Fruit acidity, a key quality trait, is primarily determined by malic acid in many fruit crops. However, the molecular machinery governing its accumulation, particularly in response to hormonal cues like auxin, remains a "black box." Here, we systematically deconstruct the regulatory pathway controlling malic acid in litchi (Litchi chinensis). We first identify the tonoplast-localized transporter LcALMT1 as the principal channel for malate sequestration into the vacuole. We then uncover its upstream regulatory module, revealing that the R2R3-MYB transcription factor LcMYB70 directly binds the LcALMT1 promoter but lacks intrinsic transcriptional activity. Instead, LcMYB70 functions as a molecular scaffold to recruit the Auxin Response Factor LcARF10, which confers transcriptional repression upon the complex. This LcMYB70-LcARF10 repressor module is negatively regulated by auxin signaling, providing a direct molecular link between the decline in endogenous auxin during fruit maturation and the attenuation of malate accumulation. Critically, we identified a single-nucleotide polymorphism in the LcARF10 gene that governs the divergence between high- and low-acid cultivars. The functional C allele in low-acid cultivars enables the formation of the repressor complex. In contrast, the T allele in high-acid cultivars contains a premature stop codon that results in a non-functional, truncated protein, preventing complex assembly and causing the high-acid phenotype. Our study defines a complete pathway from hormone to trait and its genetic basis, revealing a novel MYB-ARF scaffold module; a definitive molecular link between auxin withdrawal and metabolic reprogramming; and the functional integrity of a corepressor as a new paradigm for trait diversification.

Low-temperature stress (LTS) inhibits the growth and yield of pitaya. HuBBX1, a cold-tolerance transcription factor, was identified based on the physiological and transcriptomic analyses between two pitaya cultivars. HuBBX1 enhances plant cold tolerance via the activation of cold-regulated (COR) genes. Low-temperature stress (LTS) affects fruit quality and yields. Pitaya plants are sensitive to LTS; however, the molecular mechanisms underlying LTS in pitaya remain poorly understood. In this study, the chlorophyll fluorescence, photosynthetic system, cold tolerance-associated physiological indexes, and differentially expressed genes (DEGs) were compared using two pitaya cultivars, i.e., ‘SCAU-NH’ (resistant to low temperature) and ‘SCAU-KX’ (sensitive to low temperature) at the LTS. Six candidate transcription factors (TFs) related to LTS were identified according to their expression profiles. Yeast growth assay and GUS activity analysis showed that HubZIP6, HuOFP12, HuBBX1, HuHY5, HuBOA1, and HuERF39 could enhance cold resistance. Among them, HuBBX1 is a nuclear protein with transcriptional activation capability. Overexpression of HuBBX1 enhanced cold tolerance in Arabidopsis thaliana and tomato. Dual-luciferase reporter assays (DLR), yeast one hybrid (Y1H), and EMSA showed that HuBBX1 promotes the expression of cold-regulated (COR) genes (HuCOR15A and HuRD29A) by directly bounding to the G-box in their promoters to increase plant cold tolerance. The present work provides a foundation for further study of the molecular mechanisms of pitaya’s response to LTS.

Wampee (Clausena lansium) is a tropical and southern subtropical fruit tree whose productivity is limited by low-temperature stress (LTS). This study investigated the physiochemical and ‌molecular mechanisms by which melatonin (MLT) enhances chilling tolerance in wampee. MLT treatment improved membrane stability and reduced oxidative damage by enhancing antioxidant capacity and osmotic adjustment. Metabolomic and transcriptomic analyses revealed that differentially expressed genes and metabolites were primarily associated with flavonoid biosynthesis and starch and sucrose metabolism pathways. Eleven key candidate genes were identified, including sucrose synthase, granule-bound starch synthase, trehalose-6-phosphate synthase, chalcone-flavonone isomerase, flavonoid 3',5'-hydroxylase, and UDP-glycosyltransferase. MLT treatment also increased soluble carbohydrate levels, such as sucrose and fructose, and enhanced sucrose phosphate synthase activity. Transcription factors ClbHLH2 and ClNAC1 enhanced wampee's cold tolerance by activating the sucrose-biosynthetic genes ClSUS2 and ClSPS1, respectively. These findings provide insights into the molecular and physiological mechanisms underlying wampee adaptation to LTS and highlight MLT as a potential tool to improve wampee's cold tolerance.

