The Horticulture Journal
Online ISSN : 2189-0110
Print ISSN : 2189-0102
ISSN-L : 2189-0102
ORIGINAL ARTICLES
Change in Bitterness, Accumulation of Cucurbitacin B and Expression Patterns of CuB Biosynthesis-related Genes in Melon During Fruit Development
Deping HuaJinyu FuLi LiuXuhui YangQiaoling ZhangMeiting Xie
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Supplementary material

2019 Volume 88 Issue 2 Pages 253-262

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Abstract

Bitterness, caused by cucurbitacins, is present in some melon fruit. Although bitter compound biosynthesis and regulation in Cucurbitaceae plants have been reported, the dynamic changes in bitterness during fruit development are unknown. Bitterness severity was measured for 19 inbred melon lines, including 14 lines of Cucumis melo var. chinensis, two var. inodorus and three var. conomon, using a panel tasting method. The data showed that bitterness severity was different in several lines of var. chinensis during fruit growth and maturation. Nb46 and Nb320, two elite parental lines of var. chinensis used in melon breeding, were used as experimental materials. Bitterness was severe at stage I, but moderate and disappeared at stage II and III in the fruit of Nb46. There was non-bitterness in the fruit of Nb320 throughout the development period. Furthermore, the cucurbitacin B (CuB) content gradually decreased in Nb46, while in Nb320, the CuB content changed little and remained at a quite low level during fruit development. Different expression patterns of the genes involved in CuB biosynthesis and regulation were found between Nb46 and Nb320. The expression levels of these genes were significantly higher in Nb46 than Nb320 in the early developmental stages, and this correlated with a higher concentration of CuB in Nb46 than Nb320. These results demonstrate that bitterness severity is different in var. chinensis during fruit developmental stages, and that the CuB biosynthesis-related genes are a critical factor in this process. We hope these findings will contribute to the breeding of non-bitter melon cultivars.

Introduction

The melon (Cucumis melo L.), a highly diversified species of the Cucurbitaceae family, is an economically important fruit crop cultivated around the world. During the past decade, the average production of melon was over 29 million tons per year (FAOSTAT 2017, http://faostat.fao.org/). It is cultivated in mainly temperate and tropical countries, with the major producer regions located in Asia and China leading the world (FAOSTAT 2017, http://faostat.fao.org/). Melon has many variant types. On the basis of length and distribution of hairs on the ovary and young fruit, vegetative morphological characteristics and fruit variation, melons are subdivided into two subspecies, namely, subsp. agrestis having ovaries with short, appressed hairs, and subsp. melo having pilose or lanate ovaries with long, spreading soft hairs (Kirkbride, 1993). The former is further subdivided into five group varieties, such as var. chinensis, var. conomon and var. makuwa, and the latter is further subdivided into eleven group varieties, such as var. inodorus, var. flexuosus and var. catalupensis (Brickell et al., 2009; Hammer and Gladis, 2014). Another classification method was also reported in other studies. Using the combination of independent fruit characteristics, for example, fruit shape and size, skin colour, flesh colour, sex expression, seed size, fruit development and conservation, melons could be classified into 19 horticultural Groups (some of which are divided into sub-groups), such as Group agrestis, kachri, chito, tibish, acidulus, momordica, conomon, makuwa, chinensis, and sub-group ogon, nashi-uri, and yuki (Pitrat, 2008, 2016). Components that determine melon quality, such as fruit skin color, flesh color, sugar content, fruit weight, and shape, change significantly in different developmental stages. Bitterness is one trait that is unacceptable to customers, but very precious to researchers, and is due to cucurbitacins mainly produced in Cucurbitaceae plants, such as the cucumber (Cucumis sativus L.) (Shang et al., 2014), melon (Zhou et al., 2016), watermelon (Citrullus lanatus var. lanatus) (Davidovich-Rikanati et al., 2015), and pumpkin (Cucurbita pepo L. and C. maxima L.) (Shibuya et al., 2004). Watermelon bitterness is due to the accumulation of cucurbitacin E (CuE) and B (CuB) (Davidovich-Rikanati et al., 2015). The accumulation of cucurbitacin C (CuC) and CuB could lead to cucumbers and melons having a bitter taste, respectively (Shang et al., 2014; Zhou et al., 2016). According to previous studies, CuB was the major bitter compound isolated from melons (Lester, 1997; Zhou et al., 2016). Cucurbitacins are bitter and toxic to most organisms, and they are recognized as toxins in plant defense responses against insects and herbivores (Balkema-Boomstra et al., 2003; Tallamy et al., 1997). Cucurbitacins also have considerable pharmaceutical value. They have been used in the form of traditional herbal medicine, such as using the stem of bitter melon fruit as a traditional hepatoprotective medicine in China. Topical application of CuB resulted in significant reduction of epidermal hyperplasia and inflammatory cytokines, and ameliorated psoriatic symptoms (Li et al., 2015). Natural cucurbitacins and their derivatives have been recognized as promising antitumor compounds for several types of cancer, including Non-small cell lung cancer (Marostica et al., 2017), Osteosarcoma (Zhang et al., 2017) and gastric cancer (Liu et al., 2017).

