Metabolomic analysis of the occurrence of bitter fruits on grafted oriental melon plants.

Grafting has been widely applied to melon (Cucumis melo L.) production to alleviate obstacles of continuous cropping and control soil-borne diseases. However, grafting often leads to a decline of fruit quality. For example, sometimes bitter fruits are produced on grafted plants. However, the underlying physiological mechanism still remains unclear. This study investigated the effects of different rootstocks on the taste of fruits of the Balengcui, an oriental melon cultivar, during summer production. The results showed that all grafted plants with Cucurbita maxima Duch. rootstocks produced bitter fruits, while non-grafted plants and plants grafted onto muskmelon rootstocks produced no bitter fruits. Liquid chromatography-mass spectrometry and metabonomic analysis were performed to investigate the mechanism underlying the occurrence of bitter fruits. Metabolite comparisons of fruits from plants grafted onto Ribenxuesong rootstocks both with non-grafted plants and plants grafted onto muskmelon rootstocks showed that 17 metabolites including phospholipids, cucurbitacins and flavonoids, exhibited changes. The three Cucurbitacins, Cucurbitacin O, Cucurbitacin C, and Cucurbitacin S, increased dramatically. The 10 phospholipids PS(18:1(9Z)/18:2(9Z,12Z)), PS(P-18:0/15:0), PA(18:1(11Z)/18:1(11Z)), PE(16:0/18:0), PS(O-16:0/17:2(9Z,12Z)), PI(16:0/18:2(9Z,12Z)), PA(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PS(P-16:0/17:2(9Z,12Z)), PS(22:0/22:1(11Z)), and PA(17:1(9Z)/0:0)) were significantly decreased, while two PA (16:0/18:2 (9Z, 12Z) and 16:0/18:1 (11Z)), two flavonoids (pelargonidin 3-(6''-malonylglucoside)-5-glucoside and malvidin 3-rutinoside) significantly increased in fruits of plants grafted onto Cucurbita maxima Duch. rootstocks. These metabolites were involved in the glycerophospholipid metabolic pathway, the mevalonate pathway, and the phenylpropanoid pathway. In summary, these results showed that the bitter fruits of grafted Balengcui were caused by Cucurbita maxima Duch. rootstocks. Phospholipids, cucurbitacins, and flavonoids were the key contributors for the occurrence of bitter fruits in Balengcui melon after grafting onto Cucurbita maxima Duch. rootstocks.

Introduction

The melon (Cucumis melo L.) is an important horticultural crop [1]. In 2017, the planting area was 490,327 hectares, achieving a global yield of 17,147,817 t, according to the Food and Agriculture Organization of the United Nations (FAO) [2]. Melon production plays an important role in horticultural planting [3-5]. However, intensive production led to continuous cropping obstacles and an aggravation of soil-borne diseases [6,7]. To alleviate continuous cropping obstacles and control soil-borne diseases, grafting is being widely applied to melon production [8-10]. However, grafting sometimes leads to a decline of fruit quality. Balengcui, an oriental melon cultivar known for its crispy fruit, was commonly grafted onto Cucurbita maxima Duch. However, the grafted plants produced bitter fruits during summer cultivation.

Cucurbitacins were identified as the main compounds that caused the bitterness of Cucurbitaceae fruits [11]. Highly oxidized tetracyclic triterpenoid compounds were synthesized by the methylovalerate pathway from acetyl CoA under the catalysis of a series of enzymes [12]. Generally, cultivated Cucurbitaceae plants do not produce bitter fruits. However, several factors, such as low or high temperature, excessive nitrogen, grafting, plant growth regulators, and changes in the physical and chemical properties of soil may lead to a coordinated operation of a number of genes and induce the synthesis of cucurbitacins [13-15]. Kano and Goto reported that during cucumber growth, plants that received twice the amount of nitrogen fertilizer had a higher probability to produce bitter leaves and fruits than plants that received a normal amount of nitrogen fertilizer [16]. Excessive use of nitrogen fertilizer led to an imbalance of the N metabolism in cucumber plants, and HMG-CoA reductase activity increased, which resulted in the synthesis of cucurbitacin in cucumber plants. However, few reports addressed how grafting caused bitterness in fruits.