Cold stress severely limits the yield and quality of fruit crops, yet its regulatory mechanisms in pitaya remain poorly understood. Here, we identified a cold-inducible bZIP transcription factor, HubZIP6, that plays a central role in enhancing cold tolerance in pitaya. HubZIP6 is a nuclear-localized protein with transcriptional activation activity, and its overexpression in Arabidopsis and tomato significantly improved cold tolerance, as reflected by higher survival rates, reduced ion leakage, and lower reactive oxygen species accumulation. Mechanistically, HubZIP6 directly binds to ACGT motifs in the promoters of HuCBF1 and HuCBF3, thereby activating their expression under cold stress. In addition, HubZIP6 physically interacts with the salicylic acid-binding protein HuSABP2, which synergistically enhances the transcriptional activation of HuCBF genes. Notably, HubZIP6 also directly activates HuSABP2, forming a regulatory loop that connects CBF transcriptional control with salicylic acid signaling. Consistently, overexpression of HuSABP2 further enhances cold tolerance in transgenic plants. Collectively, these findings demonstrate that cold tolerance is enhanced through HubZIP6-mediated integration of CBF activation and salicylic acid signaling, providing a promising genetic target for improving stress resilience in fruit crops.

Seed abortion is a critical agronomic trait affecting both fruit quality and yield. Cell wall invertase (CWIN) hydrolyzes sucrose into glucose and fructose, which serve as both nutrients and signaling molecules to regulate seed development, with its activity suppressed by invertase inhibitor (INH). However, the transcriptional regulators controlling INH expression remain unknown. Here, we identify the MADS-box transcription factor LcMADS28 as a direct activator of LcINH1. LcMADS28 is highly expressed in the funicle and embryo of the abortive-seeded cultivar 'Nuomici' at 15 days after anthesis, whereas its expression is much lower in the big-seeded cultivar 'Heiye'. Further analysis reveals that LcMADS28 interacts with LcMADS27 to form a heterodimer that directly binds to the LcINH1 promoter and activates its transcription. Silencing LcMADS28 in 'Nuomici' reduces LcINH1 expression, which increases CWIN activity, promotes embryo and seed development, and lowers fruit abscission. RNA-seq analysis indicates that genes associated with auxin and sucrose metabolism are activated after LcMADS28 silencing. In addition, heterologous overexpression of LcMADS28 in tomato and Arabidopsis increases seed abortion. Together, our results indicate that LcMADS28 and LcMADS27 coordinately activate LcINH1 to regulate early seed development in lychee and provide a mechanistic framework linking MADS-box transcription factors with the invertase/INH module.

Fruit quality in wampee is strongly influenced by the accumulation of key metabolites, including anthocyanins, sugars, and organic acids, yet their metabolic dynamics during fruit development remain poorly understood. In this study, we analyzed metabolite profiles and the expression of metabolism-related genes in the pulps of two wampee cultivars, “Jixin” (JX) and “Zirou” (ZR), across multiple fruit developmental stages. Two anthocyanins, 17 sugars, and 32 organic acids were identified. Total phenolics, flavonoids, starch, and soluble sugars accumulated mainly during early fruit development. “JX” wampee exhibited higher ascorbic acid levels than “ZR” wampee. Sucrose and citric acid were the predominant sugars and organic acids in both cultivars. Transcriptomic analysis revealed 37 anthocyanin-related, 29 sugar-related, and 18 organic acid-related genes. Strong correlations between candidate gene expression and metabolite levels suggest that these genes play key roles in regulating the biosynthesis and accumulation of anthocyanins, sugars, and organic acids in wampee.