Cucurbitacins are a group of highly oxygenated tetracyclic triterpenoids, which are synthesized from 2,3-oxidosqualene through the mevalonic acid pathway (Thimmappa et al., 2014). Cucurbitadienol, one intermediate product of this pathway, which is catalyzed by Cucurbitadienol synthase, is considered the basic skeleton of cucurbitacins in the plant family Cucurbitaceae. Cucurbitacins synthesis starts with the cyclization of 2,3-oxidosqualene to cucurbitadienol, which is primarily determined by oxidosqualene cyclases (OSCs). Indeed, several sequences encoding for OSCs have been cloned and characterized in many plants (Andre et al., 2016; Calegario et al., 2016; Davidovich-Rikanati et al., 2015; Dhar et al., 2014; Pensec et al., 2016; Zheng et al., 2015). Three OSC cDNAs (CPX, CPQ and CPR) were isolated from pumpkin seedlings, and CPQ encoded cucurbitadienol synthase, which was the first committed enzyme for cucurbitacins biosynthesis in plants (Shibuya et al., 2004). Bryonolic acid, a friedooleanane-type pentacyclic triterpene from luffa (Luffa cylindrical), exhibited antiallergic activity against cutaneous anaphylaxis and contact dermatitis (Tabata et al., 1993). Hayashi et al. (2001) cloned a gene, LcIMS1, from luffa, which encoded the bryonia alkyd synthase belonging to OSC. Three OSC genes (CcCDS1, CcCDS2 and ClCDS1) were cloned from watermelons, of which only CcCDS2 possessed cucurbitadienol synthase activity (Davidovich-Rikanati et al., 2015). A bitterness gene CsBi, which encoded a cucurbitadienol synthase, was identified in cucumbers (Shang et al., 2014). Zhou et al. (2016) did a further study on the bitterness biosynthetic genes ClBi and CmBi in watermelon and melon, respectively, and summarized the metabolic synthesis process of cucurbitacins in Cucurbitaceae plants.

Gene clusters that play important functions in the biosynthesis of secondary metabolites are common in plants. For example, the DIBOA biosynthesis gene cluster in maize (Frey et al., 1997), triterpene synthesis gene cluster (Field and Osbourn, 2008) and marneral synthesis and modification gene cluster (Field et al., 2011) in Arabidopsis thaliana. The clusters co-regulate a set of genes controlling successive steps in a biosynthetic or developmental pathway. The gene clusters were also found in the cucurbitacins biosynthesis of cucumbers, melons and watermelons, respectively (Shang et al., 2014; Zhou et al., 2016). In melons, nine CuB biosynthetic genes, including an OSC (CmBi), six cytochromes P450 (CYPs, including Cm160, Cm170, Cm180, Cm710, Cm890, and Cm490), an acyltransferase (CmACT), and a fruit-specific regulator (CmBt), are co-expressed in the fruit of a wild ancestor, six of which are clustered (CmBi, four CYPs and CmACT) on chromosome 11 (Zhou et al., 2016). According to the previous study, CuB biosynthesis starts from 2,3-oxidosqualene to generate cucurbitadienol, which is catalyzed to 11-carbonyl-20β-hydroxycucurbitadienol and 11-carbonyl-2β, 20β-dihydroxycucurbitadienol. Then, CmACT acetylates cucurbitacin D into CuB in the melon (Fig. 3; Zhou et al., 2016).

