The Horticulture Journal 88 (2): 253–262. 2019. e Japanese Society for doi: 10.2503/hortj.UTD-004 JSHS Horticultural Science http://www.jshs.jp/

Change in Bitterness, Accumulation of B and Expression Patterns of CuB Biosynthesis-related Genes in During Fruit Development

Deping Hua1*, Jinyu Fu1, Li Liu2, Xuhui Yang2, Qiaoling Zhang2 and Meiting Xie1

1College of Life Sciences, Tianjin University, Tianjin, 300072, China 2School of Environmental Science and Engineering, Tianjin University, Tianjin, 300072, China

Bitterness, caused by , is present in some melon fruit. Although bitter compound biosynthesis and regulation in 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 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.

Key Words: bitterness, Cucurbitacin B (CuB), CuB biosynthesis-related genes, fruit development, melon (Cucumis melo L.).

the ovary and young fruit, vegetative morphological Introduction characteristics and fruit variation, are subdi- The melon (Cucumis melo L.), a highly diversified vided into two subspecies, namely, subsp. agrestis hav- species of the Cucurbitaceae family, is an economically ing ovaries with short, appressed hairs, and subsp. melo important fruit crop cultivated around the world. During having pilose or lanate ovaries with long, spreading soft the past decade, the average production of melon was hairs (Kirkbride, 1993). The former is further subdi- over 29 million tons per year (FAOSTAT 2017, http:// vided into five group varieties, such as var. chinensis, faostat.fao.org/). It is cultivated in mainly temperate var. conomon and var. makuwa, and the latter is further and tropical countries, with the major producer regions subdivided into eleven group varieties, such as var. located in Asia and China leading the world (FAOSTAT inodorus, var. flexuosus and var. catalupensis (Brickell 2017, http://faostat.fao.org/). Melon has many variant et al., 2009; Hammer and Gladis, 2014). Another classi- types. On the basis of length and distribution of hairs on fication method was also reported in other studies. Using the combination of independent fruit characteris- Received; June 13, 2018. Accepted; November 19, 2018. tics, for example, fruit shape and size, skin colour, flesh First Published Online in J-STAGE on January 19, 2019. colour, sex expression, seed size, fruit development and This study was supported by the National Natural Science Foundation conservation, melons could be classified into 19 horti- of China (31400236), Tianjin Research Program of Application cultural Groups (some of which are divided into sub- Foundation and Advanced Technology (14JCQNJC9700) and the Elite Scholar Program of Tianjin University (2016XR-0007). groups), such as Group agrestis, kachri, chito, tibish, * Corresponding author (E-mail: [email protected]). acidulus, momordica, conomon, makuwa, chinensis,

