Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou, and a comparison of the main constituents and related genes of Rhizoma Curcumae

Lilan Lu; Peiwei Liu; Yanfang Yang; Yuxiu Zhang; Caixia Wang; Jian Feng; Jianhe Wei

doi:10.1371/journal.pone.0242776

. 2020 Nov 30;15(11):e0242776. doi: 10.1371/journal.pone.0242776

Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou, and a comparison of the main constituents and related genes of Rhizoma Curcumae

Lilan Lu ^1,^2,^*,^#, Peiwei Liu ^1,^#, Yanfang Yang ^3,^#, Yuxiu Zhang ¹, Caixia Wang ⁴, Jian Feng ¹, Jianhe Wei ^1,^*

Editor: Christian Schönbach⁵

PMCID: PMC7703983 PMID: 33253249

Abstract

For more than a thousand years, Rhizoma Curcumae (known as E zhu), a Chinese herbal medicine, has been used to eradicate blood stasis and relieve aches. The plant Curcuma wenyujin, which is grown primarily in Wenzhou, China, is considered the best source of Rhizoma Curcumae. In this study, we sought to ascertain differences in transcript profiles of C. wenyujin grown in traditional (Wenzhou) and recently established (Haikou) production areas based on Illumina and RNA (RNA-seq) sequencing. We also examined differences in the main components of the volatile oil terpene; curcumin, polysaccharide, and starch constituents and related genes in the corresponding pathways, in C. wenyujin cultivated in the two production areas. We accordingly found that the essential oil (2.05%), curcumin (1.46%), and polysaccharide (8.90%) content in Wenzhou rhizomes was higher than that in the rhizomes of plants from Haikou (1.60%, 0.91%, and 6.15%, respectively). In contrast, the starch content of Wenzhou rhizomes (17.0%) was lower than that of Haikou rhizomes (23.8%). Furthermore, we detected significant differences in the oil components of Haikou and Wenzhou rhizomes, with curzerene (32.34%), curdione (21.35%), and germacrene B (9.39%) being the primary components of the essential oil derived from Wenzhou rhizomes, and curzerene (20.13%), curdione (14.73%), and cineole (9.76%) being the main constituents in Haikou rhizomes. Transcriptome and qPCR analyses revealed considerable differences in gene expression between Wenzhou and Haikou rhizomes. The expression of terpene, curcumin, and polysaccharide pathway-related genes in Wenzhou rhizomes was significantly up-regulated, whereas the expression of starch-associated genes was significantly down-regulated, compared with those in Haikou rhizomes. Difference in the content of terpene, curcumin, polysaccharides, and starch in rhizomes from the two production areas could be explained in terms of differences in expression of the related genes.

Introduction

Curcuma is a plant genus comprising more than 70 rhizome-producing species worldwide. In China, there are approximately 20 Curcuma species, a small number of which have been used in Chinese herbal medicines and food products for more than a thousand years. Rhizoma Curcuma is the dried rhizome of Curcuma kwangsiensis S.G. Lee & C.F. Liang, Curcuma phaeocaulis Valeton, and Curcuma wenyujin Y.H. Chen & C. Ling [1]. The essential oils of Curcuma are considered the active ingredients of these plants and have been demonstrated to have antiviral and antitumor properties [2–5]. The main constituents of the essential oils in Curcumae rhizomes are curcumol, germacrone, and curdione, which account for most of the known pharmacological effects, and are typically used as quality control markers for the essential oils [6–8]. C. wenyujin (“wen e zhu” in Chinese) from Wenzhou, Zhejiang Province, China, is widely considered to be the best source of Rhizoma Curcumae (“e zhu” [9–11]), on account of its distinctive regional characteristics and high medicinal value, and is mainly produced in Ruian, Wenzhou. The main active constituents of C. wenyujin are volatile oil and curcumin [12, 13], the former of which is composed of terpenoids and sesquiterpene derivatives, which have been reported to have anti-inflammatory, antitumor, lipid-regulating, antioxidant, antiviral, antibacterial, and other therapeutic effects [14–17].