Both internal and external factors can cause a coordinated physiological response. Metabolomics as a well-established method to characterize the plant metabolism has been widely used to study the effects of environmental factors on the plant physiological metabolism [17,18] and identify the response mechanisms of plants to biotic or abiotic stresses [19]. Meng et al. compared the metabolite profiles of high-flavonoid mutants and common Ginkgo leaves, and identified 72 different metabolites related to the flavonoid biosynthesis [20]. Metabonomics analysis indicated that both the phenylpropanoid biosynthesis pathway and lipid metabolism pathway regulated Ginkgo flavone biosynthesis. In this study, the effect of different rootstocks on the occurrence of bitter fruits of Balengcui during summer production was investigated. Liquid chromatography–mass spectrometry (LC-MS) and metabonomic analysis were performed to investigate the mechanism underlying the occurrence of bitter fruits of grafted plants.

Materials and methods

Plant materials and growth conditions

To investigate the effect of different rootstocks on the occurrence of bitter fruits, Balengcui seedlings with two cotyledons were grafted onto six Cucurbita maxima Duch. rootstocks (Yemuyixiong, Ribenxuesong, Qingshengzhenjia, Jingxinzhen3, Feichangfuzuo, and Jingyutianzhen1) and two Cucumis melo L. rootstocks (Inbred line1 and Inbred line2). Detailed information about the rootstocks is listed in S1 Table. Both grafted and non-grafted seedlings with 3–4 euphylla were planted in a greenhouse on June 20, 2018. This study used a randomized block design with three replications. The area of each plot was 10 m2 and the planting space was 70 × 35 cm. The temperature was 35°C during the day and 22°C during the night and the relative air humidity was 70–80%.

Identification of bitter fruits

During the flowering and fruit setting periods, the fruits that flowered on the same day were marked at 10–12 nodes of the main vine. At the fruit marketable mature stage, 25 fruits with the same maturity were taken per plot. Twenty tasters were selected to sample all fruits. All tasters evaluated the fruits objectively and agreed to publish their evaluation results. They rinsed their mouth after each tasting to avoid affecting their judgment of the next sample [21]. A fruit was defined as bitter when more than 10 tasters identified it as bitter. The bitter score was evaluated with a 10-point system and the average value was used.

LC-MS analysis

Bitter fruits with the highest bitter score from grafted plants that used Ribenxuesong as rootstock, non-bitter fruits from grafted plants with muskmelon Inbred Line 1 as rootstock, and non-bitter fruits from non-grafted plants were used for metabolomic analysis. Fruit samples (60 mg) with 20 μl internal standard solutions (L-2-chloro-phenylalanine, 0.3 mg/mL) and 1 mL methanol were broken into homogenate and ultrasonically extracted for 45 min. The extracts were centrifuged and the supernatants were taken for LC-MS analysis.

LC-MS was performed with a Waters UPLC I-class system, equipped with a binary solvent delivery manager and a sample manager, and a Waters VION IMS Q-TOF Mass Spectrometer with an electrospray ionization (Waters, Milford, CT, USA), which can operate in either positive or negative ion mode. An acquity BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) was used as chromatographic column and a temperature of 45°C was maintained. Mobile phases A and B were water and acetonitrile, both of which included 0.1% formic acid. The flow rate was 0.40 mL/min and the injection volume was 3.00 μl. The gradient elution program is listed in the following: 5–20% B, 0–2 min, 20–60% B, 2–8 min, 60–100% B, 8–12 min, 100% B, 2 min, 100% to 5% B, 14–14.5 min, 5% B, 1 min. The temperatures of ion source and desolvation were 120°C and 500°C, respectively. The desolvation gas flow rate was 900 L/h. The mass spectrum scan ranged from 50 to 1,000 m/z and the scanning time was 0.1 s with an interval of 0.02 s.

Data analysis

Progenesis QI (Waters Corporation, Milford, CI, USA) was used to perform baseline filtration, peak identification, search characterization, integration, retention time correction, peak alignment, and normalization. The secondary fragments of the metabolite detection were matched to the standard position in the respective database to obtain the data matrix consisting of retention time, mass-to-charge ratio (or metabolite name), and peak intensity. SIMCA-14.1 (Umetrics AB, Umea, Sweden) was used to implement the principal component analysis (PCA) and the orthogonal partial least-squares discrimination analysis (OPLS-DA) multivariate statistical analyses. The different metabolites were initially screened based on a variable importance in the projection (VIP) >1. Student t-test was performed by SPSS11.5 and metabolites were assumed to be significantly different at P < 0.05.

