The subfamily Agavoideae comprises crassulacean acid metabolism (CAM), C3, and C4 plants with a young age of speciation and slower mutation accumulation, making it a model crop for studying CAM evolution. However, the genetic mechanism underlying CAM evolution remains unclear because of lacking genomic information. This study assembled the genome of Agave hybrid NO.11648, a constitutive CAM plant belonging to subfamily Agavoideae, at the chromosome level using data generated from high-throughput chromosome conformation capture, Nanopore, and Illumina techniques, resulting in 30 pseudo-chromosomes with a size of 4.87 Gb and scaffold N₅₀ of 186.42 Mb. The genome annotation revealed 58 841 protein-coding genes and 76.91% repetitive sequences, with the dominant repetitive sequences being the I-type repeats (Copia and Gypsy accounting for 18.34% and 13.5% of the genome, respectively). Our findings also provide support for a whole genome duplication event in the lineage leading to A. hybrid, which occurred after its divergence from subfamily Asparagoideae. Moreover, we identified a gene duplication event in the phosphoenolpyruvate carboxylase kinase (PEPCK) gene family and revealed that three PEPCK genes (PEPCK3, PEPCK5, and PEPCK12) were involved in the CAM pathway. More importantly, we identified transcription factors enriched in the circadian rhythm, MAPK signaling, and plant hormone signal pathway that regulate the PEPCK3 expression by analysing the transcriptome and using yeast one-hybrid assays. Our results shed light on CAM evolution and offer an essential resource for the molecular breeding program of Agave spp.

β-amylase proteins (BAM) are important to many aspects of physiological process such as starch degradation. However, little information was available about the BAM genes in Annona atemoya, an important tropical fruit. Seven BAM genes containing the conservative domain of glycoside hydrolase family 14 (PF01373) were identified with Annona atemoya genome, and these BAM genes can be divided into four groups. Subcellular localization analysis revealed that AaBAM3 and AaBAM9 were located in the chloroplast, and AaBAM1.2 was located in the cell membrane and the chloroplast. The AaBAMs belonging to Subfamily I contribute to starch degradation have the higher expression than those belonging to Subfamily II. The analysis of the expression showed that AaBAM3 may function in the whole fruit ripening process, and AaBAM1.2 may be important to starch degradation in other organs. Temperature and ethylene affect the expression of major AaBAM genes in Subfamily I during fruit ripening. These expressions and subcellular localization results indicating β-amylase play an important role in starch degradation.

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.