Calcium-Triggered PuMYB44-PuERF118 Cascade Activates PuFAD8 to Promote Aroma Biosynthesis in Pear Fruit

Fruit aroma is a key factor shaping the commercial value of pears. MYB and ERF transcription factors (TFs) are involved in regulating the biosynthesis of fruit aroma compounds, yet the mechanisms underlying their synergistic regulation of pear aroma formation remain elusive. Calcium signaling exerts an important role in fruit aroma biosynthesis; nevertheless, how calcium signaling mediates MYB-ERF transcription factors to participate in pear aroma biosynthesis remains unclear. Here, transcriptomic profiling of calcium-treated pear fruits screened two significantly upregulated TFs, PuMYB44 and PuERF118, as candidate genes. Phylogenetic analysis and conserved domain characterization further suggested that these two genes may be involved in regulating fatty acid biosynthesis. Transient overexpression experiments in pear fruits revealed that the co-expression of PuMYB44 and PuERF118 synergistically activated a set of fatty acid biosynthesis genes, including PuFAD2, PuFAD3, PuFAD8, PuLOX5, PuLOX21, and PuSCD. Meanwhile, the co-expression markedly increased the contents of key aroma volatiles, such as ethyl acetate, 1-hexanol, ethyl 2-methylbutyrate and hexadecane. Yeast two-hybrid and luciferase complementation assays confirmed an interaction between PuMYB44 and PuERF118. Analysis of the PuFAD8 promoter sequence revealed a conserved MYB-binding motif and an ERF-responsive cis-acting element. Dual-luciferase reporter assays further confirmed that both PuMYB44 and PuERF118 can independently activate PuFAD8 transcription, and that the synergistic activation effect resulting from the co-expression of these two factors is significantly stronger than that observed when either factor is expressed alone. In summary, this study elucidated a calcium-responsive transcriptional module underlying the biosynthesis of pear aroma and provided potential molecular targets for improving the flavor quality of pear fruit.