The Nucleotides That Bind: Finding the Motif behind the Hypoxia Response.

Plants need oxygen, but they don’t always get enough of it. Under waterlogging or other hypoxic conditions, oxygen is not available to act as an electron acceptor in the oxidative phosphorylation pathway. ATP production plummets, depriving the plant of the energy it needs to survive. Fortunately,many plants have developed strategies to help them endure short bouts of hypoxia. By switching to anaerobic fermentation, plants can produce at least some ATP through glycolysis and an altered tricarboxylicacidpathway(Antonioetal.,2016). This hypoxia survival mode is accompanied by dramatic morphological and metabolic changes(VoesenekandBailey-Serres,2015). In Arabidopsis thaliana, these changes include the upregulation of 49 diverse hypoxiaresponsive genes (HRGs), many of which are inducedbygroupVII ethylene-responsive factor transcription factors (ERF-VII TFs), such as RELATED TO APETALA2.12 (RAP2.12) and RAP2.2. These TFs, in turn, are regulated by a clever oxygen-sensing mechanism.Under standard (normoxic) conditions, the bulk of these constitutively expressed RAPs undergo proteolysis. Upon exposure to hypoxia, RAPs protected from degradation by the plasma membrane are released and travel to the nucleus (along with newly synthesized RAPs), where they upregulate HRG expression (Voesenek and Bailey-Serres, 2015), most likely acting redundantly. This coordinated regulation suggests that the promoters of the HRGs share a common cis-regulatory motif. However, until recently, finding a motif common to these highly diverse genes has remained elusive, as TF binding sequences are short and sometimes variable. Two approaches are often used to search for common motifs among genes. First, the sequences of multiple, coregulated genes within a species are searched to detect overrepresented patterns. Second, homologous sequences of a single gene are compared across multiple species. However, some important motifs are not overrepresented in the genome, and promoter size varies across species, which complicates analysis. Gasch et al. (2016) combined these approaches in a powerful technique known as comparative phylogenetic footprinting, which selects for evolutionarily conserved motifs that occur in coregulated genes but are not necessarily overrepresented in the genome. First, the authors aligned the 1-kb upstream sequences of the 49 HRGs in 25 plant species and looked for conserved patterns for each gene set, with49 low-phosphate (Pi)-responsivegenes serving as controls. They then constructed adendrogramcontainingclusterswith similar promoter patterns, designating consensus sequences within each cluster as motifs. After eliminating any motifs that are also responsive to low Pi, comparing motif sequences with cis-motifs in various databases, and performing other in silico analyses, they identified a promoter region present in most HRGs that partially resembles an MYB TF binding site. Step-by-step deletion analysis examining LUCIFERASE (LUC) expression under the control of truncatedpromoter regions inRAP2.2-induced protoplasts revealed a 33-bp region containingthebioinformaticallypredicted12-bplong hypoxia-responsive promoter element (HRPE). Althoughapromoterwith thismotif was only slightly activated by HA-RAP2.2 (a stabilized version of RAP2.2) in protoplast assays, a triplicate version of this sequence was highly transactivated by HA-RAP2.2. Transgenic Arabidopsis plants harboring a triplicate HRPE-LUC construct exhibited LUC activity under hypoxia but not under normoxia (see figure). Yeast one-hybrid and chromatin immunopurification analyses demonstrated that both RAP2.2 and RAP2.12 directly interact with the HRPE. These results help confirm the identity of this crucial, evolutionarily conserved regulatory element, whose presence in diverse genes allows plants to endure hypoxia.