Functional exploration of mitochondrial carrier family proteins in faba bean for drought tolerance improvement

Drought stress is a major constraint on global faba bean production. Mitochondrial Carrier Family (MCF) proteins are crucial for the transport of metabolic substrates across the inner mitochondrial membrane, thus regulating the metabolic balance in plants for growth and adaptation in adverse conditions. In this study, we identified 37 VfMCF genes using transcription data in faba bean germinating seeds, which exhibited both diverse structures and conserved motifs. Promoter analysis revealed that these genes are enriched with abiotic stress-responsive cis-elements, hinting at their potential roles in stress adaptation. Subsequent expression profiling revealed that three VfMCF genes (VfMCF2, VfMCF15 and VfMCF29) were constitutively upregulated in the drought-tolerant cultivar (CDAS105), with expression levels further increased under drought stress. Potato virus X (PVX)-mediated transient expression demonstrated that among the three VfMCFs, ectopic expression of VfMCF2 and VfMCF29, but not VfMCF15, significantly enhanced drought tolerance in tobacco. VfMCF2, a homolog of Arabidopsis mitochondrial NAD⁺ transporter AtNDT2, elevated cellular NAD(H) and NADP(H) levels, improved activities of antioxidant enzyme POD, CAT and GPX, thereby reducing ROS accumulation in transient transgenic tobacco lines under drought stress. Furthermore, VfMCF2 orchestrated the upregulation of twelve genes involved in NAD⁺ biosynthesis. In contrast, VfMCF29 may regulate ROS scavenging in tobacco by enhancing drought-induced activities of CAT and GPX and basal POD activity, but not drought-induced POD activity. Our findings indicate the potential of VfMCF2 and VfMCF29 for future genetic improvement. SUMMARY: This study identifies 37 VfMCF genes expressed in faba bean germinating seeds. Functional analysis reveals that VfMCF2 may scavenge ROS by improving the activities of multiple antioxidant enzymes, whereas VfMCF29 may regulate a narrower range of antioxidant enzymes. Both genes are valuable candidates for improving drought resistance.