Results

Effects of different rootstocks on the occurrence of bitter fruits

The results showed that all grafted plants with Cucurbita maxima Duch. rootstocks produced bitter fruits at different proportions (Table 1). The highest proportion of bitter fruits was 72% in plants grafted onto Ribenxuesong rootstocks. However, no bitter fruits were identified in all plants grafted onto muskmelon rootstocks (Inbred line 1 and 2) and in all non-grafted plants. These data indicated that rootstocks are responsible for the occurrence of bitter fruits of Balengcui grafted plants during summer cultivation.

Table 1

Evaluation of bitter fruits.

Rootstocks	Number of bitter fruits	Total number of fruits	Rate of bitter fruit (%)
Yemuyixiong	7	25	28
Ribenxuesong	18	25	72
Qingshengzhenjia	10	25	40
Jingxinzhen 3	12	25	48
Feichangfuzuo	10	25	40
Jingyutianzhen1	13	25	52
Muskmelon Inbred line 1	0	25	0
Muskmelon Inbred line 2	0	25	0
Non-grafted	0	25	0

Identification and analysis of fruits metabolites

LC-MS ion flow maps of bitter fruits from plants grafted onto Ribenxuesong as rootstock, non-bitter fruits from plants grafted onto muskmelon Inbred Line 1 as rootstock, and non-bitter fruit from non-grafted plants are shown in Fig 1. The number and intensity of metabolites in fruits of plants grafted onto Ribenxuesong rootstocks were significantly different compared with fruits of non-grafted plants and plants grafted onto muskmelon rootstocks in positive and negative ion mode (Fig 1A and 1B). 1194 metabolites were identified (Fig 2), 27% of which were fatty acyl metabolites, 23% were glycerol phospholipids metabolites, 12% were sterol lipid metabolites, 11% were polyketide glycosides metabolites, 6% were glycerol metabolites, 5% were prenol lipid metabolites, 4% were sphingolipids metabolites, and 2% were steroid and derivative metabolites, carboxylic acid and derivative metabolites, and organic oxide metabolites. Furthermore, 1% amino acids and 5% other compounds were identified.

Fig 1

Base peak intensity (BPI) chromatograms of melon fruits from non-grafted plants and plants grafted onto either muskmelon or pumpkin rootstocks.

(A) BPI chromatogram in positive mode. (B) BPI chromatogram in negative mode.

Fig 2

Classification of identified fruit metabolites.

Different classifications of fruit metabolites are shown as different colors: Black, fatty acyls. Blue, glycerophospholipids. Orange, sterol lipids. Gray, polyketides. Green, glycerolipids. Purple, prenol lipids. Red, sphingolipids. Light blue, steroids and steroid derivatives. Yellow, carboxylicacids and derivatives. Dark red, organooxygen compounds. Pink, amino acids. Light green, other metabolites.

PCA was performed to verify the data and the PCA score map is shown in Fig 3 (R2X = 0.763, Q2 = 0.219). All samples were uniformly distributed in the T2 ellipse, indicating a lack of abnormal sample points. Sample points of the same group clearly clustered together and sample points from three types of fruit can be distinguished in the overall distribution. These results indicated the existence of significant differences in metabolites among fruits from non-grafted plants and plants grafted onto Ribenxuesong or muskmelon Inbred Line 1 as rootstocks. The model parameter was R2X = 0.763, which indicates a good fit and can be used for the screening and analysis of different metabolites.

Fig 3

PCA score plot of melon fruits from non-grafted plants and plants grafted onto muskmelon or pumpkin rootstocks.

R2X = 0.763, Q2 = 0.219.

To further analyze the metabolites of fruits, OPLS-DA models were established. The score maps are shown in Fig 4. The OPLS-DA model parameters of fruits from plants grafted onto Ribenxuesong rootstocks and non-grafted plants were R2X = 0.586, R2Y = 0.955, Q2 = 0.873 (Fig 4A). The parameters of fruits from plants grafted onto Ribenxuesong rootstocks and muskmelon Inbred Line 1 rootstocks were R2X = 0.598, R2Y = 0.972, Q2 = 0.881 (Fig 4B). The parameters of R2 and Q2 all exceeded 0.5. Six replicates per group were closely concentrated and sample points of different groups were well separated. Therefore, these models could be used to screen different metabolites.

Fig 4

OPLS-DA score plot of melon fruits from non-grafted plants and plants grafted onto muskmelon or pumpkin rootstocks.