Although the expression patterns and functions of some important genes involved in CuB biosynthesis have been identified in the melon, the fruit bitterness and expression patterns of those genes was unknown during fruit development. In this study, we found several lines of var. chinensis had a bitter taste at the early developmental stage, but did not have any bitter taste at the mature stage. Therefore, we examined the accumulation of CuB and expression patterns of CuB biosynthesis-related genes in the fruit development process, and identified genes that played an important role in the change in bitterness. This is the first report on the change in bitterness in the melon fruit development process. This study could provide valuable information to understand bitterness in melons and other Cucurbitaceae plants during the fruit development and ripening process.

Materials and Methods

Plant materials

A total of 19 melon inbred lines, including 14 lines of C. melo var. chinensis, two var. inodorus and three var. conomon (Table 1), provided by Tianjin Derit Seeds Co., Ltd., were used in this study. Seeds were soaked and germinated in an incubator with 25°C and a 16 h light/8 h dark cycle. At the 3-leaf stage, the seedlings were transplanted into a greenhouse at a temperature not higher than 33°C and under natural light conditions. Irrigation and pest control were carried out according to standard procedures. Two or three fruit per plant at maturity were kept. Fruit bitterness evaluation of these materials was carried out in autumn 2016 and spring 2017, respectively, and four important traits, including skin color, flesh color, fruit weight and fruit shape index (the ratio of fruit length to diameter) for these plants were assessed in spring 2017. A randomized block design, consisting of three replications with five plants per plot/replication for each inbred line, was adopted.

Table 1

Analysis of four traits and bitterness of the 19 inbred melon lines.

Elite parental lines that usually possess a range of excellent traits and high combining ability are important materials for hybrid breeding. Nb46 and Nb320, two parental lines with desirable traits, have been used as cultivars for breeding ‘Boyang’ lines, which are particularly excellent varieties in China nowadays.

Fruit bitterness and phenotype evaluation

The fruits were harvested at about 10 days (stage I), 20 days (stage II) and 35 days (stage III, maturation stage) after pollination, respectively, and the flesh bitterness was evaluated by three volunteers using the panel tasting method described in a previous study (Andeweg and Bruyn, 1959) in autumn 2016 and spring 2017. The tasters had to reset their taste buds by gargling with water after they tasted the bitterness of fruit. We rated the bitterness severity using a scale of 0–3, where 0 = no bitterness, 1 = light bitterness, 2 = moderate bitterness, and 3 = severe bitterness. Fruits with scores of 1 to 3 were categorized as bitterness; while those with a score of 0 were categorized as no-bitterness.

Observations and measurements of the four important traits (Table 1) and flesh soluble sugar content (Table S1), were performed for these materials following standards described in “Descriptors and data standards for melon” at the maturation stage (stage III) in spring 2017. The sugar content was measured with a portable refractometer PR101 (Atago, Japan).

Average values for bitterness and each trait for each line were calculated from three randomly selected fruits in each replication at stage I, II or III. The data were statistically analyzed using the Tukey-Kramer test following an analysis of variance, with a correlation coefficient of P < 0.05. SPSS 20.0 software (IBM, USA) was used for statistical analysis between the bitterness severity at stage I and the soluble sugar content at stage III.

