Integrated metabolomic and transcriptomic analyses elucidates the molecular mechanisms underlying cucurbitacin accumulation and bitterness development in Luffa fruits

Abstract

The fruits of Luffa in the Cucurbitaceae family are characterized by a bitter taste, which is mainly attributed to cucurbitacin. However, the types of cucurbitacins related to bitterness and the molecular mechanism of cucurbitacin accumulation during Luffa fruit growth and development remain largely unclear. Here, we identified a total of 633 metabolites in Luffa, including alkaloids, flavonoids, and terpenoids, with 364 key metabolites showing significant differences between bitter and non-bitter varieties. Cucurbitacins B and D were significantly more abundant in bitter Luffa and showed an upward trend from non-bitter to extra-bitter varieties. Transcriptome analysis revealed 25,577 differentially expressed genes (DEGs) in these Luffa varieties, including the upregulation of terpenoid biosynthetic pathways in bitter fruits. Integrative metabolite profiling and transcriptome analyses showed that LaCBS(Luffa cucurbitadienol synthase), encoding cucurbitadienol synthase, is a hub gene for the first committed step in cucurbitacin biosynthesis, located within gene clusters comprising LaP450s(Luffa cytochrome P450s) and LaACT(Luffa acetyltransferase). In addition, two LaCBS genes were located within two gene clusters, alongside LaP450s and LaACT, in the interspecific hybrid of Luffa. A total of 746 DEGs were identified as transcription factors, including 103 LabHLH family members; two LabHLHs were positively correlated with LaCBS and LaACT. Heat and abscisic acid stress activated the cucurbitacin biosynthetic pathway. These findings help to elucidate the molecular mechanism of cucurbitacin biosynthesis in bitter Luffa fruits.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08400-5.

Introduction

Luffa of the Cucurbitaceae family is a traditional vegetable crop in tropical and subtropical regions, especially in China and India, with nine distinct species identified to date. Among them, Luffa acutangula (Roxb.) and Luffa cylindrica (Roem.) are the most commonly cultivated species [1–3]. These two species exhibit substantial differences in life habits, fruit shape, fruit color, and other agronomic traits. Accordingly, there is a certain degree of reproductive isolation between L. acutangula and L. cylindrica, exemplified by traits such as poor flowering periods, pollen abortion or abnormal pollen development, and even a particularly strong bitter fruit after hybridization [4, 5]. Furthermore, exposure to abiotic stress such as drought, heat, and hormone treatment or biological stress such as insect invasion and grafting, causes the fruits of cultivated species of Cucurbitaceae to have a more bitter flavor [6, 7]. Bitterness in Cucurbitaceae plants is mainly attributed to the accumulation of cucurbitacins, which are plant-specific triterpenoid secondary metabolites [8, 9].

Terpenoid biosynthesis is accomplished via the mevalonate (MVA) pathway in animals and fungi and by the methylerythritol phosphate (MEP) pathway in prokaryotes, whereas both pathways play independent roles in terpenoid biosynthesis in higher plants [10, 11]. Cucurbitacins are highly oxidized tetracyclic triterpenoid compounds. The biosynthesis of triterpenoids is a highly complex and diversified process with over 100 different triterpenoid skeletal structures identified in nature to date. Most cucurbitacins are formed from the triterpenoid biosynthetis precursor 2,3-oxidosqualene through substrate folding, carbocation cyclization, and variations in rearrangement steps to form a wide range of carbonaceous architectures, including dammaranes, lanostanes, oleanane, cucurbitane, and other structures [12, 13]. The initial basic skeleton for the formation of cucurbitacins is the cucurbitane-type carbon skeleton cucurbitadienol. Acetyl-coenzyme A (CoA) produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) under the catalysis of a series of enzymatic reactions, including acetyl-CoA-C-acetyltransferase (ACAT), hydroxymethylglutaryl-CoA synthase (HMGCS), and hydroxymethylglutaryl-CoA reductase (HMGCR). Subsequently, farnesyl pyrophosphate synthase catalyzes the synthesis of farnesyl pyrophosphate (FPP) from two molecules of IPP and one molecule of DMAPP. Two FPP molecules are then polymerized into squalene by squalene synthase (SS); squalene acts as a major precursor for isoprenoid biosynthesis in prokaryotes. Squalene is oxidized by squalene epoxidase (SE) to form 2,3-oxidosqualene, which is a precursor of the triterpene skeleton, membrane sterols, and steroid hormone biosynthesis in higher plants, as well as a branching node of plant primary and secondary metabolites [14–16]. 2,3-Oxidosqualene is divided into two unused structures based on substrate conformation: the chair-chair-chair (C–C-C) conformation and the chair-boat–chair (C-B-C) conformation (CBC). The C–C-C conformation is catalyzed by oxidosqualene cyclase (OSC) to form the pentacyclic carbon skeletons of triterpene, such as β-amyrin and lupinol, while the C-B-C conformation is catalyzed by OSC to form membranous sterols and the tetracyclic carbon skeleton of triterpenes such as cycloartenol and cucurbitadienol [17, 18]. Subsequently, cucurbitadienol undergoes various modifications at different carbon positions by cytochrome P450 (CYP450), acetyltransferase (ACT), and glycosyltransferase (GT), ultimately leading to the formation of diversified cucurbitacins and cucurbitacin glycosides.