© 2019 The Japanese Society for Horticultural Science (JSHS), All rights reserved. 254 D. Hua, J. Fu, L. Liu, X. Yang, Q. Zhang and M. Xie and sub-group ogon, nashi-uri, and yuki (Pitrat, 2008, against cutaneous anaphylaxis and contact dermatitis 2016). Components that determine melon quality, such (Tabata et al., 1993). Hayashi et al. (2001) cloned a as fruit skin color, flesh color, sugar content, fruit gene, LcIMS1, from luffa, which encoded the bryonia weight, and shape, change significantly in different de- alkyd synthase belonging to OSC. Three OSC genes velopmental stages. Bitterness is one trait that is unac- (CcCDS1, CcCDS2 and ClCDS1) were cloned from ceptable to customers, but very precious to researchers, , of which only CcCDS2 possessed cucur- and is due to cucurbitacins mainly produced in bitadienol synthase activity (Davidovich-Rikanati et al., Cucurbitaceae plants, such as the (Cucumis 2015). A bitterness gene CsBi, which encoded a cucur- sativus L.) (Shang et al., 2014), melon (Zhou et al., bitadienol synthase, was identified in 2016), (Citrullus lanatus var. lanatus) (Shang et al., 2014). Zhou et al. (2016) did a further (Davidovich-Rikanati et al., 2015), and study on the bitterness biosynthetic genes ClBi and ( pepo L. and C. maxima L.) (Shibuya et al., CmBi in watermelon and melon, respectively, and sum- 2004). Watermelon bitterness is due to the accumula- marized the metabolic synthesis process of cucurbita- tion of cucurbitacin E (CuE) and B (CuB) (Davidovich- cins in Cucurbitaceae plants. Rikanati et al., 2015). The accumulation of cucurbitacin Gene clusters that play important functions in the C (CuC) and CuB could lead to cucumbers and melons biosynthesis of secondary metabolites are common in having a bitter taste, respectively (Shang et al., 2014; plants. For example, the DIBOA biosynthesis gene Zhou et al., 2016). According to previous studies, CuB cluster in maize (Frey et al., 1997), triterpene synthesis was the major bitter compound isolated from melons gene cluster (Field and Osbourn, 2008) and marneral (Lester, 1997; Zhou et al., 2016). Cucurbitacins are bit- synthesis and modification gene cluster (Field et al., ter and toxic to most organisms, and they are recog- 2011) in Arabidopsis thaliana. The clusters co-regulate nized as toxins in plant defense responses against a set of genes controlling successive steps in a biosyn- insects and herbivores (Balkema-Boomstra et al., 2003; thetic or developmental pathway. The gene clusters Tallamy et al., 1997). Cucurbitacins also have consider- were also found in the cucurbitacins biosynthesis of cu- able pharmaceutical value. They have been used in the cumbers, melons and watermelons, respectively (Shang form of traditional herbal medicine, such as using the et al., 2014; Zhou et al., 2016). In melons, nine CuB stem of bitter melon fruit as a traditional hepatoprotec- biosynthetic genes, including an OSC (CmBi), six cyto- tive medicine in China. Topical application of CuB re- chromes P450 (CYPs, including Cm160, Cm170, sulted in significant reduction of epidermal hyperplasia Cm180, Cm710, Cm890, and Cm490), an acyltransfer- and inflammatory , and ameliorated psoriatic ase (CmACT), and a fruit-specific regulator (CmBt), are symptoms (Li et al., 2015). Natural cucurbitacins and co-expressed in the fruit of a wild ancestor, six of which their derivatives have been recognized as promising are clustered (CmBi, four CYPs and CmACT) on chro- antitumor compounds for several types of cancer, in- mosome 11 (Zhou et al., 2016). According to the previ- cluding Non-small cell lung cancer (Marostica et al., ous study, CuB biosynthesis starts from 2,3- 2017), Osteosarcoma (Zhang et al., 2017) and gastric oxidosqualene to generate cucurbitadienol, which is cancer (Liu et al., 2017). catalyzed to 11-carbonyl-20β-hydroxycucurbitadienol Cucurbitacins are a group of highly oxygenated tetra- and 11-carbonyl-2β, 20β-dihydroxycucurbitadienol. cyclic triterpenoids, which are synthesized from 2,3- Then, CmACT acetylates cucurbitacin D into CuB in oxidosqualene through the mevalonic acid pathway the melon (Fig. 3; Zhou et al., 2016). (Thimmappa et al., 2014). Cucurbitadienol, one inter- Although the expression patterns and functions of mediate product of this pathway, which is catalyzed by some important genes involved in CuB biosynthesis Cucurbitadienol synthase, is considered the basic skele- have been identified in the melon, the fruit bitterness ton of cucurbitacins in the plant family Cucurbitaceae. and expression patterns of those genes was unknown Cucurbitacins synthesis starts with the cyclization of during fruit development. In this study, we found sever- 2,3-oxidosqualene to cucurbitadienol, which is primari- al lines of var. chinensis had a bitter taste at the early ly determined by oxidosqualene cyclases (OSCs). In- developmental stage, but did not have any bitter taste at deed, several sequences encoding for OSCs have been the mature stage. Therefore, we examined the accumu- cloned and characterized in many plants (Andre et al., lation of CuB and expression patterns of CuB 2016; Calegario et al., 2016; Davidovich-Rikanati et al., biosynthesis-related genes in the fruit development 2015; Dhar et al., 2014; Pensec et al., 2016; Zheng process, and identified genes that played an important et al., 2015). Three OSC cDNAs (CPX, CPQ and CPR) role in the change in bitterness. This is the first report were isolated from pumpkin seedlings, and CPQ en- on the change in bitterness in the melon fruit develop- coded cucurbitadienol synthase, which was the first ment process. This study could provide valuable infor- committed enzyme for cucurbitacins biosynthesis in mation to understand bitterness in melons and other plants (Shibuya et al., 2004). Bryonolic acid, a Cucurbitaceae plants during the fruit development and friedooleanane-type pentacyclic triterpene from luffa ripening process. (Luffa cylindrical), exhibited antiallergic activity Hort. J. 88 (2): 253–262. 2019. 255