High performance liquid chromatography (HPLC) analysis of CuB

To investigate a possible relationship between fruit bitterness and CuB accumulation during melon fruit development, we analyzed the CuB content of fruits in different developmental stages using HPLC analysis (Fig. 2). Flesh samples of Nb46 and Nb320 were frozen in liquid N2 and ground with a mortar and pestle. Then, the samples (0.5 g) were added to 2 mL methanol and homogenized for 15 min, followed by centrifugation at 10,000 × g for 10 min at 4°C (Shang et al., 2014). The solution was filtered through a 0.22 μm membrane and then analyzed on an HPLC system (Model2695; Waters, USA) equipped with a C18 column (5 μm, 150 × 4.6 mm) and eluted with 51% acetonitrile at 1 mL/min under a wavelength of 228 nm. The identification of CuB from samples was performed by retention time and the peak area according to a CuB reference compound (Shanghai Shifeng Biological Technology Co., LTD, China).

Quantitative RT-PCR analyses

Total RNA was isolated using a TransZolTM Plant RNA kit (Beijing TransGen Biotech Co., Ltd, China), and the first-strand cDNA was synthesized using a TranScript First-Strand cDNA Synthesis Kit (Beijing TransGen Biotech Co., Ltd, China). Then, the cDNA was used as a qRT-PCR template and PCRs were performed on the ABI 7500, according to the method described by Zhou et al. (2016). The expression patterns of these CuB biosynthetic genes were assayed and compared between bitter and non-bitter melon materials (Nb46 and Nb320) in different developmental stages. The primers used in this study are described in Table S2.

Phylogenetic analysis and three-dimensional structure prediction

OSC protein sequences of plants, humans and bacteria were retrieved from the National Center for Biotechnology Information (NCBI) or Phytozome (www.Phytozome.net). Based on a neighbor-joining method, sequence alignments and phylogenetic analyses were carried out with the Clustal X program. A phylogenetic tree was created with the Molecular Evolutionary Genetics Analysis (MEGA) 5.0 program, and the number of bootstrap replications was 1000.

Results

Traits and bitterness evaluation

Nineteen lines differed greatly in terms of morphological traits, including skin color, flesh color, fruit weight and shape index (ratio of the fruit length to diameter) (Table 1). Nb46 had a globular fruit with striped skin and orange flesh, while Nb320 had an oval oblong fruit with white skin and white flesh (Fig. 1). The weights of Nb46 and Nb320 were 472 g and 682 g respectively (Table 1). The data showed that fruits of nine lines, including four lines of var. chinensis, two inodorus and three conomon, were non-bitter during whole period of fruit development. Bitter fruits were found in 10 other lines (Table 1). Interestingly, in 10 lines of var. chinensis, including Nbs and LPys, Lvpicui, we found the bitterness severity could change during fruit development. At the early developmental stage (stage I), the fruit bitterness severity ratings of nine lines were severe (4 lines) or moderate (5 lines), decreasing at stage II, and disappearing at stage III. Lvpicui had light bitterness at stage I, but became non-bitter at stage II and III. TY-60 had severe, moderate and light bitterness at stage I, II, and III respectively. Bitterness in Nb46 fruit was severe at stage I, and gradually decreased at stage II and III, but Nb320 was non-bitter during the whole period of fruit development (Table 1).

Fig. 1

Phenotype of Nb46 and Nb320 at different periods of fruit development. (A, B, and C) represent a fruit image of Nb46 at about 10 days (stage I), 20 days (stage II) and 35 days (stage III) after pollination, respectively. (D, E, and F) represent a fruit image of Nb320 at about 10 days (stage I), 20 days (stage II), and 35 days (stage III) after pollination, respectively. Scale bars=5 cm.