Pitaya, a non-climacteric fruit commonly cultivated in tropical and subtropical regions, is valued for its nutritional benefits but is highly susceptible to chilling injury (CI) when stored below 6 °C. This study investigates the role of γ-aminobutyric acid (GABA) in mitigating CI during six weeks of cold storage at 5 ± 1 °C. Findings reveal that GABA treatment markedly suppressed CI severity up to 3 times in pitaya fruit compared with the control during cold storage. Decay incidence reached 62.3% in untreated fruit, whereas treated fruit exhibited 22.0% lower decay incidence. GABA also effectively preserved fruit quality by reducing weight loss from 10.9% in the control to 6.3% while maintaining higher total soluble solids (1.1-fold increase) and titratable acidity (0.9-fold higher than control). Moreover, GABA significantly alleviated oxidative stress, as evidenced by reduced malondialdehyde accumulation and H2O2 levels (2.7-fold lower) together with decreased superoxide anion production. These effects were accompanied by enhanced antioxidant defense, with increased activities of superoxide dismutase, peroxidase, and ascorbate peroxidase. In addition, GABA treatment promoted proline accumulation, up to 1.4-fold higher than in control, and suppressed respiration rates during storage. Transcriptomic analysis further revealed that GABA modulated the phenylpropanoid biosynthesis pathway, including key genes PAL, C4H, and 4CL, and influenced auxin, ethylene, and abscisic acid signaling pathways associated with stress responses and fruit senescence. Collectively, these results indicate that GABA mitigates chilling injury in pitaya by enhancing antioxidant capacity and regulating phenylpropanoid metabolism and hormonal signaling, thereby maintaining postharvest fruit quality during cold storage.

Safflower (Carthamus tinctorius L.) is a medicinal and edible cash crop that is widely cultivated worldwide. However, the genetic diversity of safflower germplasm resources and the reasons for the variations in safflower flower colour remain unclear. In this study, we used a combination of agronomic traits and Indel markers to assess the genetic diversity of 614 safflower germplasm resources. The results showed that most of the evaluated agronomic traits had high variability. The mean values of the Shannon’s information index (I) and polymorphism information content (PIC) in 50 pairs of Indel markers were 0.551 and 0.296, respectively. The population structure, neighbour-joining phylogeny, and principal coordinate analyses classified all genotypes into four subgroups, and 214 safflower core germplasms were constructed. Multiple analyses of genetic diversity parameters, range conformity, and the percentage of variance difference showed that the core germplasm did not differ significantly and could represent the original germplasm better. Transcriptome and metabolome analyses revealed that flavonoid synthesis-related genes, including CHS, F3H, ANS, and BZ1, were differentially expressed in different coloured safflowers. Most significantly, different genes and metabolite compounds in white safflowers were enriched upstream from the phenylpropanoid metabolic pathway to the production of naringenin, whereas those in red safflowers were concentrated in the downstream pathway from eriodictyol. Meanwhile, the preliminary quantification of anthocyanins and carotenoids extracted from red, orange, and white types of safflower showed that the level of both anthocyanins and carotenoids were highest in red types. This work provides new insights into the formation of different safflower flower colours and in the conservation and management of safflower germplasm.

Flower induction in pitaya (Hylocereus polyrhizus) is regulated by complex gene networks involving multiple signaling pathways that ensure flower bud (FB) formation, but its molecular determinants remain largely unknown. In this study, we aimed to identify key genes and pathways involved in pitaya flower induction by analyzing transcriptomics profiles from differentiating buds. Our results indicate that the flower induction process is driven by a combination of sugar, hormone, transcription factor (TF), and flowering-related genes. We found that during the FB induction period, the levels of sugar, starch, auxin (AUX), cytokinin (CTK) active forms dihydrozeatin riboside (dhZR), zeatin riboside (ZR), N6-isopentenyladenosine (iPA), and brassinosteroid (BR) increase in the late stage (LS), while active gibberellins (GA3, GA4) decrease, signaling a metabolic and hormonal shift essential for flowering. Differential gene expression analysis identified key genes involved in starch and sugar metabolism, AUX, CTK, BR synthesis, and (GA) degradation, with notable differential expression in photoperiod (COL, CDF, TCP), age-related (SPL), and key flowering pathways (FT, FTIP, AGL, SOC1). This study reveals a multidimensional regulatory network for FB formation in pitaya, primarily mediated by the crosstalk between sugar and hormone signaling pathways, providing new insights into the molecular mechanism of FB formation in pitaya.