C. wenyujin is mainly cultivated in China, wherein the quality of the rhizomes varies substantially depending on the variety, cultivation region, cultivation techniques, and extraction methods [11, 18, 19]. C. wenyujin cultivated in different regions, for example, the traditional and non-traditional producing areas, can differ notably with respect to oil components. Given that different environmental conditions can promote changes in the active constituents and alter the transcriptional regulation of genes that control the formation of secondary metabolites, the same C. wenyujin genotype can produce rhizomes with differing qualities depending on the region in and conditions under which the plants are cultivated, and consequently this can potentially present difficulties with respect to quality control.

A notable characteristic of plants is their plasticity in response to unfavorable environmental conditions [20, 21]. Plants typically initiate a series of specific biochemical responses to environmental stressors associated with diverse aspects of their biology, including development, evolution, molecular biology, anatomy, physiology, genetics, and biochemistry [22, 23], which are of particular importance from the perspectives of predicting changes in community composition, distribution, and crop productivity in response to continuously changing environments [24, 25].

A range of environmental factors (both biotic and abiotic) can affect (often adversely) medicinal plants during their growth, thereby influencing the formation and accumulation of terpenoids, ketones, phenols, alkaloids, flavonoid, flavonols, volatile oils, and other secondary metabolites [26, 27]. For example, differences in the chemical and physical properties of soils (determined by the parent material) can influence the growth and quality of plants, and in this regard, it has been found that the oil content of C. wenyujin rhizomes cultivated in alkaline soils is higher than that of rhizomes cultivated in acidic red soils [28].

Isopentenyl pyrophosphate (IPP) is an important intermediate in the biosynthesis of terpenoids and steroids in plants, and steroidal alkaloids can also be formed through other pathways. IPP is formed as a common intermediate in both the mevalonate (MVA) and 2-methyl-d-erythritol-4-phosphoric acid (MEP) pathways, and can pass through cell walls to transfer between these two pathways [29]. Abiotic environmental factors (including light, temperature, moisture, and nutrition) affect metabolism and the synthesis of metabolites, predominantly by influencing gene expression and transcriptional factors in the associated metabolic pathways (e.g., MVA and MEP) of secondary metabolites, such as terpenoids, flavonoids and flavonols [30–38]. Thus, it is speculated that variations in natural environmental conditions in the Wenzhou and Haikou regions, which are characterized by subtropical monsoon and tropical monsoon climates, respectively, may contribute to notable differences in the secondary metabolite profiles of C. wenyujin rhizomes sourced from these two regions.

In recent years, drug companies (such as Haikou Bikai Pharmaceutical Co. Ltd) in Haikou, Hainan Province, have introduced the local cultivation of C. wenyujin using plant material originally sourced from Ruian, Wenzhou, Zhejiang Province [39]. C. wenyujin is cultivated in Haikou for the production of medicines derived from this plant, with the goal of meeting the growing demand for C. wenyujin constituents (volatile oils, crude extracts, and raw materials). In this study, we sought to determine the main constituents and genetic characteristics of C. wenyujin rhizomes sourced from Wenzhou and Haikou, with the aim of assessing the qualities of the respective C. wenyujin rhizomes. The main objective was to analyze the content of essential oils (such as terpene), curcumin, polysaccharides, and starch in C. wenyujin rhizomes obtained from the traditional production areas (Wenzhou) and newly established production areas (Haikou). In this regard, although there have been a few transcriptome studies on plants in the ginger family (e.g., Curcuma zedoaria (Christm.) Roscoe) [40–42], to date there have been no studies that have examined genetic variations in the metabolic pathways of C. wenyujin. Moreover, there have been only a limited number of studies on the transcriptome of C. wenyujin and the mechanisms underlying interspecific differences in the primary and secondary metabolites that accumulate in the rhizomes of C. wenyujin rhizomes cultivated in traditional and non-traditional production areas.

In this study, using Illumina and RNA (RNA-seq) sequencing, we examined the transcript profiles of C. wenyujin rhizomes produced in the traditional production area (Wenzhou) and those derived from an area in which the plant has recently been introduced (Haikou). We also sought to determine variations in the content of the main components of the volatile oil terpene, curcumin, polysaccharides, and starch, and related genes in the corresponding pathways. The data, thus, obtained provide a basis for evaluating the quality of C. wenyujin and elucidating the molecular mechanisms underlying the biosynthetic pathways in plants cultivated in these two production areas, and hence will enable us to assess the potential medicinal value, development, and utilization of C. wenyujin in more agriculturally productive areas, such as Haikou.