CuB content in bitter and non-bitter fruits during melon development

The CuB content of fruits in different developmental stages was analyzed using HPLC analysis. Nb46, a bitter line, accumulated CuB at the first and second stages, but did not accumulate CuB at stage III (Fig. 2A, C). This coincided with the results from the tasting method for Nb46, in which bitterness was found at stage I and II, but not at stage III (Table 1). In Nb320, a non-bitter line, no CuB was detected at stage I, II, or III (Fig. 2B, C). These results showed that there was a significant positive correlation between the data from the tasting method and the content of CuB [R = 0.993 (P ≤ 0.01)]. This implied that fruit bitterness may be related to CuB content throughout the melon fruit development and ripening process.

Fig. 2

HPLC analysis of Nb46 and Nb320 fruit extracts. (A) HPLC analysis of fruit extracts from Nb46 at stage I, II, and III, respectively. (B) HPLC analysis of fruit extracts from Nb320 at stage I, II, and III, respectively. (C) Relative CuB contents in fruit of Nb46 and Nb320 at stage I, II, and III, respectively. mAU, milli-arbitary units. I, II, and III represent about 10 days (stage I), 20 days (stage II), and 35 days (stage III) after pollination, respectively.

Furthermore, the contents of total soluble sugar were measured at stage III of these melon materials. We found the sugar contents were different among various samples, ranging from 7.4% to 14.3% (Table S1). A positive correlation [R = 0.890 (P ≤ 0.01)] was found between bitterness severity at stage I and sugar content at stage III among bitter materials.

The expression patterns of bitterness-related genes during melon fruit development

Six (Cm160, Cm170, Cm180, CmBi, CmACT, and Cm710) CuB biosynthesis related genes were clustered on chromosome 11, while Cm890, Cm490, and CmBt were located on chromosomes 12, 4, and 9, respectively, in the melon genome (Fig. 3A). We found the expression patterns of both CmBi and CmBt in fruits were significantly different between Nb46 and Nb320 (Fig. 3B, C). These two genes showed much higher expression levels in Nb46 compared to that in Nb320 at stage I and II. In Nb46, the transcription levels of CmBi and CmBt gradually decreased during fruit growth and maturation. The gene expression levels were the highest at stage I, but decreased at stage II. At stage III, the expression levels of CmBi and CmBt fell to 0.1% and 8.9%, respectively, (Fig. 3B, C). In Nb320, mRNA for both genes accumulated at very low levels and showed a stable expression pattern during fruit growth and maturation (Fig. 3B, C). The data showed the expression patterns of these two genes were consistent with the change in fruit bitterness and the accumulation of CuB.

Fig. 3

Expression profiles of CuB biosynthesis-related genes in melon. (A) The model of CuB biosynthesis in the melon. A four-step enzymatic reaction is understood. Firstly, 2,3-oxidized squalene cyclase encoded by CmBi catalyzes 2,3-oxidosqualene to cucurbitadienol. Secondly, cucurbitadienol is catalyzed to 11-carbonylcucurbitadienol and 11-carbonyl-20β-hydroxycucurbitadienol by Cm890. Then, 11-carbonyl-20β-hydroxycucurbitadienol is catalyzed to 11-carbonyl-2β, 20β-dihydroxycucurbitadienol by Cm180. In the final step, CmACT acetylates cucurbitacin D into CuB. Chr, chromosome. (B and C) The expression patterns of CmBi (B) and CmBt (C) in fruits of Nb46 and Nb320 at stage I, II and III during development. (D) The expression patterns of other CuB biosynthetic-related genes (CmACT, Cm160, Cm170, Cm180, Cm490, Cm710, and Cm890) in fruits of Nb46 and Nb320 at stage I, II and III during development. Expression levels were determined by qRT-PCR. Relative gene expression levels are shown by identical scales (means ± standard error of the mean (s.e.m), n = 3 biological replicates).

The transcription of seven other CuB biosynthesis-related genes (Cm160, Cm170, Cm180, Cm710, Cm890, Cm490, and CmACT) was evaluated and compared between Nb46 and Nb320 during fruit development. Interestingly, we found that the expression patterns of these genes showed almost the opposite expression patterns between Nb46 and Nb320 (Fig. 3D). Transcript levels of these seven genes were much higher in the bitter melon Nb46, but steadily decreased during the fruit development and ripening process. These genes showed much lower and stable expression patterns in the non-bitter melon Nb320. These results were consistent with not only the CuB content, but also the change in bitterness during fruit development, suggesting that these genes may be involved in CuB biosynthesis in melons during fruit development.