ularly excellent varieties in China nowadays. Materials and Methods Plant materials Fruit bitterness and phenotype evaluation A total of 19 melon inbred lines, including 14 lines The fruits were harvested at about 10 days (stage I), of C. melo var. chinensis, two var. inodorus and three 20 days (stage II) and 35 days (stage III, maturation var. conomon (Table 1), provided by Tianjin Derit stage) after , respectively, and the flesh bit- Seeds Co., Ltd., were used in this study. Seeds were terness was evaluated by three volunteers using the soaked and germinated in an incubator with 25°C and a panel tasting method described in a previous study 16 h /8 h dark cycle. At the 3-leaf stage, the seed- (Andeweg and Bruyn, 1959) in autumn 2016 and spring lings were transplanted into a greenhouse at a tempera- 2017. The tasters had to reset their taste buds by gar- ture not higher than 33°C and under natural light gling with water after they tasted the bitterness of fruit. conditions. Irrigation and pest control were carried out We rated the bitterness severity using a scale of 0–3, according to standard procedures. Two or three fruit per where 0 = no bitterness, 1 = light bitterness, 2 = moder- plant at maturity were kept. Fruit bitterness evaluation ate bitterness, and 3 = severe bitterness. Fruits with of these materials was carried out in autumn 2016 and scores of 1 to 3 were categorized as bitterness; while spring 2017, respectively, and four important traits, in- those with a score of 0 were categorized as no- cluding skin color, flesh color, fruit weight and fruit bitterness. shape index (the ratio of fruit length to diameter) for Observations and measurements of the four impor- these plants were assessed in spring 2017. A random- tant traits (Table 1) and flesh soluble sugar content ized block design, consisting of three replications with (Table S1), were performed for these materials follow- five plants per plot/replication for each inbred line, was ing standards described in “Descriptors and data stan- adopted. dards for melon” at the maturation stage (stage III) in Elite parental lines that usually possess a range of ex- spring 2017. The sugar content was measured with a cellent traits and high combining ability are important portable refractometer PR101 (Atago, Japan). materials for hybrid breeding. Nb46 and Nb320, two Average values for bitterness and each trait for each parental lines with desirable traits, have been used as line were calculated from three randomly selected fruits cultivarsTable 1. Analysis for breeding of four traits ‘Boyang’ and bitterness lines, of whichthe 19 inbred are partic-melon lines. in each replication at stage I, II or III. The data were

Table 1. Analysis of four traits and bitterness of the 19 inbred melon lines.

