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.

NAC transcription factors are one of the largest families of transcriptional regulators in plants, and members of the gene family play vital roles in regulating plant growth and development processes including biotic/abiotic stress responses. However, little information is available about the NAC family in pitaya. In this study, we conducted a genome-wide analysis and a total of 64 NACs (named HuNAC1-HuNAC64) were identified in pitaya (Hylocereus). These genes were grouped into fifteen subgroups with diversities in gene proportions, exon–intron structures, and conserved motifs. Genome mapping analysis revealed that HuNAC genes were unevenly scattered on all eleven chromosomes. Synteny analysis indicated that the segmental duplication events played key roles in the expansion of the pitaya NAC gene family. Expression levels of these HuNAC genes were analyzed under cold treatments using qRT-PCR. Four HuNAC genes, i.e., HuNAC7, HuNAC20, HuNAC25, and HuNAC30, were highly induced by cold stress. HuNAC7, HuNAC20, HuNAC25, and HuNAC30 were localized exclusively in the nucleus. HuNAC20, HuNAC25, and HuNAC30 were transcriptional activators while HuNAC7 was a transcriptional repressor. Overexpression of HuNAC20 and HuNAC25 in Arabidopsis thaliana significantly enhanced tolerance to cold stress through decreasing ion leakage, malondialdehyde (MDA), and H₂O₂ and O₂⁻ accumulation, accompanied by upregulating the expression of cold-responsive genes (AtRD29A, AtCOR15A, AtCOR47, and AtKIN1). This study presents comprehensive information on the understanding of the NAC gene family and provides candidate genes to breed new pitaya cultivars with tolerance to cold conditions through genetic transformation.

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.