The vast diversity of naturally existing cucurbitacins can be classified into 12 main types based on their chemical structures and biological activities, namely cucurbitacins A–T, among which cuB, cuC, cuD, cuE, and cuI are the most widely recognized [19]. In cucumber, bitterness-related genes are located on chromosome 6, with OSCs (Bi) located within a gene cluster containing the CYP450 gene family and ACT genes. This cluster is involved in the biosynthesis of cuC and is regulated by two transcription factors: Bt and Bl. Similarly, in melon, OSCs located on chromosome 11 are located within a gene cluster containing CYP450 gene family and the ACT genes, which are involved in the biosynthesis of cuB and regulated by two transcription factors: Bt and Br [20–22]. However, to date, little research has been reported on the biosynthesis and molecular mechanisms of bitter substances in Luffa fruit, except for one recent study investigating the biosynthetic pathway of fruit bitterness [23]. To further clarify the main role of cucurbitacins in Luffa bitterness and their biosynthetic pathway, we performed metabolome and transcriptome analyses to compare mature fruits of non-bitter and bitter Luffa varieties, as well as a highly bitter hybrid. We also performed abiotic stress experiments and used quantitative real-time polymerase chain reaction (qRT-PCR) to validate the results from the integrated metabolome and transcriptome analysis. This work will help elucidate the detailed molecular pathways underlying cucurbitacin biosynthesis in Luffa.

Materials and methods

Plant materials

The Luffa plant materials were provided by the Vegetable Research Institute of Guangzhou Academy of Agricultural Sciences, including the non-bitter variety WK1 and the bitter variety YK1 derived from the high-generation self-inbred germplasm materials of Luffa acutangula. In addition, YK2 is a highly bitter variety from the F₁ generation obtained after interspecific hybridization of Luffa cylindrica and Luffa acutangula, with non-bitter fruits formed by both parents. The three materials were planted at the Nansha base of the Guangzhou Academy of Agricultural Sciences under open-field cultivation and managed according to local conventional agricultural practices. The seeds were sown in 21-hole trays (with a substrate ratio of coconut bran:vermiculite = 3:1) and the seedlings transplanted to the open field once they reached the two-true-leaf stage. The base fertilizer applied to the field consisted of a mixture of organic biological fertilizer and chemical fertilizer at a 5:1 ratio, with an application rate of 4000 kg/ha. Z-shaped vine training and pruning of basal branches on the main vine were carried out when the plant reached a height of approximately 1 m, with proper water and fertilizer management. Aulacophora indica was controlled by spraying the plants with a mixture of imidacloprid and spinetoram at 1500 × dilution each; the melon silk borer was controlled by spraying with broflanilide at 2500 × dilution once every 5 days; and powdery mildew was controlled by spraying with a mixture of azoxystrobin at 2000 × dilution and thiophanate-methyl at 800 × dilution. Finally, the fruits were collected 16 days after pollination, with three samples taken from each treatment, wrapped in tinfoil, and immediately frozen in liquid nitrogen. Three biological replicates were prepared from at least five fruits.

Assessment of bitterness phenotype in Luffa fruit

The bitterness of the developing fruits is concentrated primarily near the stems (2–3 cm from to the stem), with little bitterness present in other parts. The fruits grow in three stages: early (7 days after pollination), middle (12 days after pollination), and late (16 days after pollination). According to our experimental design and previous research methodologies, the three volunteers trained in bitter taste evaluation using standard reference compounds to minimize subjective bias evaluated the fruit bitter taste phenotype at the middle stage, focusing on the fruit part 2–3 cm from the stem. After tasting each sample, volunteers rinsed their mouths with deionized water to eliminate residual bitter taste interference, thus reducing tasting errors. Inter-rater reliability of the bitterness scores was analyzed and reported to validate the phenotype data [24, 25]. We divided the bitterness phenotype into four levels as follows: level 0, not bitter (sweet); level 1, slightly bitter; level 2, moderately bitter; and level 3, highly bitter. WK1 and YK1, two homozygous inbred lines of Luffa, were categorized as having fruit phenotypes of not bitter (sweet) and moderately bitter (levels 0 and 2), respectively. The phenotype of YK2, an interspecific hybrid F₁ of Luffa cylindrica and Luffa acutangula, was characterized as highly bitter (level 3).

Metabolomic analysis

The fruits (50 mg, sampled ~ 2–3 cm from the stem) were ground for 10 min in a grinder at 30 Hz after being vacuum freeze-dried. The samples were subsequently extracted at 4 °C in 1000μL of a methanol–acetonitrile solution (methanol:acetonitrile:water = 2:2:1, v/v). Samples were centrifuged at 12,000 rpm for 15 min at 4 °C before being filtered and subjected to Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC, Waters Acquity I-Class PLUS; MS, Applied Biosystems 6500 + Q TRAP) analysis for metabolite detection, which was performed at Beijing Biomarker Technologies Co., Ltd.

