The SQUAMOSA promoter binding protein-like (SPL) gene family is a unique family of plant-specific transcription factors (TFs), which plays vital roles in a variety of plant biological processes. Its role in betalain biosynthesis in Hylocereus undantus; however, is still unclear. Here, we report a total of 16 HuSPL genes from the pitaya genome, which were unevenly distributed among nine chromosomes. The HuSPL genes were clustered into seven groups, and most HuSPLs within the same group shared similar exon-intron structures and conserved motifs. Eight segment replication events in the HuSPL gene family were the main driving force behind the gene family expansion.Nine of the HuSPL genes had potential target sites for Hmo-miR156/157b. Hmo-miR156/157b-targeted HuSPLs exhibited differential expression patterns compared with constitutive expression patterns of most Hmo-miR156/157b-nontargeted HuSPLs. The expression of Hmo-miR156/157b gradually increased during fruit maturation, while the expression of Hmo-miR156/157b-targeted HuSPL5/11/14 gradually decreased. In addition, the lowest expression level of Hmo-miR156/157b-targeted HuSPL12 was detected 23rd day after flowering, when the middle pulps started to turn red. HuSPL5, HuSPL11, HuSPL12, and HuSPL14 were nucleus-localized proteins. HuSPL12 could inhibit the expression of HuWRKY40 by binding to its promoter. Results from yeast two-hybrid and bimolecular fluorescence complementation assays showed that HuSPL12 could interact with HuMYB1, HuMYB132, or HuWRKY42 TFs responsible for betalain biosynthesis. The results of the present study provide an essential basis for future regulation of betalain accumulation in pitaya.
| INTRODUCTIONPitaya/pitahaya, also known as dragon fruit, belongs to the genus Hylocereus in the family of Cactaceae. Pitaya is a commercial fruit crop widely cultivated in tropical and subtropical regions (Attar et al., 2022).Pitaya fruits contain abundant polyphenols, vitamins, oligosaccharides, ascorbic acids, and soluble dietary fiber, which have medicinal values in reducing the risk of constipation, obesity, and cancer (Jiang et al., 2021;Verona-Ruiz et al., 2020;Wichienchot et al., 2010). Besides, pitaya is a fast-return fruit crop with 8-16 separate fruiting cycles and yields as high as 75,000 kg per hectare per year. Therefore, pitaya has been a favorite of many consumers and farmers because of its good taste, unique appearance, abundant nutrients, and high yield.There are mainly three types of pitaya, Hylocereus undatus (red peel with white pulp), Hylocereus monacanthus/Hylocereus polyrhizus (red peel with red pulp), and Hylocereus megalanthus (yellow peel with