(A) OPLS-DA score plot of melon fruits from non-grafted plants and from plants grafted onto pumpkin rootstocks. R2X = 0.586, R2Y = 0.955, Q2 = 0.873. (B) OPLS-DA score plot of melon fruits from plants grafted onto muskmelon rootstocks and plants grafted onto pumpkin rootstocks. R2X = 0.598, R2Y = 0.972, Q2 = 0.881.

Effects of different rootstocks on fruit metabolites

According to the results of OPLS-DA with the screening condition of VIP > 1 and P < 0.05, 37 metabolites, including phospholipids, sterols, flavonoids, and terpenoids, significantly changed in bitter fruits of plants grafted onto Ribenxuesong as rootstock compared with fruits of non-grafted plants (S2 Table). Sixteen phospholipids were significantly decreased and six were significantly increased. For sterol metabolites, four were significantly increased and two were significantly decreased. Three flavonoid metabolites were significantly increased and one was significantly decreased. The terpenoids metabolites cucurbitacin O, cucurbitacin C, and cucurbitacin S increased 398.53, 203.33, and 238.21 times, respectively. Two further metabolites (spiramycin and monoisobutyl phthalic acid) were also significantly increased in fruits of plants grafted onto Ribenxuesong rootstocks.

Comparing metabolites of bitter fruits of the plants grafted onto Ribenxuesong as rootstock with fruits of plants grafted onto muskmelon Inbred Line 1 as rootstock identified 33 significantly changed metabolites including 16 phospholipids, four flavones, three sterols, three terpenoids, three organic acids, and four other compounds (S3 Table). Among these 16 phospholipid metabolites, PA (16:0/18:2 (9Z, 12Z), PA (16:0/18:1 (11Z), and PA (18:1 (9Z)/18:4 (6Z, 9Z, 12Z, 15Z) increased, while the other 13 glycerophospholipids decreased significantly. Among the four flavonoids, Pelargonidin 3-(6''-malonylglucoside)-5-glucoside, Malvidin 3-rutinoside, and (+)-Myristinin A increased, while 7-Prenyloxy-3',4'-dimethoxyisoflavone decreased. For the three sterols, hippuristanolide increased, while 1β, 3β, 5α, 6β-tetrahydroxyandrostan-17-one, and diginatin decreased. The three terpenoids Cucurbitacin O, Cucurbitacin C, and Cucurbitacin S increased by 89.36 times, 337.81 times, and 168.58 times, respectively. 9-tetradecynoic acid decreased. Citric acid, 26:4(11Z,14Z,17Z,20Z) and glycerol esters, DG (16:0/18:3 (9Z, 12Z, 15Z)/0:0) and DG (20:5 (5Z, 8Z, 11Z, 14Z, 17Z)/0:0/20:5 (5Z, 8Z, 11Z, 14Z, 17Z)) and two other metabolites (D-Maltose and Uridine 5'-monophosphate) increased.

Overall, compared with the non-bitter fruits produced by non-grafted plants and plants grafted onto muskmelon Inbred Line 1 rootstock, 17 metabolites showed changes, including three terpenoids, two flavonoids, and 12 phospholipids in bitter fruits of plants grafted onto Ribenxuesong as rootstock (Fig 5). Phospholipids (PS(18:1(9Z)/18:2(9Z,12Z)), PS(P-18:0/15:0), PA(18:1(11Z)/18:1(11Z)), PE(16:0/18:0), PS(O-16:0/17:2(9Z,12Z)), PI(16:0/18:2(9Z,12Z)), PA (15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PS(P-16:0/17:2(9Z,12Z)), PS(22:0/22:1(11Z)), and PA(17:1(9Z) /0:0)) were significantly decreased, while PA (16:0/18:2 (9Z, 12Z)) and PA (16:0/18:1 (11Z) were significantly increased (Fig 5A). The three terpenoid metabolites Cucurbitacin O, Cucurbitacin C, and Cucurbitacin S significantly increased (Fig 5B). Two flavonoids, pelargonidin 3-(6'-malonyl glucoside) -5-glucoside and malvidin 3-rutinoside, were significantly increased (Fig 5C). These 17 different metabolites were mapped onto KEGG and Metabo Analyst 3.0 websites. Pathway enrichment analysis was conducted by combining -log P and Impact values. The involved metabolic pathways were the glycerol phospholipid pathway, the methylovalerate pathway, and the phenylpropane pathway.