All metabolite annotations were performed against the BMK G database. Metabolite quantification was achieved via multiple reaction monitoring (MRM)-mode analysis using triple-quadrupole MS. Variations in metabolite abundance among groups was assessed using orthogonal partial least-squares discriminant analysis (OPLS-DA) with the R language package “ropls” and 200 permutation tests were performed to veridate the reliability of the model. The variable importance in projection (VIP) value of the model was calculated using multiple cross-validations. Differentially accumulated metabolites (DAMs) were screened using the criteria of fold change (FC) > 1, P value < 0.05, and VIP > 1 of the OPLS-DA model. The functions of the DAMs were annotated based on significant enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways via a hypergeometric distribution test.

Transcriptome analysis

Total RNA was extracted from the nine samples (in the middle stages, three replicates of WK1, YK1, and YK2) using the RNAprep Pure Plant Kit (Tiangen, Beijing, China), following the manufacturer's instructions. The RNA quality was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and the concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The cDNA libraries were sequenced on an Illumina NovaSeq platform, generating 150-bp paired-end reads that were processed using Trimmomatic [26]. Clean reads were mapped to the Luffa reference genome using Hisat2 software to determine their genomic positions [27]. The reads from the comparison were then combined with StringTie [28] to reconstruct the transcriptome for further investigation. Differentially expressed genes (DEGs) were screened using the differential analysis program DESeq2 [29] according to the thresholds of |log2 (FC)|≥ 1 and false discovery rate < 0.01 [30]. Functional annotation and analysis of the DEGs were performed using Gene Ontology (GO) [31], Kyoto Encyclopedia of Genes and Genomes (KEGG) [32], and Cluster of Orthologous Groups of proteins (COG).

Heat stress and abscisic acid (ABA) treatment

For heat stress, Luffa seedlings were cultivated at 20–25 °C. The 14-day-old seedlings were then exposed to 45 °C/28 °C (day/night) with a 12 h/12 h light/dark cycle in a climate-controlled chamber; control plants were cultivated under normal conditions. The leaves were sampled after 5 days of treatment. For the ABA treatment, the 14-day-old seedlings were separated into two groups: the treatment group was sprayed with a 100 μM ABA solution, while the control plants received water. The leaves were sampled 6 days after treatment. Each experimental treatment comprised five experimental samples and three biological replicates.

Validation with qRT-PCR

Total RNA was extracted from the samples using the RNAprep Pure Plant Kit, following the manufacturer's instructions (Tiangen, China). Total RNA (0.2 μL) was reverse-transcribed into cDNA with SynScript® III RT SuperMix. The qRT-PCR analysis was performed using the ArtiCan^CEO SYBR qPCR Mix. The 18S rRNA (gene ID = 58,119,177) was used as an internal control. The 2^−△△Ct method was used to calculate the relative expression level of the target genes. Three biological replicates were performed. The primers used for qRT-PCR are listed in Supplementary Table S1.

Statistical analysis

Statistical analysis was performed using the independent samples t-test in SPSS 22.0 (SPSS Inc., USA), with *p ≤ 0.05 and **p < 0.01. The data were expressed as mean ± SD. Each analysis was performed based on three biological replicates.

Results

Variation in metabolites among Luffa fruits with different bitterness phenotypes

Samples were extracted from Luffa fruits with three distinct bitterness phenotypes, and qualitative and quantitative analyses of bitter-related metabolites were performed using UPLC-MS/MS. A total of 633 metabolites were detected, including 572 in WK1, 573 in YK1, and 576 in YK2 (Supplementary Table S2). These metabolites were assigned to 20 functional categories, with the main categories being others (17.38%); ketones, aldehydes, and acids (15.80%); alkaloids (12.48%); flavonoids (8.37%); and terpenoids (7.42%) (Fig. 1A, Supplementary Table S3). Sesquiterpenes (31.91%) and triterpenes (25.53%) accounted for the predominant components among the identified terpenoids.

Fig. 1.

Characterization of compounds identified in Luffa fruits. A General classification of metabolic substances. B Principal component analysis showing clusters of Luffa fruit samples (n = 3). C Venn diagram of differentially accumulated metabolites from three comparisons between fruits with different levels of bitterness. D KEGG pathway enrichment of differentially accumulated metabolites (DAMs)

Principal component analysis (PCA) of the detected metabolites was performed to identify differential metabolites. As shown in Fig. 1B, significant differences in metabolites were observed among the non-bitter, moderately bitter, and highly bitter Luffa fruits. A total of 364 metabolites were identified as DAMs, including 162, 242, and 249 DAMs in the WK1 vs. YK1, YK1 vs. YK2, and WK1 vs. YK2 comparisons, respectively (Fig. 1C; Supplementary Tables S4–S6). Among the DAMs, a total of 62, 151, and 128 metabolites were up-regulated, whereas 100, 91, and 121 metabolites were down-regulated in the WK1 vs. YK1, YK1 vs. YK2, and WK1 vs. YK2 comparisons, respectively. Further analysis revealed that the types of compounds in fruits without bitterness were not significantly different those with bitterness, and some even showed a decreasing trend with increasing bitterness. However, the number of terpenoid compounds increased when moving from no bitterness (31 terpenoids) to moderately bitter (37 terpenoids) and then to highly bitter (40 terpenoids) (Supplementary Table S7).