Materials and methods

Plant materials

Samples of the traditionally cultivated (Wenzhou) C. wenyujin (WZ) were purchased from Taoshan Town, Ruian, Wenzhou, Zhejiang Province, on December 5, 2017, whereas those of the introduced (Haikou) C. wenyujin (HK) were obtained on the same date from cultivation fields at the Haikou branch of the Institute of Medicinal Plants. The cultivation management of C. wenyujin in the two areas is similar, and details of the environmental and weather conditions of these areas are presented in S1 Table. The Curcuma samples were subsequently identified as C. wenyujin Y.H. Chen & C. Ling. by Li-Rongtao, Associate Professor at the Institute of Medicinal Plants, Chinese Academy of Medical Sciences. For the purposes of the present study, we selected the rhizomes of the C. wenyujin plants as the experimental materials.

RNA isolation, quantification, and qualification

Total RNA was isolated from each sample (rhizomes) using a Quick RNA isolation kit (Bioteke Corporation, Beijing, China). Three biological replicates per experimental group were used for sequencing. Agarose gel (1%) electrophoresis was used to assess the contamination and degradation of RNA. The purity and concentration of the isolated RNA were determined using a Nano spectrophotometer (Photometer^®; IMPLEN, CA, USA) and a Qubit^®2.0 Fluorometer Kit (RNA Assay, Life Technologies, CA, USA), respectively, whereas an Agilent 2100 Bioanalyzer system (Nano 6000; Agilent Technologies, CA, USA) was used to evaluate RNA integrity.

Library preparation and sequencing

An RNA library was prepared using a total of 3 μg RNA per sample, which was analyzed using high-throughput Illumina sequencing technology at the Beijing BioMarker Technologies Company (Beijing, China). An RNA Library Prep Kit (NEB for Illumina, USA) was used for generating sequencing libraries, in accordance with the manufacturer’s recommendations, and the sequences of each sample were attributed using index codes. mRNA was extracted from the total RNA using Poly-T oligo-attached magnetic beads. Fragmentation of the sequences was facilitated by crushing the samples in the presence of divalent cations in a NEBNext first-chain synthetic buffer (5×) at an elevated temperature. First-chain cDNA synthesis was performed using RNase-H (Invitrogen, USA), and Polymerase I (Invitrogen, USA) and RNase-H (Invitrogen, USA) were used for second-chain cDNA synthesis. The remaining overhangs were blunt-ended based on polymerase or exonuclease reactions. Subsequent to adenylation of the DNA fragments (3ʹ ends), the junction (NEBNext), with a hairpin loop structure, was ligated in preparation for hybridization. To preferentially select cDNA fragments of 150–200 bp in length, we used an XP system (AMP, Beckman Coulter, Beverly, USA) to separate the DNA fragments. Prior to PCR, 3 μL USER enzyme (NEB, USA) was added to the samples containing the size-selected adaptor-ligated cDNA, which were heated at 37°C for 15 min, followed by 95°C for 5 min. PCR was carried out using DNA polymerase (Phusion High-Fidelity), Index (X), and Universal Primers (PCR), and the XP system (AMP, Beckman Coulter, Beverly, USA) was applied to purify the PCR products. Library quality was evaluated using an Agilent 2100 Bioanalyzer system (Nano 6000; Agilent Technologies, CA, USA).

Data analysis and assembly

An internal Perl script was initially used to handle the raw read data in fastq (fq) format. Clean reads were acquired by deleting reads including adapters, poly-N sequences, and low-quality reads from the raw data. Moreover, we statistically assessed the sequence repetition, GC content, and Q20 and Q30 levels of clean read data. For all library samples, the left read1 and right read2 files were placed in two larger files (left fq and right fq, respectively). Assembly of the transcriptome was performed based on left/right fq data using the Trinity method, with min_kmer_cov set to 2 and all other parameters automatically set to default [43, 44].

Sequence annotation and functional classification

Genes with unique sequences (unigenes) were annotated and functionally classified based on Clusters of Orthologous Groups of proteins (COG), KOG, and eggNOG protein sequence data libraries for manual annotation and reviewed against Swiss-Prot. Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases [45].

