Characterization of CXE genes in pineapple and their aroma-related expression during fruit ripening

Carboxylesterases (CXEs) are ester hydrolyzing enzymes closely associated with the degradation of volatile esters and aroma release in fruit. Pineapple possesses a strong, ester-rich aroma, yet its <i>CXE</i> gene family has not been characterized. Here, we performed a genome-wide identification and analysis of the pineapple <i>CXE</i> family, detected 20 members, and pinpointed <i>AcCXE4</i> and <i>AcCXE7</i> as key negative regulators of aroma accumulation and prime candidates for aroma-oriented breeding. Phylogenetic comparison with <i>CXEs</i> from <i>Arabidopsis</i>, tomato, strawberry, Nanguo pear, and peach resolved five clades, in which <i>AcCXE4</i> grouped with <i>PuCXE15</i>, a reported aroma-related gene in Nanguo pear, while <i>AcCXE7</i> clustered with tomato <i>SlCXE1</i> and <i>AcCXE3</i> with apple <i>MdCXE1</i>, suggesting these members participate in ester metabolism in pineapple and are potential contributors to fruit-aroma formation. Besides, although <i>AcCXE13</i> and <i>AcCXE20</i> show conserved gene structure and sequence, their exon numbers and motif architectures differ from other <i>AcCXEs</i>, implying functional specialization. Comparative genomics indicated that family expansion in pineapple was driven primarily by tandem duplication and large segmental duplication. Integrating cis-regulatory element profiling, transcriptome analyses, and experimental validation, we found that most <i>AcCXEs</i> are likely responsive to light and hormone signaling (including the jasmonate pathway) and to abiotic stress cues. Several <i>AcCXE</i> genes exhibited decreasing expression across cultivars, tissues, and developmental stages, showing negative correlations with aroma accumulation, with <i>AcCXE4</i> and <i>AcCXE7</i> displaying the strongest association with pineapple aroma formation. Collectively, this work systematically defines the pineapple <i>CXE</i> family and highlights priority targets to inform molecular improvement of fruit aroma.