Metabolite KEGG enrichment analysis revealed that the most significant enrichment was observed for carbohydrate metabolic pathways, followed by membrane transport and amino acid metabolism pathways (Fig. 1D). The content of triterpenoids in bitter fruits was significantly higher than that in non-bitter fruits and showed an increasing trend, with the highest content found in YK2, including the cucurbitacins cuA, cuB, and cuD (Supplementary Table S2). Furthermore, seven DAMs were enriched in various alkaloid biosynthetic pathways (ko00996): Salicylic Acid, 2- (Methylamino)Benzoic Acid, Nsc 12,465, Vanillylamine, Vasicine, cuB and cuD (Supplementary Table S15). Enrichment of the metabolites cuB and cuD was commonly observed in all three comparisons of WK1 vs. YK1, YK1 vs. YK2, and WK1 vs. YK2 (Supplementary Tables S4–S6).

Transcriptome assembly and functional annotation

The global transcriptomes of the Luffa fruits with three distinct bitterness phenotypes were profiled via RNA-seq. A total of 19.42 Gb, 18.36 Gb, and 18.20 Gb clean reads were obtained from WK1, YK1, and YK2, respectively. The percentage of bases with quality scores ≥ 20 (Q20) and (Q30) exceeded 91.61% and 91.08%, respectively. The GC content of the clean reads ranged from 44.80 to 45.72% (Supplementary Table S8). Following quality control of the raw sequences, the transcriptome data were deemed suitable for subsequent downstream analysis.

A total of 57,454 genes (88.3%) were functionally annotated, including 5725 genes (8.8%) commonly annotated in eight transcriptome sequencing databases (Supplementary Table S9). The top five species matched in the non-redundant (NR) database, in terms of proportion, were Cucumis melo (12,865, 23%), Momordica charantia (11,047, 19%), Cucurbita moschata (7,877, 14%), Cucurbita pepo (7186, 13%), and Cucumis sativus (6211, 11%), indicating their close relationship with Luffa (Fig. 2). GO analysis further classified these genes into 7884 GO terms, which were categorized into biological process, cellular component, and molecular function categories. Metabolic process and cellular process were the most significant gene-enriched terms in the biological process category. In the cellular component category, cellular anatomical entity had the highest level of gene enrichment, whereas catalytic activity and binding were the top two gene-enriched terms in the molecular function category (Supplementary Table S10). According to the KEGG pathway analysis, 35,851 genes were divided into five expression branches and 136 metabolic pathway maps. The five branches were metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems (Supplementary Table S11).

Fig. 2.

Annotation and functional analysis of unigenes from Luffa fruits. Species annotations of unigene homologs

Gene expression and identification of DEGs

Comparison analysis of transcriptome data from Luffa samples with three distinct bitterness phenotypes identified a total of 25,577 significant DEGs, including 7877 DEGs between WK1 and YK1, 20,942 DEGs between YK1 and YK2, and 18,657 DEGs between WK1 and YK2 (Fig. 3A). Therefore, a larger number of DEGs was observed between moderately bitter (YK1) and highly bitter (YK2) Luffa fruits (Fig. 3B).

Fig. 3.

Gene expression and functional classification of differentially expressed genes (DEGs) in Luffa fruits with different bitterness phenotypes. A Venn diagram of DEGs from the three comparisons between fruits with different bitterness levels. B Histogram of up- and downregulated genes in the three comparisons. C KEGG enrichment analysis of DEGs from the three comparisons

The DEGs were further subjected to KEGG enrichment analysis and functional classification, and we detected subtle changes in gene expression related to bitterness in Luffa. From non-bitter (WK1) to moderately bitter (YK1) fruits, up- and down-regulated DEGs were mapped to 128 and 127 metabolic pathways, respectively. These DEGs were enriched in 135 and 128 pathways from moderately bitter (YK1) to highly bitter (YK2), indicating more active expression of differential genes in bitter Luffa (Supplementary Table S16). The DEGs were clearly different in the three different comparisons. For example, compared with the DEGs obtained in the comparison of WK1 vs. YK2, the DEGs in the comparison of YK1 vs. YK2 were more actively involved in pathways such as terpenoid skeleton biosynthesis (68), sesquiterpenoid and triterpenoid biosynthesis (20), and diterpenoid biosynthesis (29). Few DEGs overall were found in the comparison of WK1 vs. YK1 (Fig. 3C). Interestingly, the number of these DEGs related to bitterness was consistent with the number of metabolites in the metabolic pathways of samples in the three comparisons. Therefore, these DEGs may be key genes involved in the metabolism of different bitter taste compounds.