Quantification of gene expression

RSEM was applied to analyze the levels of gene expression in each sample [46]. Clean read data were mapped onto the assembled transcriptome, and the read counts per gene were obtained from the transcriptome database. For the analysis of differential gene expression, we used the DESeq package of R (1.10.1). P-value results were assessed using the Benjamin and Hochberg method (1995) to control for the false discovery rate (FDR), and genes with a P-value < 0.05 were defined as being differentially expressed (DEGs) [45, 47].

GO enrichment analysis

For analysis of DEG enrichment, we used the R package topGO, based on the Kolmogorov–Smirnov test [45, 48].

KEGG pathway enrichment analysis

KEGG pathway enrichment with DEGs was determined using KOBAS 2.0 software [47, 49].

Sample preparation and Gas Chromatography-Mass Spectrometry (GC-MS) analysis

Sample materials were obtained from large rhizomes of C. wenyujin, which had been boiled for 1 h at 100°C and 0.1 MPa, and then cut into 1–2 cm slices and dried for 24 h at 50°C. The volatile oil content of samples was then extracted in accordance with Chinese Pharmacopoeia guidelines [1]. For high-performance liquid chromatography (HPLC) we used an Agilent 7890A gas chromatograph under the following chromatographic conditions: DB-5MS column (0.25 mm × 30 m, 0.25 μm), helium carrier gas, EI ion ionization method, 200°C source temperature, 70 eV electron energy, 250°C interface temperature, 150 μA emission current, 35 to 455 mass range, and a 0.4 s scan cycle. Xcalibur 1.2 was used as a data processing system, and the map library was NIST (Version 1.7). The heating program used was as follows: an initial temperature of 60°C held for 1 min, followed by an increase from 60°C to 100°C at 4°C·min^-1, from 100°C to 120°C at 2°C·min^-1, from 120°C to 180°C at 1°C·min^-1, and from 180°C to 230°C at 23°C·min^-1, where the temperature was held for 10 min. Other parameters were as follows: 53 kPa column pressure, 1.04°C·min^-1 flow rate, and 0.2 μL sample volume. A 20 μL volume of eugenol:n-hexane (20:1) was added to a 2 mL brown gas-phase flask, to which was added 1000 μL of n-hexane. The solution was mixed by vortexing for 1 min and then filtered. To obtain the total ion chromatograms of different volatile oils, we used an Agilent 7890A gas chromatograph coupled to a 5975 C quadrupole mass spectrometer. The relative value (percentage) of each chromatogram peak was calculated using the peak area normalization method, and following a computer search and manual analysis, the compounds were identified with reference to the literature.

Quantitative reverse-transcription PCR (qRT-PCR) analysis

qPCR analysis was performed using an IQ5 Multicolor (CFX96) PCR detection system (Bio-Rad, Bole, USA) and a Power PCR Master Mix (SYBR green) (Takara, Beijing, China). RNase-free DNase (TaKaRa, Beijing, China) was used to extract the total RNA from the C. wenyujin rhizomes, and 15 primers (Oligod-T) and a SuperScript (III) RT kit (Invitrogen, USA) were used for the reverse transcription (RT) reaction. For each RT reaction, we used 20 μL reaction mixtures containing 10 μL of master mix reagent (2× SYBR Green Master) (TaKaRa, Beijing, China), 10 ng of sample (cDNA), and 200 nM of gene-specific primers. The cycle conditions were as follows: an initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 34 s. The amplification program was then followed by a 55°C–85°C melting curve analysis, with each temperature maintained for 5 s. For PCR amplification, we used gene-specific primers (S2 Table), the annealing efficiency of which was assessed using the primer program (PRIMER 3.0) [50]. As an internal control gene, we used c121677 against which the average amplification of three replicate samples was normalized. The relative expression values of the target genes were calculated by comparing the target gene cycle thresholds (CTs) with those of the c121677 housekeeping gene using the 2^-ΔΔCT method [51, 52].