Fig 5

Significantly changed fruit metabolites identified via comparison between plants grafted onto pumpkin rootstocks and non-grafted plants or plants grafted onto muskmelon rootstocks.

Seventeen significantly changed fruit metabolites were identified including phospholipids (A), cucurbitacin (B), and flavonoids (C). Numbers 1–12 represent PS(18:1(9Z)/18:2(9Z,12Z)), PS(P-18:0/15:0), PA(18:1(11Z)/18:1(11Z)), PE(16:0/18:0), PA(16:0/18:1(11Z)), PA(16:0/18:2(9Z,12Z)), PS(O-16:0/17:2(9Z,12Z)), PI(16:0/18:2(9Z,12Z)), PA(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PS(P-16:0/17:2(9Z,12Z)), PS(22:0/22:1(11Z)), PA(17:1(9Z)/0:0), respectively. Numbers 13–15 represent Cucurbitacin C, Cucurbitacin S, and Cucurbitacin O, respectively. Numbers 16 and 17 represent pelargonidin 3-(6''-malonylglucoside)-5-glucoside and malvidin 3-rutinoside, respectively. Lowercase letters represent P < 0.05 and capital letters represent P < 0.01. The letter before "/" indicates the significance between plants grafted onto pumpkin rootstocks and non-grafted plants. The letter after "/" indicates the significance between plants grafted onto pumpkin rootstocks and muskmelon rootstocks.

Discussion

Bitterness sometimes occurs in fruits of cucurbitaceae plants such as melon, cucumber, and gourd, which severely affected the fruit quality. Low or high temperature, malnutrition, plant growth regulators, and rootstock could all lead to bitter fruits in cucurbitaceae plants [14, 15]. In this study, all Balengcui plants grafted onto Cucurbita maxima Duch. produced bitter fruits, but bitter fruits were not produced on the non-grafted plants and plants grafted onto muskmelon rootstocks. These Cucurbita maxima Duch. rootstocks were also often used as Balengcui rootstocks by farmers during winter; however, these grafted plants did not produce bitter fruits. Why bitter fruits were only produced in summer remained unknown.

LC-MS was performed for metabonomic analysis to investigate the mechanism underlying the occurrence of bitter fruits. Metabolites comparisons of fruits from the plants grafted onto Ribenxuesong rootstocks both with non-grafted plants and plants grafted onto muskmelon rootstocks showed that 17 metabolites exhibited the same changes (Fig 5). Three cucurbitacins dramatically increased hundreds of times in the fruits of plants grafted onto Ribenxuesong rootstocks. Cucurbitacins, as secondary metabolites of highly oxidized tetracyclic triterpenoids, were the main compounds that caused bitterness in fruits of cucurbitaceous plants such as cucumber, pumpkin, and squash [22-24]. These metabolites were the direct cause of bitterness of fruits from plants grafted onto Ribenxuesong rootstocks.

This increase of cucurbitacins was accompanied by a significant decrease of 10 phospholipids, and increases of two PA (PA (16:0/18:1 (11Z) and PA (16:0/18:2 (9Z, 12Z)) and two flavonoids (pelargonidin 3-(6''-malonylglucoside)-5-glucoside and malvidin 3-rutinoside) in the fruits of plants grafted onto Ribenxuesong rootstocks (Fig 5A and 5C). Phospholipids are important membrane components, which are essential for the maintenance of cell stability and for the protection of plants against stress [25]. PAs are also important lipid signaling molecules that can be found in plants, which involved mediating the production and reaction of peroxides and affecting the accumulation of oxidized lipids [26-28]. Several PAs increased under abiotic stress in plants [29]. Flavonoids, as secondary metabolites of phenolic compounds, play an important role in the plant response to environmental signals. Flavonoids can protect plant tissues from damage of high-energy wavelengths (ultraviolet-A and ultraviolet-B) under high temperature and strong light stress [30, 31].

