Red fruit peel is an attractive target for pineapple breeding. Various pineapple accessions with distinct red coloration patterns exist; however, the precise molecular mechanism accounting for these differences remains unknown, which hinders the pineapple breeding process from combining high fruit quality with red peel. In this study, we characterized a transcription factor, AcMYB266, which is preferentially expressed in pineapple peel and positively regulates anthocyanin accumulation. Transgenic pineapple, Arabidopsis, and tobacco plants overexpressing AcMYB266 exhibited significant anthocyanin accumulation. Conversely, transient silencing of this gene led to decreased anthocyanin accumulation in pineapple red bracts. In-depth analysis indicated that variations of AcMYB266 sequences in the promoter instead of the protein-coding region seem to contribute to different red coloration patterns in peels of three representative pineapple varieties. In addition, we found that AcMYB266 was located in a cluster of four MYB genes exclusive to and conserved in Ananas species. Of this cluster, each was proved to regulate anthocyanin synthesis in different pineapple tissues, illustrating an interesting case of gene subfunctionalization after tandem duplication. In summary, we have characterized AcMYB266 as a key regulator of pineapple red fruit peel and identified an MYB cluster whose members were subfunctionalized to specifically regulate the red coloration of different pineapple tissues. The present study will assist in establishing a theoretical mechanism for pineapple breeding for red fruit peel and provide an interesting case for the investigation of gene subfunctionalization in plants.

A large number of pineapple (Ananas comosus) fruits are discarded in China every year due to softening. However, the underlying molecular mechanism is still unknown. AcoPG3 (GenBank accession number: XM020243935), a pineapple gene of polygalacturonase, was found to be the major gene responsible for the softening of pineapple fruit. Fruit of AcoPG3-overexpressing tomato (Solanum lycopersicum var. Jingfan 101) begins to soften 9 days earlier than that transformed with a net vector. Fruit of AcoPG3-overexpressing pineapple (APG3-2) begins to soften 6 days earlier than that transformed with a net vector. Fruit of MPG3-1, a pineapple line in which AcoPG3 is mutated, begins to soften 31 days later than that transformed with a net vector. The sequence of polygalacturonase activities in fruit from the highest to the lowest was APG3-2, wild type, MPG3-1. The same sequences were also found in the liquid content of apoplast and the electrolyte leakage of pineapple pulp. The order of methyl-esterified pectin content in the pulp cell wall, from the highest to the lowest, was MPG3-1, wild type, and APG3-2. The same order was also observed for the contents of non-methyl-esterified homogalacturonan and rhamnogalacturonan-I in the pulp cell wall. The AcoPG3 mutation resulted in a decrease in polygalacturonase activity in pineapple fruit, decreasing the degradation of methyl-esterified pectin, non-methyl-esterified homogalacturonan, galactan and rhamnogalacturonan-I in the pulp cell wall. Fruit softening can be deferred, and the shelf life can be extended by mutating the AcoPG3 gene.

Ananas comosus var. bracteatus (Ac. bracteatus) is a typical leaf-chimeric ornamental plant. The chimeric leaves are composed of central green photosynthetic tissue (GT) and marginal albino tissue (AT). The mosaic existence of GT and AT makes the chimeric leaves an ideal material for the study of the synergistic mechanism of photosynthesis and antioxidant metabolism. The daily changes in net photosynthetic rate (NPR) and stomatal conductance (SCT) of the leaves indicated the typical crassulacean acid metabolism (CAM) characteristic of Ac. bracteatus. Both the GT and AT of chimeric leaves fixed CO₂ during the night and released CO₂ from malic acid for photosynthesis during the daytime. The malic acid content and NADPH-ME activity of the AT during the night was significantly higher than that of GT, which suggests that the AT may work as a CO₂ pool to store CO₂ during the night and supply CO₂ for photosynthesis in the GT during the daytime. Furthermore, the soluble sugar content (SSC) in the AT was significantly lower than that of GT, while the starch content (SC) of the AT was apparently higher than that of GT, indicating that AT was inefficient in photosynthesis but may function as a photosynthate sink to help the GT maintain high photosynthesis activity. Additionally, the AT maintained peroxide balance by enhancing the non-enzymatic antioxidant system and antioxidant enzyme system to avoid antioxidant damage. The enzyme activities of reductive ascorbic acid (AsA) and the glutathione (GSH) cycle (except DHAR) and superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were enhanced, apparently to make the AT grow normally. This study indicates that, although the AT of the chimeric leaves was inefficient at photosynthesis because of the lack of chlorophyll, it can cooperate with the GT by working as a CO₂ supplier and photosynthate store to enhance the photosynthetic ability of GT to help chimeric plants grow well. Additionally, the AT can avoid peroxide damage caused by the lack of chlorophyll by enhancing the activity of the antioxidant system. The AT plays an active role in the normal growth of the chimeric leaves.

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 CXE gene family has not been characterized. Here, we performed a genome-wide identification and analysis of the pineapple CXE family, detected 20 members, and pinpointed AcCXE4 and AcCXE7 as key negative regulators of aroma accumulation and prime candidates for aroma-oriented breeding. Phylogenetic comparison with CXEs from Arabidopsis , tomato, strawberry, Nanguo pear, and peach resolved five clades, in which AcCXE4 grouped with PuCXE15 , a reported aroma-related gene in Nanguo pear, while AcCXE7 clustered with tomato SlCXE1 and AcCXE3 with apple MdCXE1 , suggesting these members participate in ester metabolism in pineapple and are potential contributors to fruit-aroma formation. Besides, although AcCXE13 and AcCXE20 show conserved gene structure and sequence, their exon numbers and motif architectures differ from other AcCXEs , 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 AcCXEs are likely responsive to light and hormone signaling (including the jasmonate pathway) and to abiotic stress cues. Several AcCXE genes exhibited decreasing expression across cultivars, tissues, and developmental stages, showing negative correlations with aroma accumulation, with AcCXE4 and AcCXE7 displaying the strongest association with pineapple aroma formation. Collectively, this work systematically defines the pineapple CXE family and highlights priority targets to inform molecular improvement of fruit aroma.