Determination of curcumin content

For HPLC analysis of curcumin, we used an Agilent Extend C18 column (4.6 × 250 mm, 5 μm) under the following chromatographic conditions: a mobile phase of 4% acetonitrile and glacial acetic acid solution (45:55), a flow rate of 1.0 mL·min^-1, a detection wavelength of 420 nm, a column temperature of 25°C, and an injection volume of 10 μL. The number of theoretical plates was not less than 4,000. The curcumin reference standard was accurately weighed (6.35 mg) after ensuring that it had been dried to a constant weight [CAS: 110823–201706; China National Institute of Food and Drug Control (CNIFDC), China], and was then placed into a 25 mL brown volumetric flask, dissolved with methanol to 25 mL, shaken, and filtered as the reference stock solution. A curcumin control solution (25.4 μg·mL^-1) was prepared by diluting a 1 mL aliquot of this stock solution with methanol in a 10 mL brown volumetric flask. Dried powdered rhizome (0.5 g) was placed into a 50 mL conical flask to which 20 mL methanol was added. The mixture was sonicated for 1 h, and then centrifuged. To the resulting supernatant, 15 mL of methanol was added, followed by sonication for 40 min and subsequent filtering. The residual material was rinsed with a small amount of methanol and refiltered. The filtrate, thus, obtained was added to the initial extraction supernatant solution, and the combined filtrate was then made up to 20 mL using methanol at a temperature below 20°C.

Determination of polysaccharide content

The polysaccharide content of rhizomes was determined in accordance with the methodology of the “Chinese Pharmacopoeia” (Appendix VA; 2015) [1]. The extracted polysaccharides were analyzed using a UV1601 UV-Vis spectrophotometer (Shimadzu, Japan) at a wavelength of 490 nm, with an appropriate amount of anhydrous glucose being used as the reference material (reference d-anhydroglucose; CAS: 0833–9501; CNIFDC, China). The analytical chemicals, apparatus, conditions, and preparation of standard and sample solutions have previously been described in detail by Chen [53].

Determination of starch content

Starch content was analyzed using a UV1601 UV-Vis spectrophotometer (Shimadzu, Japan) at a wavelength of 600 nm, with a soluble starch standard (chromatographic grade, CAS: 140602; CNIFDC, China). The analytical chemicals, apparatus, analytical conditions, standard preparation, starch extraction, and preparation of C. wenyujin rhizomes samples have previously been described in detail by Zhang et al. [54].

Statistical analysis

Average values of each parameter were calculated from three replicate analyses, and standard errors of the mean values were obtained. Univariate analysis (analysis of variance) of variance was applied to determine the significance of the results among different treatments, with a P-value < 0.05 being considered significant. SPSS V.13 was used for statistical analyses.

Results

Analysis of rhizome constituent and volatile oil compositions

Our analyses revealed notable differences in the rhizome (E zhu) structure and volatile oil color and content of the Curcuma wenyujin samples obtained from the two different regions. The volatile oil content of C. wenyujin rhizomes derived from Wenzhou was found to be higher than that in the rhizomes from Haikou, and these oils were characterized by bright and dark yellow colors, respectively (Fig 1). Paraffin sections of rhizome tissue, stained using the periodic acid-Schiff (PAS) reaction and potassium iodide, revealed the cellular localization of oil and the quantity of starch granules, respectively (Fig 2). The oil (2.05%), curcumin (1.46%), and polysaccharide (8.90%) content of C. wenyujin rhizomes from Wenzhou was significantly higher than that of rhizomes from Haikou (1.60%, 0.91%, and 6.15%, respectively), whereas the starch content of rhizomes from Wenzhou (17.0%) was significantly lower than that of rhizomes from Haikou (23.8%) (Fig 3). We also detected significant differences in the compositions of the volatile oils derived from the rhizomes of plants obtained from the two cultivation regions, with the content of curzerene and curdione being higher in those from Wenzhou (S3 Table). Curzerene (32.34%), curdione (21.35%), and germacrene B (9.39%) were found to be the major components of the essential oil of rhizomes from Wenzhou, whereas in the rhizomes harvested from Haikou, curzerene (20.13%), curdione (14.73%), and cineole (9.76%) were identified as the main essential oil constituents. Thus, the chemical constituents of essential oils in the rhizomes of Wenzhou- and Haikou-cultivated C. wenyujin appear to be quite different.