Exogenous GA3 lowers the C:N ratio, depleting starch/sucrose and suppressing flowering, while untreated control plants maintain normal C:N, ample carbohydrates, and higher floral-promoter expression, supporting floral initiation. This study elucidates the regulatory role of gibberellin-3 (GA3) in nitrogen (N) and carbon (C) metabolism and its association with bud dormancy in pitaya (Hylocereus polyrhizus). Exogenous GA3 application completely inhibited floral bud development, maintaining dormancy, whereas untreated control plants progressed to active flowering. GA3-treated plants exhibited elevated nitrogen content but reduced carbon allocation, alongside significant declines in sucrose, glucose, fructose, total sugars, and starch compared with controls. Transcriptomic profiling identified numerous differentially expressed genes (DEGs) linked to N/C metabolism, starch/sucrose pathways, and aligning with observed trends in nitrogen, carbon and sugar level changes. Key flowering-promoting transcription factors (TFs) (e.g., PHYB, CRY, VIN3-like, TCP) and floral integrators (e.g., FY, FLK, AGL, FTIP) were downregulated under GA3, while N-assimilation genes and dormancy-associated TFs (e.g., CDF) and floral inhibitors (e.g., SOC1) were upregulated. These results demonstrate that GA3 disrupts the metabolic transition from N-to-C utilization necessary for floral activation, likely through coordinated suppression of flowering-promoting networks and enhancement of nutrient metabolism pathways. Our findings provide mechanistic insights into GA3-mediated dormancy and highlight its potential application in synchronizing pitaya cultivation cycles.

Wampee (Clausena lansium), a tropical evergreen fruit from the Rutaceae family renowned for its rich nutrient profile and bioactive compounds, presents a fascinating case study in fruit coloration. However, changes in anthocyanins, and expressions of metabolism-associated genes during fruit maturation of red-pericarp wampee ('ZR') are not documented. In this study, metabolic and gene expression profiles of anthocyanin across different fruit developmental stages of red and yellow-pericarp wampees were analyzed. A total of 38 distinct anthocyanins were identified from the comparison of 'ZR3' and 'JX3' wampees and categorized into 17 differential anthocyanin metabolites (DAMs). Among these DAMs, fifteen were up-regulated in 'ZR3', while two were down-regulated compared with 'JX3'. The delphinidin 3-[6-(4-(caffeoylrhamnosyl)glucoside)] was the predominant anthocyanins in 'ZR' wampee. A total of 1135 metabolics mainly including amino acid metabolites and flavonoids were detected in the 'ZR' and 'JX' wampees. Significant differences were mainly concentrated in the biosynthesis of secondary metabolites in terms of flavonoid biosynthesis, ABC transporters, and anthocyanin biosynthesis. According to the combined analyses of qRT-PCR and transcriptome, the transcript levels of PAL1, PAL2, CHS1 and UFGT1 in 'ZR' wampee were two to eight-fold higher than those in 'JX' wampee during fruit pigmentation. Our study offers valuable insights into the mechanisms of anthocyanin accumulation in the red pericarp of wampee which is helpful to regulate fruit coloration of wampee.