CmBi belongs to a novel subgroup of the OSC family

2,3-oxidized squalene cyclise, a member of the OSC family, encoded by CmBi, catalyzes the cyclizing of 2,3-oxidosqualene into cucurbitadienol, which is the first committed step of CuB biosynthesis in melons. Based on the difference in the synthesized molecules, the members of the OSC family are mainly divided into several subgroups such as β-amyrin synthase (β-AS) (Kushiro et al., 1998), dammarenediol synthase (DS) (Huang et al., 2015), cycloartenol synthase (CAS) (Basyuni et al., 2007), lupeol synthase (LUS) (Husselsteinmuller et al., 2001) lanosterol synthase (LS) (Husselsteinmuller et al., 2001), and α-amyrin synthase (α-AS). Using amino acid sequences of CmBi protein with other OSC members from plants, humans and bacteria, a phylogenetic analysis was performed based on the neighbour joining method (Fig. 4). The data showed that CmBi with CsBi, ClBi, and CPQ were clustered in the same sub-clade, a novel subgroup named Cucurbitadienol synthase. Furthermore, we found other members of the OSC family in melons: MELO3C002943, MELO3C002945, MELO3C024271, MELO3C004329, MELO3C004327, and MELO3C024270 were not clustered in the same sub-clade with CmBi. The former five members belonged to the β-AS subgroup, and the last one belonged to the CAS subgroup (Fig. 4). A comparison of deduced amino acid sequences of CmBi with other OSCs, including CsBi of cucumbers, ClBi of watermelons, CPQ of pumpkins, WsOSCs of Withania somnifera and LAS of humans, showed that this protein had DCTAE (501–505) as a conserved motif that was necessary for OSCs activity (Fig. S1). OSCs share a unique sequence fingerprint, namely a QW repeat, with the consensus Q-X25-G-X-W, which is unique to this enzyme class. We found this conserved motif occurred three times (126–132, 175–181, and 617–623) in CmBi, and four and five times in WsOSCs and LAS, respectively (Fig. S1).

Fig. 4

Phylogenetic tree of OSCs. Phylogenetic analysis using MEGA5.0 software based on the neighbour-joining method (gene accessions provided in Table S3). Nodes (>20% statistical support) are labelled with the percentage of bootstrap iterations. OSCs grouped into several subgroups, such as beta-Amyrin Synthase (β-AS), Dammarenediol Synthase (DS), Cycloartenol Synthase (CAS), Lupeol Synthase (LUS), and Lanosterol Synthase (LS) and cucurbitadienol synthase. CmBi clustered into the subgroups of cucurbitadienol synthase. A total of 68 protein sequences were used for analysis. Two OSCs, SHC from a bacterium (Alicyclobacillus acidocaldarius) and human LAS, the crystal structures of which have been reported, were also used in the phylogenetic analysis.

Discussion

In this study, four economic traits and soluble sugar content were measured and observed for different melon lines at the fruit maturation stage. We conducted bitterness analysis using a panel tasting method in 19 melon inbred lines. The bitterness decreased gradually during fruit development in several materials of var. chinensis, such as LPy and TY lines, the fruit of which had severe or moderate bitterness at stage I, but light bitterness or no-bitterness at stage II and III. For example, the fruit of Nb46 had severe, moderate and non-bitterness at stage I, II, and III, respectively. These data showed that the severity of bitterness could change at different periods of fruit development. To the best of our knowledge, these findings have not been reported in previous studies. However, we also found that some melons, such as Nb320, had non-bitter fruits throughout the growth and maturation process.