mRNA sequencing, assembly, functional annotation, and classification

To analyze the dynamics of mRNA expression in C. wenyujin from different production areas, we constructed and sequenced mRNA-seq libraries for C. wenyujin derived from Haikou (introduced cultivation) (HK) and Wenzhou (traditional cultivation) (WZ). A comparison of three replicates of the two experimental groups verified that they do not differ significantly at the sequence level (S4 and S6 Tables). Sequencing of a total of six transcriptome samples (HK1, HK2, HK3, WZ1, WZ2, and WZ3) yielded a total of 41.74 Gbp of clean data (reaching up to at least 6.37 Gbp for each sample), with Q30 percentages (number of samples with Phred quality score higher than 30) of more than 89.15% in the mRNA-seq libraries of the six samples (S4 Table). In total, we acquired 185,006 transcripts, representing 99,942 unigenes, with N50 values of 1,796 and 1,406 bp for transcripts and unigenes, respectively, and 28,375 unigenes being larger than 1 kb (S5 and S6 Tables). Searches for all unique sequences in public databases using BLASTX (E values < 1.00⁻⁵) [55] and subsequent functional annotation implemented based on the NR, Swiss-Prot, COG, GO, and KEGG database, enabled us to confirm the identity of 51,609 unigenes (51.6%), whereas we were unable to similarly identify the remaining 48,333 unique sequences (48.4%) (S7 Table, S1 Fig).

The functions of the unique sequences extracted from C. wenyujin were classified based on the three GO terms cellular component (CC), biological process (BP), and molecular function (MF). Compared with HK, 15,151 unigenes and 924 DEGs in WZ were classified as CC; 19,906 unigenes and 1,276 DEGs were classified as BP; and 21,535 unigenes and 1,456 DEGs were classified as MF. The DEGs were mainly assigned to the categories “cell, go: 0005623,” “cell part, go: 0044464,” “organelle, go: 0043226,” “membrane, go: 0016020,” “cellular process, go: 0009987,” “metabolic process, go: 0008152,” “single-organism process, go: 0044699,” “binding, go: 0005488,” and “catalytic activity, go: 0003824” (Fig 4). The majority of the GO terms identified in the present study are in accordance with the GO categories defined previously [52, 56].

Fig 4 — The red, green, and blue histograms represent the differentially expressed genes (DEGs) in the different sub-categories, whereas the light red, green, and blue histograms represent the annotated unigenes. The right-hand Y-axis represents the number of annotated genes or DEGs in the main categories, and the left-hand Y-axis represents the percentage of annotated unigenes or DEGs in the main categories.

With respect to KEGG pathways, we identified 1,597 unique sequences (Fig 5). The predominantly enriched pathways appeared to include the following: endocytosis (36; 3.5%) and phagosome (35; 3.4%) in cellular processes; endoplasmic reticulum protein processing (70; 7.09%), spliceosome (77; 8.0%), ribosome (89; 9.09%), and RNA transport (57; 5.5%) in genetic information processing; and carbon metabolism (72; 7.12%), and biosynthesis of amino acids (58; 5.6%) in metabolism.

Differential gene expression

We used the fragments per kilobase per million reads mapped (FPKM) procedure to calculate the expression level of genes [57], and DESeq (R package, version 1.10.1) was used for DEG analysis, The latter revealed that 4,620 genes were significantly differentially expressed in WZ compared with those in HK, of which 3,978 were up-regulated and 642 were down-regulated (Fig 6).

Fig 6 — Green and red dots denote genes with significantly different expression (FDR < 0.01), with green indicating down-regulated genes, red indicating up-regulated genes, and black indicating those genes showing no significantly different expression.

To determine similarities and differences in the transcriptional changes of genes in WZ and HK C. wenyujin, we performed hierarchical clustering, which accordingly revealed differences in the expression patterns of genes in the two sources of C. wenyujin (Fig 7). A subset of transcripts, together with their respective fold changes and false discovery rate-corrected P-values, for both clusters, are presented in S8 Table. Among these, genes related to terpene, curcumin, and polysaccharide pathways were found to be up-regulated in C. wenyujin from Wenzhou, where genes associated with starch and sugar pathways were up-regulated in C. wenyujin from Haikou.