Bitterness Materials Variety Skin colory Flesh color Fruit weight (g) Shape indexx Iw II III LPy72 C. melo var. chinensis green green 0.868 ± 37 bz 2.5 ± 0.1 bc 2 1 0 LPy73 C. melo var. chinensis green green 566.7 ± 32.1 def 2.6 ± 0.1 bc 2 1 0 LPy75 C. melo var. chinensis white green 0.533 ± 55 efg 2.4 ± 0.1 bcd 3 1 0 LPy95 C. melo var. chinensis white green 0.411 ± 39.9 hi 0.2 ± 0.2 d 3 2 0 LPy383 C. melo var. chinensis white green 0.455 ± 32.8 gh 0.2 ± 0.1 d 2 1 0 Lvpicui C. melo var. chinensis green white 0.315 ± 22.9 i 2.3 ± 0.1 cd 1 0 0 Nb45 C. melo var. chinensis stripe white 0.625 ± 39.9 cde 0.1 ± 0.1 e 2 1 0 Nb46 C. melo var. chinensis stripe white 472.7 ± 25.2 fgh 0.1 ± 0.1 e 3 2 0 TY-58 C. melo var. chinensis white orange 0.940 ± 10 b 1.3 ± 0.1 e 2 1 0 TY-60 C. melo var. chinensis white orange 733.3 ± 40.4 c 1.3 ± 0.1 e 3 2 1 TY-61 C. melo var. chinensis white orange 546.7 ± 55 efg 0.1 ± 0.1 e 0 0 0 Nb320 C. melo var. chinensis white white 682.7 ± 17 c 3.1 ± 0.2 b 0 0 0 Qingwowang C. melo var. chinensis green orange 656.7 ± 30.1 cd 2.8 ± 0.3 b 0 0 0 Huacaigua C. melo var. chinensis stripe orange 658.3 ± 35.3 c 2.2 ± 0.2 c 0 0 0 HZMG C. melo var. inodorus green green 906.7 ± 36.7 b 1.2 ± 0.1 e 0 0 0 HCM C. melo var. inodorus yellow white 503.3 ± 28.4 fgh 2.1 ± 0.4 cd 0 0 0 Qingcaigua C. melo var. conomon green white 1328.3 ± 43.7 a 0.5 ± 0.2 a 0 0 0 Balengcui C. melo var. conomon green green 316.7 ± 20.9 a 2.4 ± 0.2 bcd 0 0 0 Heipishaogua C. melo var. conomon black green 1043.3 ± 45.4 b 3.5 ± 0.1 b 0 0 0 z Values of fruit weight and shape index represent the mean ± SD of 19 lines. Mean values within a row followed by the same letter are not signifi- cantly different at the 5% level by the Tukey–Kramer test. y The traits of fruit skin color, flesh color, fruit weight and fruit shape were observed and measured at 35 days (stage III) after pollination. x Shape index represents the ratio of fruit length to diameter. w “I, II and III” represent fruit bitterness measured at about 10 days (stage I), 20 days (stage II) and 35 days (stage III) after pollination, respectively. “0, 1, 2, and 3” represent the bitterness severity: 0, no bitterness; 1, light bitterness; 2, moderate bitterness; and 3, severe bitterness. 256 D. Hua, J. Fu, L. Liu, X. Yang, Q. Zhang and M. Xie statistically analyzed using the Tukey-Kramer test fol- Results lowing an analysis of variance, with a correlation coef- ficient of P < 0.05. SPSS 20.0 software (IBM, USA) Traits and bitterness evaluation was used for statistical analysis between the bitterness Nineteen lines differed greatly in terms of morpho- severity at stage I and the soluble sugar content at stage logical traits, including skin color, flesh color, fruit III. weight and shape index (ratio of the fruit length to diameter) (Table 1). Nb46 had a globular fruit with High performance liquid chromatography (HPLC) striped skin and orange flesh, while Nb320 had an oval analysis of CuB oblong fruit with white skin and white flesh (Fig. 1). To investigate a possible relationship between fruit The weights of Nb46 and Nb320 were 472 g and 682 g bitterness and CuB accumulation during melon fruit respectively (Table 1). The data showed that fruits of development, we analyzed the CuB content of fruits in nine lines, including four lines of var. chinensis, two different developmental stages using HPLC analysis inodorus and three conomon, were non-bitter during (Fig. 2). Flesh samples of Nb46 and Nb320 were frozen whole period of fruit development. Bitter fruits were in liquid N2 and ground with a mortar and pestle. Then, found in 10 other lines (Table 1). Interestingly, in 10 the samples (0.5 g) were added to 2 mL and lines of var. chinensis, including Nbs and LPys, homogenized for 15 min, followed by centrifugation at Lvpicui, we found the bitterness severity could change 10,000 × g for 10 min at 4°C (Shang et al., 2014). The during fruit development. At the early developmental solution was filtered through a 0.22 μm membrane and stage (stage I), the fruit bitterness severity ratings of then analyzed on an HPLC system (Model2695; nine lines were severe (4 lines) or moderate (5 lines), Waters, USA) equipped with a C18 column (5 μm, decreasing at stage II, and disappearing at stage III. 150 × 4.6 mm) and eluted with 51% acetonitrile at Lvpicui had light bitterness at stage I, but became non- 1 mL/min under a wavelength of 228 nm. The identifi- bitter at stage II and III. TY-60 had severe, moderate cation of CuB from samples was performed by reten- and light bitterness at stage I, II, and III respectively. tion time and the peak area according to a CuB Bitterness in Nb46 fruit was severe at stage I, and grad- reference compound (Shanghai Shifeng Biological ually decreased at stage II and III, but Nb320 was non- Technology Co., LTD, China). bitter during the whole period of fruit development (Table 1). Quantitative RT-PCR analyses Total RNA was isolated using a TransZolTM Plant CuB content in bitter and non-bitter fruits during melon RNA (Beijing TransGen Biotech Co., Ltd, China), development and the first-strand cDNA was synthesized using a The CuB content of fruits in different developmental TranScript First-Strand cDNA Synthesis Kit (Beijing stages was analyzed using HPLC analysis. Nb46, a bit- TransGen Biotech Co., Ltd, China). Then, the cDNA ter line, accumulated CuB at the first and second stages, was used as a qRT-PCR template and PCRs were per- but did not accumulate CuB at stage III (Fig. 2A, C). formed on the ABI 7500, according to the method de- This coincided with the results from the tasting method scribed by Zhou et al. (2016). The expression patterns of these CuB biosynthetic genes were assayed and com- pared 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 bacte- ria 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 analy- ses were carried out with the Clustal X program. A phylogenetic tree was created with the Molecular Fig. 1. Phenotype of Nb46 and Nb320 at different periods of fruit Evolutionary Genetics Analysis (MEGA) 5.0 program, development. (A, B, and C) represent a fruit image of Nb46 at and the number of bootstrap replications was 1000. 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. Hort. J. 88 (2): 253–262. 2019. 257