Genes involved in terpenoid and triterpenoid biosynthesis

We identified that the genes involved in the triterpenoid biosynthetic pathway were differentially expressed in fruits exhibiting varying levels of bitterness (Fig. 4A). Based on the gene expression levels (fragments per kilobase of transcript per million mapped reads values), we classified the gene characteristics and expression levels of enzymes involved in various metabolic processes (Fig. 4B; Supplementary Tables S12–S13). In the MVP pathway, 17 genes encoding six enzymes (ACAT, HMGCS, HMGCR, MVK, PMVK, and MVD) were predicted to participate in triterpenoid metabolite biosynthesis via homology analysis with characterized genes and pathways from Luffa [23]. Three ACAT-encoding genes catalyze acetyl-CoA to acetoacetyl-CoA, which is then converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by two HMGCS genes. Subsequently, five HMGCR genes reduce HMG-CoA to mevalonate. Two MVK-encoding genes transform mevalonate to mevalonate-5-phosphate, and two MVD-encoding genes catalyze its conversion to isopentenyl 5-diphosphate. Thirteen genes encoding seven enzymes were identified in the MEP pathway: six for 1-deoxy-d-xylulose-5-phosphate synthase, two for 1-deoxy-d-xylulose-5-phosphate reductoisomerase, and one each for the other five related enzymes. Additionally, four genes encode isopentenyl diphosphate delta-isomerase, while five and four genes encode geranyl pyrophosphate synthase and farnesyl diphosphate synthase, respectively. Furthermore, seven genes related to squalene synthase (SS) and 2,3-oxidosqualene cyclase (SE) were enriched.

Fig. 4.

Genes involved in terpenoid and triterpenoid biosynthesis in Luffa. A KEGG enrichment of the top ten genes and metabolites. B Expression heatmaps of genes associated with terpenoid biosynthesis from Luffa of three different fruits varying in bitterness, the columns of rectangles represent genes in triterpenoid and terpenoid biosynthesis. C Phylogenetic tree constructed from multiple sequence alignment of amino acid sequences of Luffa oxidosqualene cyclases (LaOSCs) and OSCs from other plants; the red boxes indicate the LaOSCs

OSC is involved in the first committed step in the modification of plant triterpenoids from a linear to cyclic form. In this study, two Luffa genes (Lac05g013070 and GeneMaker00013650) were annotated as cucurbitadienol synthase (LaCBS1 and LaCBS2) and two genes (Lac08g011790 and GeneMaker00025502) were annotated as β-amyrin synthase (LaBAS1 and LaBAS2). The coding sequence of LaCBS1, with 2717 nucleotides exhibited 97.1% sequence similarity to TcCBS of Trichosanthes cucumerina, 89.2% similarity to SgCBS of Siraitia grosvenorii, and 88.9% similarity to McCBS of Momordica charantia. Similarly, LaCBS2 with 2199 nucleotides showed 99.5% similarity to TcCBS, 91.0% similarity to McCBS, and 89.0% sequence similarity to CpCBS of Cucurbita pepo. LaBAS1, with a coding sequence of 2890 nucleotides, showed 9.3%, 90.1%, and 89.0% similarity to TcCBS, McCBS, and CpCBS, respectively, while LaBAS2, with a coding sequence of 2424 nucleotides, showed 99.8%, 90.4%, and 89.7% similarity to TcCBS, McCBS, and CpCBS, respectively. Consistently, these four OSCs, LaCBS1, LaCBS2, LaBAS1, and LaBAS2, were phylogenetically categorized into the crades of cucurbitadienol synthase and β-amyrin synthase (Fig. 4C).

Genes involved in the biosynthesis of the bitter substances

A BLAST nucleotide search and comparison with sequences from other Cucurbitaceae species such as cucumber and melon revealed that Luffa exhibited similar expression patterns of the related gene clusters involved in cucurbitacin biosynthesis. Interestingly, no cucurbitacin synthesis-related genes were expressed in WK1. One gene cluster was involved in cucurbitacin biosynthesis in YK1, while two gene clusters were detected in YK2. These two clusters were co-expressed with the cucurbitadienol synthase module. Combined with metabolomics analysis, they may contribute to cuB and cuD biosynthesis. First, LaBi (Lac05g013070) encodes the enzyme catalyzing 2,3-oxidosqualene cyclization into cucurbitadienol, the first committed step in cucurbitacin biosynthesis. Second, several CYP450 enzymes (encoded by Lac05g013040, Lac07g007990, Lac05g013060, Lac05g013090, Lac06g003140, Lac02g004580) drive the formation of cucurbitadienol intermediates and the production of cuD. Finally, cuD undergoes acetylation under the action of LaACT (Lac05g013080) to ultimately yield cuB (Shang et al., 2014; Shibuya et al., 2004). Notably, both parents of YK2 show a non-bitter phenotype. These findings thus suggest that the highly bitter phenotype that emerged after hybridization is related to a shift toward a similar expression pattern to that of the moderately bitter fruit YK1. Another gene cluster (one LaBi (2): GeneMaker00013650; six CYP450s: NEW 5702, GeneMaker00001220, NEW 5703, GeneMaker00014196, GeneMaker00036685, GeneMaker00038613; and one LaACT(2): GeneMaker00015020) was introgressed from Luffa cylindrica, and the two parental gene clusters were originally silent. After hybridization, these two gene clusters were activated and co-expressed, leading to increased accumulation of cuD and cuB (Fig. 5). This explains why hybrid fruits derived from non-bitter Luffa parents show strong bitterness.

