Genome-Wide Identification and Characterization of the mTERF Gene Family in Spinach and the Role of SomTERF5 in Response to Heat Stress

Spinach (<i>Spinacia oleracea</i> L.), a globally consumed, nutrient-dense vegetable, contains diverse vitamins and minerals. However, elevated temperatures can constrain yield by interrupting leaf development and photosynthetic efficiency. The mitochondrial transcription termination factor (mTERF) family, which regulates organellar gene expression, plays crucial roles in plant growth and photosynthetic regulation. Thus, characterization of the spinach mTERF (SomTERF) family is critical for elucidating thermotolerance mechanisms in this crop. In this study, we systematically identified 31 <i>SomTERF</i> genes from the spinach genome, which are distributed across five chromosomes and nine unassembled genomic scaffolds. Subcellular localization predictions indicated that these proteins predominantly target chloroplasts and mitochondria. Conserved domain analyses confirmed that all SomTERF proteins possess canonical mTERF domains and ten conserved motifs. Phylogenetic clustering segregated these proteins into nine distinct subgroups (I-IX), with significant divergence observed in gene copy numbers among subgroups. Cis-element screening identified an abundance of heat-, cold-, and hormone-responsive motifs within <i>SomTERF</i> promoter regions. Notably, seven members (including <i>SomTERF5</i>) exhibited pronounced enrichment of heat shock elements (HSEs). Organ-specific expression profiling revealed preferential leaf expression of these seven genes. Comparative RT-qPCR in heat-sensitive (Sp73) and heat-tolerant (Sp75) cultivars under thermal stress demonstrated genotype-dependent expression dynamics. Functional validation of <i>SomTERF5</i> was achieved through cloning, and transgenic <i>Arabidopsis</i> overexpressing <i>SomTERF5</i> showed significantly enhanced thermotolerance, as evidenced by improved survival rates following heat treatment. Yeast two-hybrid (Y2H) assays further revealed physical interaction between SomTERF5 and SopTAC2. This study provides a comprehensive foundation for understanding mTERF-mediated developmental regulation and advanced molecular breeding strategies for developing heat-resilient spinach varieties.