pression patterns of both CmBi and CmBt in fruits were significantly different between Nb46 and Nb320 (Fig. 3B, C). These two genes showed much higher ex- pression 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 ex- pression 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 ma- turation (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. The transcription of seven other CuB biosynthesis- related genes (Cm160, Cm170, Cm180, Cm710, Cm890, Cm490, and CmACT) was evaluated and com- pared between Nb46 and Nb320 during fruit develop- ment. Interestingly, we found that the expression patterns of these genes showed almost the opposite ex- pression patterns between Nb46 and Nb320 (Fig. 3D). Transcript levels of these seven genes were much high- er in the bitter melon Nb46, but steadily decreased dur- Fig. 2. HPLC analysis of Nb46 and Nb320 fruit extracts. (A) ing the fruit development and ripening process. These HPLC analysis of fruit extracts from Nb46 at stage I, II, and III, genes showed much lower and stable expression pat- respectively. (B) HPLC analysis of fruit extracts from Nb320 at stage I, II, and III, respectively. (C) Relative CuB contents in terns in the non-bitter melon Nb320. These results were fruit of Nb46 and Nb320 at stage I, II, and III, respectively. consistent with not only the CuB content, but also the mAU, milli-arbitary units. I, II, and III represent about 10 days change in bitterness during fruit development, suggest- (stage I), 20 days (stage II), and 35 days (stage III) after polli- ing that these genes may be involved in CuB biosynthe- nation, respectively. sis in melons during fruit development.

