Systematic Analysis of Dof Gene Family in Prunus persica Unveils Candidate Regulators for Enhancing Cold Tolerance

Late-spring frost events severely damage low-chill peach blossoms, causing significant yield losses. Although 5-aminolevulinic acid (ALA) enhances cold tolerance through the PpC3H37-PpWRKY18 module, the regulatory mechanism of ALA on <i>PpC3H37</i> remains to be elucidated. Using yeast one-hybrid screening with the <i>PpC3H37</i> promoter as bait, we identified PpDof9 as a key interacting transcription factor. A genome-wide analysis revealed 25 <i>PpDof</i> genes in peaches (<i>Prunus persica</i>). These genes exhibited variable physicochemical properties, with most proteins predicted as nuclear-localized. Subcellular localization experiments in tobacco revealed that <i>PpDof9</i> was localized to the nucleus, consistent with predictions. A synteny analysis indicated nine segmental duplication pairs and tandem duplications on chromosomes 5 and 6, suggesting duplication events drove family expansion. A conserved motif analysis confirmed universal presence of the <i>Dof</i> domain (Motif 1). Promoter cis-element screening identified low-temperature responsive (LTR) elements in 12 <i>PpDofs</i>, including <i>PpDof1</i>, <i>PpDof8</i>, <i>PpDof9</i>, and <i>PpDof25</i>. The quantitative real-time PCR (qRT-PCR) results showed that <i>PpDof1</i>, <i>PpDof8</i>, <i>PpDof9</i>, <i>PpDof15</i>, <i>PpDof16</i>, and <i>PpDof25</i> were significantly upregulated under low-temperature stress, and this upregulation was further enhanced by ALA pretreatment. Our findings demonstrate ALA-mediated modulation of specific <i>PpDof</i> TFs in cold response and provide candidates (<i>PpDof1</i>, <i>PpDof9</i>, <i>PpDof8</i>, <i>PpDof25</i>) for enhancing floral frost tolerance in peaches.