements predict iron deficiency responses in Arabidopsis roots

Abstract

Plant iron deficiency (2Fe) activates a complex regulatory network that coordinates root Fe uptake and distribution to sink tissues. In Arabidopsis (Arabidopsis thaliana), FER-LIKE FE DEFICIENCY–INDUCED TRANSCRIPTION FACTOR (FIT), a basic helix-loop-helix (bHLH) transcription factor (TF), regulates root Fe acquisition genes. Many other 2Fe-induced genes are FIT independent, and instead regulated by other bHLH TFs and by yet unknown TFs. The cis-regulatory code, that is, the cis-regulatory elements (CREs) and their combinations that regulate plant 2Fe-responses, remains largely elusive. Using Arabidopsis root transcriptome data and coexpression clustering, we identified over 100 putative CREs (pCREs) that predicted 2Fe-induced gene expression in computational models. To assess pCRE properties and possible functions, we used large-scale in vitro TF binding data, positional bias, and evolutionary conservation. As one example, our approach uncovered pCREs resembling IDE1 (iron deficiency-responsive element 1), a known grass 2Fe response CRE. Arabidopsis IDE1-likes were associated with FIT-dependent gene expression, more specifically with biosynthesis of Fe-chelating compounds. Thus, IDE1 seems to be conserved in grass and nongrass species. Our pCREs matched among others in vitro binding sites of B3, NAC, bZIP, and TCP TFs, which might be regulators of 2Fe responses. Altogether, our findings provide a comprehensive source of cis-regulatory information for 2Fe-responsive genes that advance our mechanistic understanding and inform future efforts in engineering plants with more efficient Fe uptake or transport systems.