Litchi chinensis Sonn. is an important economic fruit tree in tropical and subtropical regions. Regrettably, the efficiency of plant regeneration via somatic embryogenesis in litchi is typically low due to the poor conversion of embryos to plants. The purpose of this study was to establish a regeneration system via somatic embryogenesis from immature embryos explants in 'Heiye' cultivar of litchi. Our results demonstrated that MS medium supplemented with 2.0 mg L-1 2,4-D was optimal for callus induction. For somatic embryo (SE) induction, MS medium containing 0.5 g L-1 activated charcoal (AC) was the most effective, while the use of zeatin (ZT) and thidiazuron (TDZ) resulted in abnormal somatic embryos. The rooting and regeneration rate of 2.15% and 17.5%, respectively, were achieved using MS medium supplemented with 0.5 g L-1 AC. Furthermore, transcriptome analysis was performed on embryogenic callus (EC), globular embryo (GE), and heart embryo (HE) to explore the molecular mechanisms of early somatic embryogenesis. 2,587 common differentially expressed genes (DEGs) between EC_vs_GE and EC_vs_HE were identified, and the expression patterns of these common DEGs were separated into twelve major clusters. GO annotation and KEGG pathway analysis revealed that these common DEGs were implicated in plant hormone signal transduction, auxin-activated signaling pathway, and other biological processes. Additionally, differentially expressed transcription factors were identified, and the function of LcBBM2 which is specifically highly expressed during early somatic embryogenesis was verified. Overexpression of LcBBM2 in tomato promotes callus and shoot formation. Therefore, this study can provide a theoretical basis and technical support for genetic breeding improvement of litchi.

Basic helix-loop-helix (bHLH) proteins have been recognized as activators of anthocyanin in many plant species, but their role as inhibitors of anthocyanin is seldom reported. In this study, LcbHLH107 was identified as a transcription repressor regulating anthocyanin biosynthesis in litchi. LcbHLH107 was up-regulated in the red hairy roots overexpressing LcMYB1, which was the key transcription activator in litchi anthocyanin biosynthesis. Dual luciferase assay and yeast one-hybrid assay confirmed that LcbHLH107 was regulated by LcMYB1. LcbHLH107 could suppresses anthocyanin accumulation through transient transformation in petunia leaves and overexpression in litchi callus. Moreover, LcbHLH107 directly bound to the promoter of LcDFR, and inhibited its expression. Furthermore, protein sequence mutation and functional verification revealed that the EAR motif was essential for the inhibitory function of LcbHLH107. In summary, LcbHLH107 is a repressor that inhibits anthocyanin biosynthesis in litchi, providing a negative feedback mechanism to regulate anthocyanin accumulation at transcriptional level.

Pitaya is an important perennial herbaceous fruit tree. The color of fruit determines pitaya nutritive (and attractive) value, which is considered as an important objective in breeding improvement. In this study, we reported the first telomere-to-telomere (T2T) gap-free genome of "Shuangse No. 1" pitaya (Hylocereus polyrhizus; red peel). Two high-quality genomes for "Dahong" (H. polyrhizus; red peel) and "Honghuaqinglong" (H. stenopterus; stay-green) were further assembled, aiming to explore the genetic diversity of pitaya genomes. In further analysis, we noticed a high proportion of viral contamination in pitaya tissues, which hindered the efficient utilization of transcriptomic data. To address this issue, we analyzed 111 pitaya transcriptome data from different geographic regions to characterize and separate viral components. Then we developed an efficient, novel, and universal transcript purification system for pitaya transcriptomes by applying it to 27 samples from different tissues and species, thereby enhancing the utility for transcriptomic and broader biological research. Combining the purified transcriptomic data with comparative genomic analyses, we identified HuERF72, a transcription factor (TF) that potentially regulates chlorophyll degradation in pitaya. Interaction assays and plant transformation elucidated that HuERF72 acts as a repressive TF by directly binding to the promoter of HuSGR1, a key structural gene in the chlorophyll degradation pathway. This study provides high-quality genomic resources and novel methodologies for molecular investigations in pitaya. Additionally, the proposed regulatory network advances our understanding of the transcriptional regulatory mechanisms underlying chlorophyll degradation, offering valuable insights into the genetic improvement of pitaya.