The decrease of a large number of phospholipids and the increase of two PAs in the fruits of plants grafted onto Ribenxuesong rootstocks indicated that fruits were subjected to stress (high temperature and strong light in summer), which led to the occurrence of and membrane lipid peroxidation. The flavonoid increase was also in response to this stress. Wu et al. reported that grafted muskmelon plants with pumpkin rootstocks exhibited lower antioxidant enzyme activity and a higher level of reactive oxygen species (ROS) [32, 33]. ROS lead to membrane lipid peroxidation, which destroyed the cell membrane structure and physiologic integrity [34, 35]. Pumpkins are biologically less resistant to high temperatures than melons. During summer cultivation, the prevailing high temperature and strong light stress may have caused an imbalance of the ROS metabolism system in fruits of plants grafted onto pumpkin rootstocks. This then resulted in membrane lipid peroxidation and phospholipid decomposition into fatty acids [36]. These fatty acids were further oxidized and produced large amounts of acetyl coenzyme A, which (as a precursor) entered the methylovalerate pathway and Cucurbitacins were synthesized [37]. However, the molecular mechanism underlying the signal transduction and gene regulation of the cucurbitacin synthesis through the methylovalerate pathway and how the rootstocks affect the fruit metabolism still requires further research.

Conclusions

Pumpkin (Cucurbita maxima Duch.) rootstocks yileded bitter fruits in grafted melon plants during summer production. In bitter fruits, dramatic increases of cucurbitacins were accompanied by significant decreases of a large number of phospholipids and increases of several PAs and flavonoids. These results indicated that during summer cultivation, oxidation and decomposition of phospholipids affected the fruits of plants grafted onto pumpkin rootstocks and the methylovalerate pathway was activated to synthesize cucurbitacins. This study enables a better understanding of the mechanisms of how bitter fruits are produced on plants grafted onto specific rootstocks. Grafting onto pumpkin rootstocks for summer production should be avoided and other special rootstocks should be selected and bred.

Information of different rootstocks.

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Comparison of metabolites in fruits of plants grafted onto Ribenxuesong rootstocks and non-grafted plants.

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Comparison of metabolites in fruits of plants grafted onto Ribenxuesong and muskmelon rootstocks.

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30 Aug 2019

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PONE-D-19-19662

Metabolomic analysis for the occurrence of bitter fruits of grafting oriental melon plants

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Reviewer #1: Comments

1. The manuscript presents some interesting information that may be published after necessary revision.

2. Generally document read well but requires language editing before publication, the word grafting and grafted are used in a misappropriate way, please check. Similarly there are several other language mistake and formatting errors, please check carefully

3. Please check the following

L 1. The word “grafting” should be replaced with “grafted”

L 24-25. “For example, bitter fruits sometimes occurred on grafted plants.” Replace with “For example, sometimes bitter fruits are produced on grafted plants.”

L 164-166. Please check the word “ion”, this do not seems appropriate word for description of metabolites figure.

4. Please add a conclusion sentence at the end of abstract.

5. L 55. Following recent references may be cited

1. Mu Xiong, Xuejun Zhang, Sergey Shabala, Lana Shabala, Yanjun Chen, Chengli Xiang, Muhammad Azher Nawaz, Zhilong Bie, Haibo Wu, Hongping Yi, Mingzhu Wu, Yuan Huang. 2018. Evaluation of salt tolerance and contributing ionic mechanism in nine Hami melon landraces in Xinjiang, China. Scientia Horticulturae. 237:277-286.

https://www.sciencedirect.com/science/article/pii/S0304423818302723

2. Muhammad Azher Nawaz, Muhamamd Imtiaz, Qiusheng Kong, Cheng Fei, Waqar Ahmed, Yuan Huang and Zhilong Bie. (2016). Grafting: a technique to modify ion accumulation in horticultural crops. Frontiers in Plant Science. 7: 1457. (IF: 4.298). http://journal.frontiersin.org/article/10.3389/fpls.2016.01457/full

3. Zhilong Bie, Muhammad Azher Nawaz, Yuan Huang，Jung-Myung Lee and Giuseppe Colla. 2017. Introduction to Vegetable Grafting. In. Giuseppe Colla, Francisco Perez Alfocea, Dietmar Schwarz (Eds.). Vegetable Grafting. Principles and Practices. CABI Publishing, UK. pp. 1-21.

6. L 57. “Grafted on” should be replaced with “grafted onto”, please check and replace this throughout the manuscript.

7. Discussion section requires improvement.

8. Conclusion section just seems a summary of results, please improve and add a conclusive statement and future aspects of research in this field.