Fig. 5.

Genes involved in the biosynthesis of the bitter substances. The distribution and expression of cucurbitacin biosynthesis genes are shown by the fragments per kilobase of transcript per million (FPKM) values

Basic helix–loop helix (bHLH) transcription factors involved in the biosynthesis of cucurbitacin

In cucurbit plants, bHLH transcription factors play important roles in plant growth, development, and physiological processes, and some bHLH transcription factors are even directly involved in regulating cucurbitacin biosynthesis [33]. Some of these are present in gene clusters, acting as bHLH transcriptional activators to regulate the biosynthesis of cuC, cuB, and cuE in Cucumis sativus, Cucumis melo, and Citrullus lanatus, respectively. A total of 746 transcription factors were identified as DEGs in Luffa, with 103 of them being bHLH transcription factors. Furthermore, 286 transcription factors were identified as DEGs in the comparison of WK1 vs. YK1, including 42 bHLH transcription factors. In contrast, 632 and 564 transcription factors were identified in the comparisons of YK1 vs. YK2 and WK1 vs. YK2, respectively, of which 78 and 74 were bHLH transcription factors. These findings suggested that the difference appeared to arise from the differential expression of the transcription factors (Fig. 6A).

Fig. 6.

Gene number and expression analysis. A Number of transcription factors and bHLH genes among differentially expressed genes in different comparisons. B Relative expression levels of up-regulated genes under heat stress compared with the control group. C Relative expression levels of up-regulated genes under aba treatment compared with the control group. Error bars represent the standard deviation (SD) of three independent biological replicates (n = 3). Statistical significance was determined by Student’s t-test between treatment and control groups; *P < 0.05, **P < 0.01

Compared with the Bt transcription factor of cucumber and melon, the homologous gene LaBt (Lac02g016030) was not detected in any of the three samples. However, LaBt is located within a gene cluster containing two other bHLH transcription factors (Lac02g016040, Lac02g016080), and Lac02g016040 was also not detected in the three samples. In contrast, Lac02g016080 had significantly higher expression in the bitter fruits (YK1 and YK2) than in the non-bitter fruit (WK1) and showed an up-regulated trend. In addition, GeneMaker00003045 encodes a specific transcription factor found only in the bitter samples (YK2), which is highly homologous to Lac02g016080 (Supplementary Table 14). Referring to the cucumber transcription factor CsBt, this gene encodes a fruit-specific transcription factor. During artificial domestication, Bt underwent changes from the original wild bitter ancestor to non-bitter cultivated varieties. In this study, LaBt was not detected in the three Luffa samples, which may be because LaBt is only expressed in wild species, whereas all three samples examined here are cultivated or hybrid varieties. Therefore, the two bHLH transcription factors Lac02g016080 and GeneMaker00003045 may participate in the biosynthesis of cuB and cuD.

Abiotic stress and qRT-PCR validation of representative DEGs

Finally, we used Luffa seedlings to investigate whether heat stress and ABA treatment activated cucurbitacin biosynthesis genes. The LaBi (Lac05g013070), LaACT (Lac05g013080), and LaCYP450 (Lac05g013040, Lac07g007990, Lac05g013050, Lac05g013060, Lac02g004580) genes in the cucurbitacin biosynthesis pathway were selected among the DEGs identified in the RNA-seq data for validation with qRT-PCR analysis. The results showed that all of the selected genes for cucurbitacin biosynthesis were significantly differentially expressed between the heat stress and control groups, with up-regulated expression under stress conditions. Similarly, for ABA treatment, all cucurbitacin synthesis-related genes showed significantly higher expression levels compared to those of the control group (Fig. 6B, C). These results indicated that abiotic stresses could activate cucurbitacin biosynthesis genes, leading to increased production of cucurbitacin, confirming the trends found in the RNA-seq data.

Discussion

Main triterpenoid metabolites of Luffa fresh fruits

Cucurbitaceae plants contain a special and representative class of metabolic compounds known for their bitterness and toxicity called cucurbitacins. As the name implies, cucurbitacins were initially discovered only in Cucurbitaceae plants; however, they have also been extracted from plants in other families such as Brassicaceae, Malvaceae, and Primulaceae [34, 35]. There are many types of cucurbitacins, which vary according to modification of the synthesis backbone (cucurbitadienol) at different positions, including oxidation, hydroxylation, and acetylation. Cucurbitacins are classified into 12 types, named cucurbitacin A to T [36]. Therefore, different Cucurbitaceae plants may contain different bitter substances. For instance, cuC is the main bitter compound found in cucumber [20], whereas the main bitter compound in melon is cuB [22], and the bitter taste in watermelon is mainly attributed to cuE [15, 37]. In this study, we performed quantitative and qualitative analysis of bitter metabolites in Luffa via UPLC-MS/MS, which enabled the identification of 633 metabolites including 17 triterpenoids and triterpenoid glycosides (Supplementary Table S2). Among the triterpenoid compounds, cuA, cuB, and cuD were detected, and quantitative analysis revealed that the contents of these three cucurbitacins in bitter fruits (YK1 and YK2) were significantly higher than those in the non-bitter fruit sample (WK1). Similarly, Zhao et al. [23] discovered several cucurbitacins (including cuA, cuD, cuF, and iso-cuB) in bitter Luffa fruits. In this study, the content of cuA was relatively lower in the three Luffa samples than in other Cucurbitaceae species [23]. CuA is a toxic and bitter medicinal compound that exhibits effective activity against ovarian cancer cells [38]. CuB and cuD were more abundant in bitter Luffa fruits, particularly in YK2. CuD is synthesized from the triterpene precursor 2,3-oxidosqualene to form the cucurbitacin skeleton cucurbitadienol, which is subsequently oxidized by CYP450 family members (CYP87D, CYP81Q32, and CYP705A5) to generate cuD. CuD is then acetylated to form cuB, and the two cucurbitacins can be interconverted under the action of CoA and ACT [22].