Chlorophyll is vital for plants, giving them their green color and playing indispensable crucial role in photosynthesis. Chlorophyll-deficient mutants serve as classic models for studying plant pigment metabolism and typically exhibit chlorotic or albino phenotypes, resulting in major impacts on photosynthetic efficiency and growth development of plants. Understanding the mechanisms behind chlorophyll deficiency not only advances basic plant biology but also supports crop breeding strategies aimed at improving yield, stress tolerance, and adaption. This article provides a comprehensive review of recent research on the molecular mechanisms underlying chlorophyll metabolism, chloroplast structure, photosynthetic systems, relevant transcription factors, and the effects of external environmental factors on chlorophyll-deficient mutants. It provides a theoretical basis for improving plant pigment metabolism and crop breeding.

Betalains, a distinctive group of nitrogen-containing pigments exclusive to the Caryophyllales order, possess diverse biological activities such as antioxidant, anti-inflammatory, and antimicrobial properties, making them highly valuable for applications in food, nutraceutical, and pharmaceutical industries. This Review provides a comprehensive analysis of betalain biosynthesis, structural diversity, and ecological significance, highlighting their roles in enhancing stress resilience, adaptation mechanisms, and plant evolutionary strategies. The evolutionary development of betalains is explored, revealing their emergence through gene duplication events and providing insights into their mutual exclusivity with anthocyanins. This study utilizes comparative genetics and advanced molecular tools to uncover the intricate regulatory networks involving transcription factors such as MYB, bHLH, WRKY, and SPL, which govern betalain biosynthesis. Furthermore, the Review discusses innovative transgenic studies that introduce betalains into non-native species, demonstrating their potential to enhance stress tolerance and boost agricultural productivity. While significant progress has been made in understanding betalain biosynthesis pathways, the evolutionary relationships with anthocyanins and the specific ecological functions of betalains in plants remain areas of ongoing exploration. Future research directions include integrating chemotaxonomic studies, molecular phylogenetics, and multiomics approaches to unravel the full spectrum of betalain functions and regulatory mechanisms. Such studies are essential to deepening our understanding of these vibrant pigments and their evolutionary implications, offering new opportunities for biotechnological innovations and sustainable agricultural practices. This Review stands out by combining genetic, ecological, and evolutionary perspectives, providing novel insights into the multifunctionality of betalains and their potential to drive future advancements in plant science and biotechnology.

Pitaya canker disease, caused by Neoscytalidium dimidiatum, is the primary threat to pitaya cultivation, significantly compromising fruit quality and reducing yield. WRKY transcription factors are essential regulators in plant pathogen recognition and defense mechanisms, yet their specific roles in the development of pitaya canker disease remain largely unexplored. In this study, five genes (HmeWRKY33, HmeWRKY51, HmePR1, HmeHsp70, and HmeSERK) associated with pitaya canker disease were identified through RNA-Seq analysis. The expression levels of HmeWRKY33 and HmeWRKY51 were upregulated following N. dimidiatum infection. Transient transformation revealed that these five genes negatively influenced the resistance of Nicotiana benthamiana leaves to canker disease while promoting the accumulation of reactive oxygen species and inducing cell death. Yeast one-hybrid and dual luciferase reporter assays revealed that HmeWRKY33 directly activated the expression of HmeSERK, while HmeWRKY51 directly inhibited the expression of HmePR1 and HmeHsp70, coparticipating in regulating the susceptibility of 'Youcihuanglong' pitaya to canker. These findings provide a theoretical basis for breeding new canker-resistant pitaya varieties through genetic transformation.

