Polygalacturonase (PG) is a key enzyme in cell wall metabolism and fruit ripening. Atemoya (Annona cherimola Mill. × A. squamosa L.) is a high-value tropical fruit that undergoes rapid postharvest softening at room temperature. However, the role of the atemoya PG gene family in this process remains unknown. This study determined that storing atemoya at 28 °C significantly reduced fruit firmness and the total pectin content but increased water-soluble pectin (WSP) and PG activity compared to storage at 15 °C. Genome-wide identification of the AaPG gene family in atemoya revealed that 40 AaPG genes were unevenly distributed across seven chromosomes. Nineteen genes were located within six tandem duplication clusters. AaPG proteins exhibited clade-specific differences: Clades B-E contained the polysaccharide lyase family 6 (PL-6) superfamily domain, while Clade A harbored the Aspergillus niger polygalacturonase 1 (Pgu1) domain and lacked several conserved motifs. Expression profiling and reverse transcription quantitative polymerase chain reaction (RT-qPCR) showed that AaPG19, AaPG21, AaPG23 and AaPG24 were specifically induced at 28 °C. Subcellular localization confirmed that these four proteins were located on the plasma membrane. These findings provide insights into the evolution and temperature-dependent regulation of the AaPG family, identifying candidate genes responsible for the rapid softening of atemoya fruit.

Quantitative real-time PCR (qRT-PCR) is a widely used technique for quantifying gene expression. However, accurate data normalization requires stable reference genes that exhibit constant expression levels across different experimental conditions. To date, no suitable reference genes have been validated for atemoya fruits (Annona cherimola Mill × A. squamosa L.). This study aimed to evaluate the expression stability of candidate reference genes in atemoya across distinct tissues, postharvest storage stages, and various treatment conditions. To this end, we designed specific primers for six reference genes: ubiquitin carrier-like protein (UBC), actin 7 (ACT7), actin 11 (ACT11), elongation factor 1α (EF1α), 18S ribosomal RNA (18S), and β-tubulin (TUB). Total RNA was extracted from atemoya tissues (e.g., petals, young fruits, and roots), for pulp across three post-harvest time points (0, 2, and 4 days) and under different conditions: 28 °C, 15 °C, ethylene, or 1-MCP treatment. The geNorm tool was subsequently used to identify the most stable reference transcripts under each experimental condition. ACT7 was the most stable reference gene across atemoya tissues, while UBC ranked first under all postharvest conditions tested (28 °C storage, 15 °C cold storage, ethylene, and 1-MCP). Furthermore, the suitability of these reference genes under ethylene and 1-MCP treatments was validated by examining the expression patterns of AaPG and AaERF. Collectively, the combined use of ACT7, UBC, and 18S proved to be the most reliable normalization strategy across all experimental conditions. This validated multi-gene approach provides a robust foundation for future investigations into gene expression and the postharvest ripening mechanisms of atemoya.