Triterpenoids are highly diverse natural compounds present throughout the Plant Kingdom, with their diversity stemming from rich carbon skeletons and various oxidation and glycosylation modifications at different skeleton positions [39, 40]. Almost all triterpenoid skeletons are derived from the biosynthetic precursor 2,3-oxidosqualene, which undergoes cyclization by OSCs to form various triterpene skeleton forms [41]. Moreover, multiple different triterpene skeletons can coexist in the same plant or tissue, which are catalyzed by distinct OSCs encoded by different genes [42]. In this study, we identified two common triterpenoid compounds, β-amyrin and lupeol, and their respective triterpene synthases: β-amyrin synthase and lupeol synthase. β-amyrin is catalyzed by two OSCs: LaBAS1 (Lac08g011790) and LaBAS2 (GeneMaker00025502), which catalyze the cyclization of 2,3-oxidosqualene to form the tetracyclic dammarenyl cation, followed by further oxidation and rearrangement to form the pentacyclic lupeol cation, and then another cyclization to form β-amyrin. Lupeol is catalyzed by one OSC (LUP: Lac08g011750), directly forming lupeol after formation of the intermediate lupeol cation. β-amyrin was only detected in the fruit of YK2, while lupeol was only found in WK1, and both metabolites were present at relatively low levels. β-amyrin can serve as a precursor of triterpenoids such as triterpene glycosides, oleanolic acid, and other triterpenoid compounds with various pharmacological effects. Lupeol, as a component of the cell membrane structure, participates in the growth and development of plants.

Identification of the biosynthesis pathway of bitter substances cuB and cuD in Luffa

Cucurbits show specificity in the composition of metabolic compounds that are associated with the bitter taste of the Cucurbitaceae family, which are biosynthesized via folding of 2,3-oxidosqualene into the C-B-C conformation to form their core skeleton [16, 43]. Based on our gene expression analysis, we hypothesized that LaCBS1 (Lac05g013070) and LaCBS2 (GeneMaker00013650) contribute critically to bitterness formation in Luffa fruits. The two cucurbitacin metabolites cuB and cuD were found to be significantly up-regulated in the metabolite quantification analysis of bitter fruits and were barely detectable in non-bitter fruits. Thus, CuB and CuD likely represent the main metabolites responsible for Luffa fruit bitterness, making their biosynthetic pathways worthy of in-depth investigation.

2,3-Oxidosqualene is a key branch point between membrane sterol primary metabolism and secondary metabolism, serving as a vital intermediate for researchers in biology and chemistry. Bi/CBS is a critical enzyme in the biosynthetic pathway, situated within a gene cluster that includes other functionally related genes involved in cucurbitacin biosynthesis. For example, gene clusters harboring one Bi/CBS, 7–10 CYP450, and one ACT gene have been identified in cucumber, melon, and watermelon [21, 22]. In Luffa, the biosynthesis of cuB and cuD is predicted to be initiated by the cucurbitadienol synthase gene (Bi/CBS) based on transcriptional and metabolic analyses, which first cyclizes 2,3-oxidosqualene to form cucurbitadienol as the first step in cucurbitacin synthesis. CBS shows high specificity and criticality, and provides the core cucurbitadienol backbone for all other triterpenoids. Subsequently, CYP450 members (e.g., CYP87D18) catalyze C-11 oxidation, C-3β hydroxylation and C-20β hydroxylation, whereas CYP81Q59 mediates C-2β hydroxylation, generating 11-carbonyloxy-2β,20β-dihydroxycucurbitadienol. C-2 oxidation yields CuD, which is further acylated by ACT to form CuB. Notably, we identified two gene clusters involved in cucurbitacin biosynthesis in Luffa. In bitter YK1 fruit, one gene cluster involved in CuB and CuD biosynthesis was identified, harboring one LaBi (Lac05g013070), six LaP450s (Lac05g013040, Lac07g007990, Lac05g013060, Lac05g013090, Lac06g003140, Lac02g004580) and one LaACT (Lac05g013080). Among them, Lac05g013090 and Lac06g003140 belonded to the CYP87A family, Lac05g013040 and Lac05g013060 to the CYP81Q subfamily; and Lac02g004580 to the CYP705A family. In bitter YK2 fruit, two such gene clusters were characterized. The additional cluster contained one LaBi2 (GeneMaker00013650), six LaP450s and one LaACT2 (GeneMaker00015020). These two clusters may act synergistically to enhance cucurbitacin accumulation, contributing to the highly bitter phenotype of YK2 fruits.