Glycosylation of anthocyanin plays an important role in increasing its stability and diversity in plants. Here, we identified a glucosyltransferase gene responsible for the anthocyanin components in Litchi chinensis Sonn. Cyanidin-3-rutinoside is the main anthocyanins of pericarps and red hairy roots overexpressing MYB transcription factor LcMYB1 in the late maturing cultivars. However, in the early maturing cultivars, the anthocyanins in the pericarps is dominated by cyanidin-3-glucoside, and the anthocyanins in the red hairy roots overexpressing LcMYB1 is dominated by cyanidin-3-rutinoside. Enzyme assays highlighted notable differences in flavonoid 3-O-rhamnosyltransferase (F3RT) activity between the pericarps and red hairy roots overexpressing LcMYB1 in the early maturing cultivars. Two differentially expressed genes (DEGs), LcF3RT1 and LcF3RT2, were significantly up-regulated in the red hairy roots overexpressing LcMYB1. Yeast one-hybrid and dual luciferase reporter assays revealed that LcMYB1 could bind to the promoter of LcF3RT2 and significantly activate its expression. Functional validation showed that LcF3RT2 could catalyze the conversion of cyanidin-3-glucoside into cyanidin-3-rutinoside, leading to the differences on anthocyanin components in pericarps and red hairy roots of early maturing litchi cultivars. Our results will provide insights into the regulation and glycosylation modification of anthocyanins in litchi as well as in other plants.

BACKGROUND:Flowering is a complex, finely regulated process involving multiple phytohormones and transcription factors. However, flowering regulation in pitaya (Hylocereus polyrhizus) remains largely unexamined. This study addresses this gap by investigating gibberellin-3 (GA3) effects on flower bud (FB) development in pitaya. Our findings reveal that GA3 application induces significant bud dormancy and suppresses FB formation, highlighting GA3's role in modulating flowering in this species. RESULTS:GA3 application during peak flowering period significantly altered hormone levels, reducing auxin (AUX), cytokinin (CTK) active forms dihydrozeatin riboside (dhZR), zeatin riboside (ZR), N6-isopentenyladenosine (iPA), and brassinosteroid (BR), while increasing jasmonic acid (JA), GA3, and gibberellin-4 (GA4) levels, with abscisic acid (ABA) levels remaining unchanged compared to control. Conversely, FB formation was associated with increased levels of AUX, dhZR, ZR, iPA, ABA, and JA, and decreased GA3 and GA4 levels. Transcriptomic analysis revealed batches of differentially expressed genes (DEGs) associated with phytohormone signal transduction, aligning with observed hormone changes. Notably, except four CONSTANS-like (CO) (HU06G02633, HU10G00019, HU04G00234, and HU02G01458), all other CO genes were preferentially active in GA3-treated buds. GA3 treatment inhibited genes linked to the ABC model (AP1, AP2, MADS-box, AGL, SPL) and floral identity genes (LFY, FT), favoring dormancy and clean sweep of FB formation. CONCLUSION:These findings underscore the potential of GA3 as a powerful modulator of flowering and bud dormancy in pitaya. By elucidating the hormonal and genetic responses to GA3 treatment, this study contributes to our understanding of flowering regulation in pitaya and highlights the significant impact of GA3 on bud developmental pathways.

Yellow pitaya (Selenicereus megalanthus, 2n = 4x = 44) breeding remains severely hindered due to the lack of a reference genome. Here, we present a high-quality chromosome-level genome assembly of yellow pitaya using PacBio HiFi sequencing and Hi-C scaffolding technologies. We identify yellow pitaya as an autotetraploid with a genome size of 1.79 Gb, harboring 27,246 high-confidence genes probably from diploid ancestors, red pitaya (S. undatus). By comparative analysis of the 3D chromatin architecture, we identify varying number of compartment A/B, topologically associated domains (TADs), and structural variations in diploid (red pitaya) and polyploid (yellow pitaya) species. We find that TAD boundaries are enriched with transcription factor motifs in both species. We find significant alterations in expression of genes in the betalain biosynthesis pathway in both species. We detect differential expression of genes encoding key regulators of pericarp color within the TAD regions of polyploid pitaya and diploid pitaya. We also identify the expression differences in candidate genes that likely influence betacyanin and betaxanthin synthesis in both species. Our findings suggest that differential 3D genome organization, especially differences in TAD boundaries, may impact gene expression, which may further lead to different trait formation in different pitaya species. This provides theoretical implications for fast-forward breeding.