9. Please supply a high quality Figures particularly Figure 1.

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Reviewer #1: Yes: Muhammad Azher Nawaz

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20 Sep 2019

Dear editor,

We appreciate these comments and revised the manuscript as suggested by the academic editor and reviewer. We hope that the revised manuscript has fully addressed the comments so that it is now suitable for publication in PLOS ONE. Details were listed:

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

---Response：We appreciate this comment. We have revised our manuscript and addressed each point raised during the review process.

We would appreciate receiving your revised manuscript by Oct 14 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

---Response：Thank you.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

---Response：Thank you. No changes were made to our financial disclosure.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

---Response：We appreciate this comment. However, the methods used in this study were routine metabolomics analysis and there were no new approaches or insights. Therefore, we chose not deposit our laboratory protocols in protocols.io. If we develop new protocols in the future, we'd like to share it to protocols.io.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

---Response：Thank you. All above-mentioned files were submitted.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

---Response：Thank you. We are willing to make the peer review history publicly available.

Journal Requirements:

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1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

---Response：Thank you. We have proofread our manuscript carefully to meet PLOS ONE's style requirements.

2. We understand that no formal ethical approval was obtained for this research even though it included the participation of 20 tasters.

Please clarify if your institutional review board (IRB) waived the need to obtain formal approval and consent from participants. If participant consent was obtained, please amend your methods section to state this and specify whether the consent was informed and if verbal or written.

---Response：We appreciate this comment. Our institutional review board don’t need to obtain formal approval and consent from participants and all tasters have known that the evaluate results may be published. We added the sentence “All tasters evaluated the fruits objectively and agreed to publish their evaluation results.” at methods section.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

---Response：Thank you. We appreciate this comment and checked the whole manuscript seriously. We have also asked a colleague who is a native English speaker to carefully proofread the manuscript. We believe that all grammar problems have been corrected.

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments

1. The manuscript presents some interesting information that may be published after necessary revision.

---Response：We appreciate this comment and have carefully revised our manuscript.

2. Generally document read well but requires language editing before publication, the word grafting and grafted are used in a misappropriate way, please check. Similarly there are several other language mistake and formatting errors, please check carefully

---Response：We appreciate this comment. In revised manuscript, the word grafting and grafted were used in an appropriate way. We have carefully checked each sentence in the manuscript and corrected all language mistake and formatting errors. For example:

L18. “The cause of bitterness produced after grafting” was replaced with “The cause of bitterness of fruits on grafted plants”.

L198 “In order to further analyze the fruits metabolites, the orthogonal partial least-squares discrimination analysis (OPLS-DA) models were established.” was replaced with “To further analyze the metabolites of fruits, OPLS-DA models were established.”

L463. “Information of different grafted rootstocks” was replaced with “Information of different rootstocks”.

3. Please check the following

L 1. The word “grafting” should be replaced with “grafted”

L 24-25. “For example, bitter fruits sometimes occurred on grafted plants.” Replace with “For example, sometimes bitter fruits are produced on grafted plants.”

L 164-166. Please check the word “ion”, this do not seems appropriate word for description of metabolites figure.

---Response：We appreciate this comment and revised as suggested.

L 1. The word “grafting” was replaced with “grafted”

L 24-25. “For example, bitter fruits sometimes occurred on grafted plants.” was replace with “For example, sometimes bitter fruits are produced on grafted plants.”

L 164-166. “Total ion current maps” was replaced with “Base peak intensity (BPI) chromatograms” after referring to other researches.

4. Please add a conclusion sentence at the end of abstract.

---Response：We appreciate this comment and added the conclusion sentence “In summary, these results showed that the bitter fruits of grafted Balengcui were caused by Cucurbita maxima Duch. rootstocks. Phospholipids, cucurbitacins, and flavonoids were the key contributors for the occurrence of bitter fruits in Balengcui melon after grafting onto Cucurbita maxima Duch. rootstocks.” at the end of abstract.