Effects of interspecific hybridization on the bitterness of Luffa fruit

Hybrid vigor or heterosis is a common phenomenon in nature, which has sparked great interest among scholars studying hybrid genetic relationships and biological genetic variation [44, 45]. When the kinship between two parents is too distant, a barrier to hybridization occurs, resulting in reproductive isolation [46]. Interspecific hybridization in Luffa can also lead to hybrid vigor, significantly enhancing the growth and development process of Luffa, increasing fruit retention ability, yield, and resistance to adversity and disease, and even introducing the advantageous traits of wild species into cultivated varieties, thereby improving targeted traits for genetic breeding and providing a wide range of interspecific materials for Luffa breeding and molecular marker development [5]. However, interspecific hybridization in Luffa also encounters some hybridization barriers such as the failure of flowering, pollen sterility, and even very bitter fruits. In previous research work, it was found that almost all F₁ hybrids of Luffa acutangula and Luffa cylindrica had highly bitter fruits, whereas neither parent had a bitterness phenotype [3, 47]. Therefore, we sought to use the fruits of F₁ hybrids as materials to investigate whether the bitterness traits of other Luffa fruits (moderately bitter) differ from those of F₁ hybrids (highly bitter), and explore how interspecific hybridization in Luffa affects fruit bitterness, aiming to elucidate the biosynthesis and molecular mechanism of bitterness regulation in fruits.

In the current study, metabolomic analysis revealed a total of 633 metabolites for the three samples. In the comparisons of WK1 vs. YK1 and YK1 vs. YK2, there were 162 and 242 DAMs, respectively, of which 62 and 151 DAMs were up-regulated, respectively. Furthermore, the bitter compounds cuA, cuB, and cuD were significantly up-regulated. Additionally, metabolic analysis showed that the contents of cuB and cuD in the interspecific F₁ hybrid YK2 (with a highly bitter fruit) were markedly increased compared to those of YK1 (with moderate bitterness fruit). The gene clusters involved in the cucurbitacin biosynthesis of Luffa (LaBi/CBS1, P450, and LaACT) were all up-regulated. The expression levels of bitter genes in YK2 were significantly higher than those in YK1. This indicates that interspecific hybridization of Luffa has a significant impact on the growth and development of the fruit, resulting in stronger bitterness. According to previous studies, interspecific hybridization between Luffa acutangula and Luffa cylindrica does not cause any fruit set issues, but reproductive isolation still occurs [3]. Interspecific hybridization within the Luffa genus may result in introgression of genetic material from Luffa cylindrica into hybrid progeny.

Conclusion

We investigated the molecular mechanisms underlying bitter substance biosynthesis in three Luffa samples with different bitterness phenotypes, using an integrated metabolomic and transcriptomic approach. Metabolomic data revealed that several DAMs were strongly associated with the biosynthesis of sesquiterpenes and triterpenes. Transcriptomic data showed that significantly up-regulated genes in bitter fruits were enriched in the biosynthetic pathways of terpene skeletons, sesquiterpenes and triterpenes, and cucurbitacins. Correlation analysis showed that the biosynthesis of cuB and cuD was significantly correlated with the genes associated with bitterness, such as LaBi, LaACT, and LaP450, and that cuB and cuD were significantly up-regulated in the bitter fruits of Luffa. Furthermore, LaBi1, LaACT1, and LaP450 are present in gene clusters, which were also significantly up-regulated in bitter fruits, indicating their potential involvement in the regulation of cucurbitacin biosynthesis. In addition, interspecific hybridization between Luffa acutangula and Luffa cylindrica introgressed other gene clusters (LaBi2, LaACT2, LaP450), leading to a significant increase in cucurbitacin synthesis in the hybrid F₁ fruit, resulting in a particularly strong bitter taste. These findings provide potential biological support for the development of medicinal cucurbitacin and its industrial application through interspecific hybridization, as well as offer a better understanding of the molecular processes and regulatory network of cucurbitacin biosynthesis in Luffa.

Authors’ contributions

**Zhu Dening:** Writing-original draft preparation, Supervision, Writing-review and editing. **Wu Yujun:** Resources. **Yu Bingwei, Peng Jiazhu:** Methodology, Investigation, Validation. **Li Lianfang:** Supervision, Writing-review and editing.

Funding

This work was supported by Guangzhou Science and Technology Bureau project (202201010733), Key Field Research and Development Project in Guangdong Province (2022B0202080003), Guangdong Province Rural Revitalization Strategy Special Fund Seed Industry Revitalization Project (2022-NPY-00–026); Guangzhou Agricultural Financial Fund Project (24103411).

Data availability

The processed transcriptomic and metabolomic datasets generated and analysed during the current study have been deposited in the BIG Submission and are publicly available at https://ngdc.cncb.ac.cn/gsa/s/ju96fW1U.

Declarations

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Competing interest

The authors declare no competing interests.