CmBi belongs to a novel subgroup of the OSC family for Nb46, in which bitterness was found at stage I and 2,3-oxidized squalene cyclise, a member of the OSC II, but not at stage III (Table 1). In Nb320, a non-bitter family, encoded by CmBi, catalyzes the cyclizing of line, no CuB was detected at stage I, II, or III (Fig. 2B, 2,3-oxidosqualene into cucurbitadienol, which is the C). These results showed that there was a significant first committed step of CuB biosynthesis in melons. positive correlation between the data from the tasting Based on the difference in the synthesized molecules, method and the content of CuB [R = 0.993 (P ≤ 0.01)]. the members of the OSC family are mainly divided into This implied that fruit bitterness may be related to CuB several subgroups such as β-amyrin synthase (β-AS) content throughout the melon fruit development and (Kushiro et al., 1998), dammarenediol synthase (DS) ripening process. (Huang et al., 2015), cycloartenol synthase (CAS) Furthermore, the contents of total soluble sugar were (Basyuni et al., 2007), lupeol synthase (LUS) measured at stage III of these melon materials. We (Husselsteinmuller et al., 2001) lanosterol synthase found the sugar contents were different among various (LS) (Husselsteinmuller et al., 2001), and α-amyrin samples, ranging from 7.4% to 14.3% (Table S1). A synthase (α-AS). Using amino acid sequences of CmBi positive correlation [R = 0.890 (P ≤ 0.01)] was found protein with other OSC members from plants, humans between bitterness severity at stage I and sugar content and bacteria, a phylogenetic analysis was performed at stage III among bitter materials. based on the neighbour joining method (Fig. 4). The data showed that CmBi with CsBi, ClBi, and CPQ were The expression patterns of bitterness-related genes dur- clustered in the same sub-clade, a novel subgroup ing melon fruit development named Cucurbitadienol synthase. Furthermore, we Six (Cm160, Cm170, Cm180, CmBi, CmACT, and found other members of the OSC family in melons: Cm710) CuB biosynthesis related genes were clustered MELO3C002943, MELO3C002945, MELO3C024271, on chromosome 11, while Cm890, Cm490, and CmBt MELO3C004329, MELO3C004327, and were located on chromosomes 12, 4, and 9, respective- MELO3C024270 were not clustered in the same sub- ly, in the melon genome (Fig. 3A). We found the ex- clade with CmBi. The former five members belonged to 258 D. Hua, J. Fu, L. Liu, X. Yang, Q. Zhang and M. Xie

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 cu- curbitacin 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 repli- cates). the β-AS subgroup, and the last one belonged to the melon lines at the fruit maturation stage. We conducted CAS subgroup (Fig. 4). A comparison of deduced bitterness analysis using a panel tasting method in 19 amino acid sequences of CmBi with other OSCs, in- melon inbred lines. The bitterness decreased gradually cluding CsBi of cucumbers, ClBi of watermelons, CPQ during fruit development in several materials of var. of , WsOSCs of Withania somnifera and LAS chinensis, such as LPy and TY lines, the fruit of which of humans, showed that this protein had DCTAE (501– had severe or moderate bitterness at stage I, but light 505) as a conserved motif that was necessary for OSCs bitterness or no-bitterness at stage II and III. For exam- activity (Fig. S1). OSCs share a unique sequence finger- ple, the fruit of Nb46 had severe, moderate and non- print, namely a QW repeat, with the consensus Q-X2–5- bitterness at stage I, II, and III, respectively. These data G-X-W, which is unique to this enzyme class. We found showed that the severity of bitterness could change at this conserved motif occurred three times (126–132, different periods of fruit development. To the best of 175–181, and 617–623) in CmBi, and four and five our knowledge, these findings have not been reported in times in WsOSCs and LAS, respectively (Fig. S1). previous studies. However, we also found that some melons, such as Nb320, had non-bitter fruits throughout Discussion the growth and maturation process. In this study, four economic traits and soluble sugar Cucurbitacins, as the main compounds for bitterness content were measured and observed for different in Cucuibitaceae plants, are arbitrarily divided into 12 Hort. J. 88 (2): 253–262. 2019. 259

