The MYB (myeloblastosis) superfamily constitutes one of the most abundant transcription factors (TFs) regulating various biological processes in plants. However, the molecular characteristics and functions of MYB TFs in pitaya remain unclear. To date, no genome-wide characterization analysis of this gene family has been conducted in the Cactaceae species. In this study, 105 R2R3-MYB members were identified from the genome data of Hylocereus undatus and their conserved motifs, physiological and biochemical characteristics, chromosome locations, synteny relationship, gene structure and phylogeny were further analyzed. Expression analyses suggested that three up-regulated HuMYBs and twenty-two down-regulated HuMYBs were probably involved in fruit ripening of pitaya. Phylogenetic analyses of R2R3-MYB repressors showed that seven HuMYBs (HuMYB1, HuMYB21, HuMYB48, HuMYB49, HuMYB72, HuMYB78 and HuMYB101) were in clades containing R2R3-MYB repressors. HuMYB1 and HuMYB21 were significantly down-regulated with the betalain accumulation during fruit ripening of ‘Guanhuahong’ pitaya (H. monacanthus). However, only HuMYB1 had R2 and R3 repeats with C1, C2, C3 and C4 motifs. HuMYB1 was localized exclusively to the nucleus and exhibited transcriptional inhibition capacities. Dual luciferase reporter assay demonstrated that HuMYB1 inhibited the expression of betalain-related genes: HuADH1, HuCYP76AD1-1 and HuDODA1. These results suggested that HuMYB1 is a potential repressor of betalain biosynthesis during pitaya fruit ripening. Our results provide the first genome-wide analyses of the R2R3-MYB subfamily involved in pitaya betalain biosynthesis and will facilitate functional analysis of this gene family in the future.

Betalains are water-soluble nitrogen-containing pigments with multiple bioactivities. Pitaya is the only large-scale commercially grown fruit containing abundant betalains for consumers. However, the upstream regulators in betalain biosynthesis are still not clear. In this study, HmoWRKY40, a novel WRKY transcription factor, was obtained from the transcriptome data of pitaya (Hylocereus monacanthus). HmoWRKY40 is a member of the Group IIa WRKY family, containing a conserved WRKY motif, and it is located in the nucleus. The betalain contents and expression levels of HmoWRKY40 increased rapidly during the coloration of pitaya and reached their maximums on the 23rd day after artificial pollination (DAAP). Yeast one-hybrid and transient expression assays showed that HmoWRKY40 could bind and activate the promoter of HmoCYP76AD1. Silencing the HmoWRKY40 gene resulted in a significant reduction of betacyanin contents. These results indicate that HmoWRKY40 transcriptionally activates HmoCYP76AD, which is involved in the regulation of pitaya betalain biosynthesis. The results of the present study provide new regulatory networks related to betalain biosynthesis in pitaya.

The WRKY gene family is a plant-specific transcription factor (TF) that regulates many physiological processes and (a) biotic stress responses. Despite this, little is known about the molecular properties and roles of WRKY TFs in pitaya betalain biosynthesis. Here we report the identification of 70 WRKY in Hylocereus undatus, their gene structure, locations on each chromosome, systematic phylogenetic analysis, conserved motif analysis, and synteny of HuWRKY genes. HmoWRKY42 is a Group IIb WRKY protein and contains a coiled-coil motif, a WRKY domain and a C₂H₂ zinc-finger motif (CX₅CX₂₃HXH). Results from yeast one-hybrid and transient dual-luciferase assays showed that HmoWRKY42 was a transcriptional repressor and could repress HmocDOPA5GT1 expression by binding to its promoter. Yeast two-hybrid assays showed that HmoWRKY42 could interact with itself to form homodimers. Knocking out the coiled-coil motif of HmoWRKY42 prevented its self-interaction and prevented it from binding to the HmocDOPA5GT1 promoter. Knocking out the WRKY domain and C₂H₂ zinc-finger motif sequence of HmoWRKY42 also prevented it from binding to the HmocDOPA5GT1 promoter. The coiled-coil motif, the WRKY domain and the C₂H₂ zinc finger motif are key motifs for the binding of HmoWRKY42 to the HmocDOPA5GT1 promoter. HmoWRKY42 is localized in the nucleus and possesses trans-activation ability responsible for pitaya betalain biosynthesis by repressing the transcription of HmocDOPA5GT1. As far as we know, no reports are available on the role of HmoWRKY42 in pitaya betalain biosynthesis. The results provide an important foundation for future analyses of the regulation and functions of the HuWRKY gene family.