Cucurbitacins, as the main compounds for bitterness in Cucuibitaceae plants, are arbitrarily divided into 12 categories (Mei et al., 2016; Thimmappa et al., 2014). CuB, one important category of cucurbitacins, is the major bitter compound isolated from melons (Lester, 1997) and it is present in many cucurbit plants (Davidovich-Rikanati et al., 2015; Shang et al., 2014). The qualitative and quantitative detection of cucrbitacins using HPLC or liquid chromatography-mass spectrometry (LC-MS) analysis have been reported in previous studies (Feng et al., 2007; Zhao et al., 2016). In order to investigate a possible relationship between fruit bitterness and CuB accumulation during fruit development, we analysed the CuB contents in Nb46 and Nb320 using HPLC equipment. Our findings clearly showed that Nb46 accumulated CuB at stage I and II, but no CuB was detected at stage III. In Nb320, the CuB content remained at a non-detectable level during throughout the fruit development period. These data were coincident with a previous report (Zhou et al., 2016), which indicated that the bitterness perceived in melon fruits was related to the accumulation of CuB.

Based on the hypothesis that bitterness is correlated to the presence of CuB in melon fruits, we then examined the expression patterns of nine genes (one OSC gene CmBi; six CYP genes Cm160, Cm170, Cm180, Cm710, Cm890, and Cm490; a cyltransferase gene CmACT; and a transcription regulator CmBt) involved in CuB biosynthesis during fruit development. CmBt, controlled the bitterness of fruit by regulating the expression levels of genes involved in CuB biosynthesis (Zhou et al., 2016), encodes a transcription factor that belongs to the basic helix-loop-helix (bHLH) family, which constitutes one of the largest families of plant transcription factors (Heim et al., 2003). CmBi could cyclize 2,3-oxidosqualene to generate cucurbitadienol in yeast (Zhou et al., 2016). Interestingly, the data showed that expression levels of these genes were different between bitter and non-bitter fruits. In Nb46, the expression levels of most genes were very high at stage I, and gradually decreased at stage II and III. This result was not reported in previous studies. The expression profiles of these genes remained at quite a low level and changed little throughout the fruit development period in Nb320. This finding concurred with previous reports (Zhou et al., 2016). The different expression patterns of these genes that were consistent with not only the CuB content, but also the severity of bitterness in bitter and non-bitter fruits, suggested these genes were involved in CuB biosynthesis during melon fruit development. In melons, the expression patterns of other two CuB biosynthetic genes, Cm510, and CmBr, were not measured in this study, because these two genes had almost identical expression patterns, with high expression in roots and quite low expression in fruit of both wild and cultivated melon lines (Zhou et al., 2016). The association between apple russetting and specific skin triterpene composition at maturity has been observed in a previous study (Andre et al., 2013) and the gene expression patterns of two OSCs indicate that OSCs are key genes in apple fruit triterpene biosynthesis (Andre et al., 2016). Triterpene saponins, known as ginsenosides, are the major pharmacological compounds in P. ginseng. Ginsenosides changed in different development stages and the expression patterns of genes related to ginsenoside biosynthesis have a similar trend (Kim et al., 2014).

Phylogenetic clustering grouped CmBi in the subgroup of cucurbitadienol synthase. This coincided with a previous study (Zhou et al., 2016). Although a QW repeat occurred five times in some 2,3-oxidosequence cyslases and up to eight times in squalene cyclises (Poralla et al., 1994; Tippelt et al., 1998), our date showed that this repeat only appeared three times in CmBi (Fig. S1). Using the crystal structure of human LAS (PDB ID 1w6k) as a template, a three-dimensional protein model of CmBi showed similar architecture to LAS (Data not shown), this indicated CmBi may have a similar function to other OSCs in a previous study (Dhar et al., 2014).