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. categories (Mei et al., 2016; Thimmappa et al., 2014). to the presence of CuB in melon fruits, we then exam- CuB, one important category of cucurbitacins, is the ined the expression patterns of nine genes (one OSC major bitter compound isolated from melons (Lester, gene CmBi; six CYP genes Cm160, Cm170, Cm180, 1997) and it is present in many cucurbit plants Cm710, Cm890, and Cm490; a cyltransferase gene (Davidovich-Rikanati et al., 2015; Shang et al., 2014). CmACT; and a transcription regulator CmBt) involved The qualitative and quantitative detection of cucrbita- in CuB biosynthesis during fruit development. CmBt, cins using HPLC or liquid chromatography-mass spec- controlled the bitterness of fruit by regulating the ex- trometry (LC-MS) analysis have been reported in pression levels of genes involved in CuB biosynthesis previous studies (Feng et al., 2007; Zhao et al., 2016). (Zhou et al., 2016), encodes a transcription factor that In order to investigate a possible relationship between belongs to the basic helix-loop-helix (bHLH) family, fruit bitterness and CuB accumulation during fruit de- which constitutes one of the largest families of plant velopment, we analysed the CuB contents in Nb46 and transcription factors (Heim et al., 2003). CmBi could Nb320 using HPLC equipment. Our findings clearly cyclize 2,3-oxidosqualene to generate cucurbitadienol showed that Nb46 accumulated CuB at stage I and II, in yeast (Zhou et al., 2016). Interestingly, the data but no CuB was detected at stage III. In Nb320, the showed that expression levels of these genes were dif- CuB content remained at a non-detectable level during ferent between bitter and non-bitter fruits. In Nb46, the throughout the fruit development period. These data expression levels of most genes were very high at stage were coincident with a previous report (Zhou et al., I, and gradually decreased at stage II and III. This result 2016), which indicated that the bitterness perceived in was not reported in previous studies. The expression melon fruits was related to the accumulation of CuB. profiles of these genes remained at quite a low level and Based on the hypothesis that bitterness is correlated changed little throughout the fruit development period 260 D. Hua, J. Fu, L. Liu, X. Yang, Q. Zhang and M. Xie in Nb320. This finding concurred with previous reports recombinant yeast system (Davidovich-Rikanati et al., (Zhou et al., 2016). The different expression patterns of 2015; Zhou et al., 2016). Nevertheless, to date, to the these genes that were consistent with not only the CuB best of our knowledge, there have been no reports on content, but also the severity of bitterness in bitter and the degradation of bitterness during fruit development. non-bitter fruits, suggested these genes were involved 23,24-dihydrocucurbitacin C, a novel compound, was in CuB biosynthesis during melon fruit development. In clearly identified and regarded as the next metabolite of melons, the expression patterns of other two CuB bio- CuC in cucumber leaves (Qing et al., 2014). However, synthetic genes, Cm510, and CmBr, were not measured why bitter compounds or CuB in stage I or stage II of in this study, because these two genes had almost iden- bitter fruit disappeared at stage III, or were translocated tical expression patterns, with high expression in roots into other organs (such as leaves or roots) or trans- and quite low expression in fruit of both wild and culti- formed into other compounds (such a sugars) at stage vated melon lines (Zhou et al., 2016). The association III, has not been elucidated for melons. The mechanism between apple russetting and specific skin triterpene by which bitterness components degrade or transport is composition at maturity has been observed in a previ- still not understood, and requires further research. ous study (Andre et al., 2013) and the gene expression Quantitative analysis of the triterpenoid content in patterns of two OSCs indicate that OSCs are key genes neem (Azadirachta indica) indicated that there was in apple fruit triterpene biosynthesis (Andre et al., tissue-specific variation in terms of abundance 2016). Triterpene saponins, known as ginsenosides, are (Pandreka et al., 2015). Oleanolic acid (OA) and ursolic the major pharmacological compounds in P. ginseng. acid (UA), the main triterpene acids in persimmon fruit, Ginsenosides changed in different development stages changed during fruit development (Zhou et al., 2012). and the expression patterns of genes related to ginseno- Limonin and nomilin, as the predominant limonoids in side biosynthesis have a similar trend (Kim et al., a group of chemically related triterpene derivatives in 2014). the Rutaceae and Meliaceae plant families, increased at Phylogenetic clustering grouped CmBi in the sub- first and then gradually decreased in some citrus species group of cucurbitadienol synthase. This coincided with during growth and maturation when the limomoid a previous study (Zhou et al., 2016). Although a QW UDP-glucosyltransferase regulated the conversion of repeat occurred five times in some 2,3-oxidosequence limonoid to glucoside (Kita et al., 2000). CuB accumu- cyslases and up to eight times in squalene cyclises lation in fruit is a negative trait for general consumers, (Poralla et al., 1994; Tippelt et al., 1998), our date but soluble sugar content is an important component of showed that this repeat only appeared three times in melon fruit quality, and is a primary target for melon CmBi (Fig. S1). Using the crystal structure of human improvement (Argyris et al., 2017; Burger et al., 2006). LAS (PDB ID 1w6k) as a template, a three-dimensional Soluble sugars in fruits include sucrose, fructose and protein model of CmBi showed similar architecture to glucose (Zhu et al., 2017). We measured the soluble LAS (Data not shown), this indicated CmBi may have a sugar contents of these compounds at stage III, and the similar function to other OSCs in a previous study results showed that the sugar contents ranged from (Dhar et al., 2014). 7.4% to 14.3% (Table S1). We found there was a posi- Furthermore, the CuB biosynthetic genes had quite tive correlation [R = 0.890 (P ≤ 0.01)] between bitter- low transcription levels in Nb320 or at stage III in ness severity at stage I and sugar contents at stage III Nb46, suggesting CuB biosynthesis did not happen in among bitter fruits. This result suggested degradation of the fruits of Nb320 or Nb46 at stage III, which was bitter compounds may be related to the accumulation of shown by the fact that no CuB was detected in these soluble sugar in melon during fruit development. Previ- samples. In a previous study, the cucurbitacin ous studies have established biosynthesis and regulation biosynthesis-related genes showed very low express in of bitterness in the plant family cucurbitaceae that in- the fruit in cultivated lines of cucumbers, melons and cludes cucumbers, melons and watermelons (Shang watermelons, consistent with the cucurbitacin content et al., 2014; Zhou et al., 2016), and the activities of in the different plant fruits (Zhou et al., 2016). Some cucurbitadienol synthase using recombinant yeast fruits, such as apples and watermelons, accumulate high (Davidovich-Rikanati et al., 2015; Shibuya et al., 2004). levels of organic acids in the early development stages. However, there is relatively little reported research on However, in fruit at later development stages, the or- the degradation of bitter compounds in fruit. Soluble ganic acid concentrations steadily decline (Gao et al., sugar accumulation in fruits is a complex quantitative 2018; Zhang et al., 2010). The accumulation and me- trait due to the high number of genes that must be coor- tabolism of organic acid in the fruit may be correlated dinately modulated to produce different compounds with the TCA cycle and glycolysis (Sweetlove et al., during fruit developmental stages (Gao et al., 2018). To 2010). Bitterness biosynthesis and metabolism are com- date, how the bitterness and soluble sugar interconvert plex biological processes. A set of genes related to bit- each other remains unclear. Additional research is terness biosynthesis and regulation were detected by needed to further understand the transformation of bit- mapping, comparative analyses of plant genomes and a ter compounds during fruit development. Hort. J. 88 (2): 253–262. 2019. 261

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