5. L 55. Following recent references may be cited

1. Mu Xiong, Xuejun Zhang, Sergey Shabala, Lana Shabala, Yanjun Chen, Chengli Xiang, Muhammad Azher Nawaz, Zhilong Bie, Haibo Wu, Hongping Yi, Mingzhu Wu, Yuan Huang. 2018. Evaluation of salt tolerance and contributing ionic mechanism in nine Hami melon landraces in Xinjiang, China. Scientia Horticulturae. 237:277-286.

https://www.sciencedirect.com/science/article/pii/S0304423818302723

2. Muhammad Azher Nawaz, Muhamamd Imtiaz, Qiusheng Kong, Cheng Fei, Waqar Ahmed, Yuan Huang and Zhilong Bie. (2016). Grafting: a technique to modify ion accumulation in horticultural crops. Frontiers in Plant Science. 7: 1457. (IF: 4.298). http://journal.frontiersin.org/article/10.3389/fpls.2016.01457/full

3. Zhilong Bie, Muhammad Azher Nawaz, Yuan Huang，Jung-Myung Lee and Giuseppe Colla. 2017. Introduction to Vegetable Grafting. In. Giuseppe Colla, Francisco Perez Alfocea, Dietmar Schwarz (Eds.). Vegetable Grafting. Principles and Practices. CABI Publishing, UK. pp. 1-21.

---Response：We appreciate this comment and cited above-mentioned references as suggested.

6. L 57. “Grafted on” should be replaced with “grafted onto”, please check and replace this throughout the manuscript.

---Response：Thank you. We replaced “grafted on” with “grafted onto” in revised manuscript and checked the whole manuscript carefully as suggested.

7. Discussion section requires improvement.

---Response：We appreciate this comment. Discussion section was modified and improved in revised manuscript.

8. Conclusion section just seems a summary of results, please improve and add a conclusive statement and future aspects of research in this field.

---Response：We appreciate this comment and improved the conclusion section. Especially, We add a conclusive statement “These results indicated that during summer cultivation, oxidation and decomposition of phospholipids affected the fruits of plants grafted onto pumpkin rootstocks and the methylovalerate pathway was activated to synthesize cucurbitacins.”and future aspects of research in this field “This study enables a better understanding of the mechanisms of how bitter fruits are produced on plants grafted on specific rootstocks. Grafting onto pumpkin rootstocks for summer production should be avoided and other special rootstocks should be selected and bred.” in Conclusion section.

9. Please supply a high quality Figures particularly Figure 1.

---Response：We appreciate this comment and high quality Figures were supplied in revised manuscript.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Muhammad Azher Nawaz

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

---Response：Thank you. All figure files were uploaded to PACE and no Image Problems were discovered. In revised manuscript, all figures were adjusted according to PACE Adjustments.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Sep 2019

PONE-D-19-19662R1

Metabolomic analysis of the occurrence of bitter fruits on grafted oriental melon plants

PLOS ONE

Dear Dr. Zhao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Nov 09 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Yuan Huang

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Line 337-338: Please replace "grafted on specific" with "grafted onto specific"

Please revise acknowledgement section "technical assistance and stimulating discussions" may be replaced with "technical assistance and encouragement"

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Muhammad Azher Nawaz

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Sep 2019

Dear editor,

We appreciate these comments and revised the manuscript as suggested. We hope that the revised manuscript has fully addressed the comments so that it is now suitable for publication in PLOS ONE.

Details were listed:

“Dear Dr. Zhao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Nov 09 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Yuan Huang

Academic Editor

PLOS ONE”

---Response：We appreciate these comments. We have revised our manuscript and addressed each point raised during the review process. No changes were made to our financial disclosure and we chose not deposit our laboratory protocols in protocols.io. All needed files including 'Response to Reviewers', 'Revised Manuscript with Track Changes' and 'Manuscript' were submitted and we are willing to make the peer review history publicly available if the revised manuscript is accepted.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

________________________________________

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Line 337-338: Please replace "grafted on specific" with "grafted onto specific"

Please revise acknowledgement section "technical assistance and stimulating discussions" may be replaced with "technical assistance and encouragement"

---Response：We appreciate these comments and revised as suggested.

Line 337-338: "grafted on specific" was replaced with "grafted onto specific".

Line 341-342: "technical assistance and stimulating discussions" was replaced with "technical assistance and encouragement" in acknowledgement section.

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Muhammad Azher Nawaz

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

---Response：Thank you. All figure files were uploaded to PACE and no Image Problems were discovered. In revised manuscript, all figures were adjusted according to PACE Adjustments.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 Sep 2019

Metabolomic analysis  of the occurrence of bitter fruits on grafted oriental melon plants

PONE-D-19-19662R2

Dear Dr. Zhao,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Yuan Huang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

2 Oct 2019

PONE-D-19-19662R2

Metabolomic analysis of the occurrence of bitter fruits on grafted oriental melon plants

Dear Dr. Zhao:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Yuan Huang

Academic Editor

PLOS ONE