Pitaya (Selenicereus) is a kind of novel fruit with a delicious taste and superior horticulture ornamental value. The potential economic impact of the pitaya lies in its diverse uses not only as agricultural produce and processed foods but also in industrial and medicinal products. It is also an excellent plant material for basic and applied biological research. A comprehensive database of pitaya would facilitate studies of pitaya and the other Cactaceae plant species. Here, we constructed pitaya genome and multiomics database, which is a collection of the most updated and high-quality pitaya genomic assemblies. The database contains various information such as genomic variation, gene expression, miRNA profiles, metabolite and proteomic data from various tissues and fruit developmental stages of different pitaya cultivars. In PGMD, we also uploaded videos on the flowering process and planting tutorials for practical usage of pitaya. Overall, these valuable data provided in the PGMD will significantly facilitate future studies on population genetics, molecular breeding and function research of pitaya.

Sugar and organic acids are important factors determining pitaya fruit quality. However, changes in sugars and acids, and expressions of metabolism-associated genes during fruit maturation of yellow-peel pitayas are not well-documented. In this study, metabolic and expression analyses in pulps of different fruit developmental stages of ‘Wucihuanglong’ (‘WCHL’, Hylocereus undatus) and ‘Youcihuanglong’ pitaya (‘YCHL’, Hylocereus megalanthus) were used to explore the sugar and organic acid metabolic process. Total phenols and flavonoids were mainly accumulated at S1 in pitaya pulps. Ascorbic acid contents of ‘WCHL’ pitaya were higher than that of ‘YCHL’ pitaya during fruit maturation. Starch was mainly accumulated at early fruit development stages while soluble sugars were rich in late stages. Sucrose, fructose, and glucose were the main sugar components of ‘YCHL’ pitaya while glucose was dominant in ‘WCHL’ pitaya. Malic and citric acids were the main organic acids in ‘WCHL’ and ‘YCHL’ pitayas, respectively. Based on the transcriptome analyses, 118 genes involved in pitaya sugar and organic acid metabolism were obtained. Results from the correlation analyses between the expression profiling of candidate genes and the contents of sugar and organic acid showed that 51 genes had a significant correlation relationship and probably perform key role in pitaya sugar and organic acid metabolism processes. The finding of the present study provides new information for quality regulation of pitayas.

Betalains are water-soluble nitrogen-containing pigments with multiple bioactivities. Pitayas are the only at large-scale commercially grown fruit containing abundant betalains for consumers. Currently, the key genes involved in betalain biosynthesis remain to be fully elucidated. Moreover, genome-wide analyses of these genes in betalain biosynthesis are not available in betalain-producing plant species. In this study, totally 53 genes related to betalain biosynthesis were identified from the genome data of Hylocereus undatus. Four candidate genes i.e., one cytochrome P-450 R gene (HmoCYP76AD1), two L-DOPA 4,5-dioxygenase genes (HmoDODAα1 and HmoDODAα2), and one cyclo-DOPA 5-O glucosyltransferase gene (HmocDOPA5GT) were initially screened according to bioinformatics and qRT-PCR analyses. Silencing HmoCYP76AD1, HmoDODAα1, HmoDODAα2 or HmocDOPA5GT resulted in loss of red pigment. HmoDODAα1 displayed a high level of L-DOPA 4,5-dioxygenase activity to produce betalamic acid and formed yellow betaxanthin. Co-expression of HmoCYP76AD1, HmoDODAα1 and HmocDOPA5GT in Nicotiana benthamiana and yeast resulted in high abundance of betalain pigments with a red color. These results suggested that HmoCYP76AD1, HmoDODAα1, and HmocDOPA5GT play key roles in betalain biosynthesis in Hylocereus. The results of the present study provide novel genes for molecular breeding programs of pitaya.