Furthermore, the CuB biosynthetic genes had quite low transcription levels in Nb320 or at stage III in Nb46, suggesting CuB biosynthesis did not happen in the fruits of Nb320 or Nb46 at stage III, which was shown by the fact that no CuB was detected in these samples. In a previous study, the cucurbitacin biosynthesis-related genes showed very low express in the fruit in cultivated lines of cucumbers, melons and watermelons, consistent with the cucurbitacin content in the different plant fruits (Zhou et al., 2016). Some fruits, such as apples and watermelons, accumulate high levels of organic acids in the early development stages. However, in fruit at later development stages, the organic acid concentrations steadily decline (Gao et al., 2018; Zhang et al., 2010). The accumulation and metabolism of organic acid in the fruit may be correlated with the TCA cycle and glycolysis (Sweetlove et al., 2010). Bitterness biosynthesis and metabolism are complex biological processes. A set of genes related to bitterness biosynthesis and regulation were detected by mapping, comparative analyses of plant genomes and a recombinant yeast system (Davidovich-Rikanati et al., 2015; Zhou et al., 2016). Nevertheless, to date, to the best of our knowledge, there have been no reports on the degradation of bitterness during fruit development. 23,24-dihydrocucurbitacin C, a novel compound, was clearly identified and regarded as the next metabolite of CuC in cucumber leaves (Qing et al., 2014). However, why bitter compounds or CuB in stage I or stage II of bitter fruit disappeared at stage III, or were translocated into other organs (such as leaves or roots) or transformed into other compounds (such a sugars) at stage III, has not been elucidated for melons. The mechanism by which bitterness components degrade or transport is still not understood, and requires further research. Quantitative analysis of the triterpenoid content in neem (Azadirachta indica) indicated that there was tissue-specific variation in terms of abundance (Pandreka et al., 2015). Oleanolic acid (OA) and ursolic acid (UA), the main triterpene acids in persimmon fruit, changed during fruit development (Zhou et al., 2012). Limonin and nomilin, as the predominant limonoids in a group of chemically related triterpene derivatives in the Rutaceae and Meliaceae plant families, increased at first and then gradually decreased in some citrus species during growth and maturation when the limomoid UDP-glucosyltransferase regulated the conversion of limonoid to glucoside (Kita et al., 2000). CuB accumulation in fruit is a negative trait for general consumers, but soluble sugar content is an important component of melon fruit quality, and is a primary target for melon improvement (Argyris et al., 2017; Burger et al., 2006). Soluble sugars in fruits include sucrose, fructose and glucose (Zhu et al., 2017). We measured the soluble sugar contents of these compounds at stage III, and the results showed that the sugar contents ranged from 7.4% to 14.3% (Table S1). We found there was a positive correlation [R = 0.890 (P ≤ 0.01)] between bitterness severity at stage I and sugar contents at stage III among bitter fruits. This result suggested degradation of bitter compounds may be related to the accumulation of soluble sugar in melon during fruit development. Previous studies have established biosynthesis and regulation of bitterness in the plant family cucurbitaceae that includes cucumbers, melons and watermelons (Shang et al., 2014; Zhou et al., 2016), and the activities of cucurbitadienol synthase using recombinant yeast (Davidovich-Rikanati et al., 2015; Shibuya et al., 2004). However, there is relatively little reported research on the degradation of bitter compounds in fruit. Soluble sugar accumulation in fruits is a complex quantitative trait due to the high number of genes that must be coordinately modulated to produce different compounds during fruit developmental stages (Gao et al., 2018). To date, how the bitterness and soluble sugar interconvert each other remains unclear. Additional research is needed to further understand the transformation of bitter compounds during fruit development.

In summary, bitterness of fruit was measured for 19 inbred melon lines at different developmental stages. The results showed that bitterness severity was different not only among various lines, but also during different development periods. Fruit bitterness was different in some materials of var. chinensis during fruit development. CuB accumulation and expression patterns of CuB biosynthesis related genes were correlated with bitterness changes in melons. here, we report an important first insight into bitterness during fruit development. These novel findings will help us understand bitterness and cucurbitacins biosynthesis in melons and other cucurbits.

Acknowledgements

We gratefully acknowledge support from Tianjin Derit Seeds Co. Ltd.

Literature Cited
 
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