Fig 7 — Red and green represent up- and down-regulated genes, respectively, in Wenzhou (WZ) rhizomes compared with those of Haikou (HK). The heatmap was produced based on fragments per kilobase per million (FPKM) data.

Functional enrichment analysis of DEGs

In the present study, we used topGO to analyze the functional enrichment of DEGs with respect to the three GO functional categories, CC, BP, and MF (enrichment significance, K_S < 0.05), the details of which are shown in S9 Table. Among all DEGs, plastid and chloroplast (CC); methylerythritol 4-phosphate (MEP) pathway, isopentenyl diphosphate biosynthetic process, and thylakoid membrane organization (BP); and protein serine/threonine kinase activity and protein kinase activity (MF) were enriched in WZ rhizomes compared with that in HK rhizomes. Among the up-regulated DEGs, the GO terms pollen tube tip (CC); leaf morphogenesis (BP); and polyamine oxidase activity, isomerase activity, ion channel activity, and phospholipase activity (MF) were significantly enriched in WZ compared with those in HK, whereas for down-regulated DEGs, cell wall (CC); defense responses to fungus (BP); and peroxidase activity, copper ion binding, sucrose synthase activity, heat shock protein binding, and catalytic activity (MF) were significantly enriched in WZ compared with those in HK (S1 File).

Fig 8 shows the results of our COG functional classifications of the consensus sequences of genes that were differentially expressed between WZ and HK. The 1,698 genes that were differentially expressed between the rhizomes obtained from these two sources can primarily be divided into the following eight categories: general function prediction only (R) (481, 28.33%); transcription (K) (230, 13.55%); replication, recombination, and repair (L) (208, 12.25%); signal transduction mechanisms (T) (190, 11.19%); translation ribosomal structure and biogenesis (J) (189, 11.13%); posttranslational modification protein turnover chaperones (O) (178, 10.48%); amino acid transport and metabolism (E) (153, 9.01%); and carbohydrate transport and metabolism (G) (140, 8.24%). The corresponding numbers and percentages of the annotated genes in these eight categories are as follows: R (2,427, 19.82%), K (415, 55.42%), L (512, 40.63%), T (405, 46.91%), J (1,426, 13.25%), O (1,373, 12.96%), E (892, 17.15%), and G (765, 18.30%). Thus, the genes associated with these eight categories can be assumed play vital roles in the growth of C. wenyujin in the Wenzhou traditional production area.

Similarly, functional analyses of the DEGs based on the KEGG classification of enriched pathways revealed that with respect to all genes differentially expressed between WZ and HK, 179 pathways showed enrichment, among which there were 117 and 62 pathways associated with up- and down-regulated DEGs, respectively (S10 Table). For these pathways, we analyzed the significance of the enrichment factor and Q values, and in Fig 9, we present the first 20 minimal Q value pathways. In total, 980 DEGs were assigned to KEGG pathways, among which, 844 and 136 were up- and down-regulated, respectively. The dominant and significantly enriched pathways are listed in S10 Table. With respect to all DEGs, the predominantly enriched KEGG pathways associated with the following functional areas: ribosome, spliceosome, endoplasmic reticulum protein processing, ubiquitin-mediated proteolysis, RNA transport, phagosome, glyoxylate and dicarboxylate metabolism, and glutathione metabolism. Pathways predominantly enriched with the up-regulated DEGs included spliceosome, carbon metabolism, ubiquitin-mediated proteolysis, proteasome, citrate cycle (TCA cycle), aminoacyl-tRNA biosynthesis, arginine and proline metabolism, and glutathione metabolism, whereas those significantly enriched the down-regulated DEGs included ribosome, protein export, riboflavin metabolism, endoplasmic reticulum protein processing, plant hormone signal transduction, starch and sucrose metabolism.

Fig 9 — Pathway name and enrichment intensity are shown in the right-hand legend. The Q value is the corrected P-value (false discovery rate). The enrichment factor is the ratio of the number of differentially expressed genes (DEGs) in a pathway to the number of all genes in that pathway. (a, all DEGs; b, up-regulated DEGs; c, down-regulated DEGs).

Verification of DEGs

To establish the reliability of the FPKM procedure, we selected the sequences of 15 of the DEGs that are involved in terpene, curcumin, polysaccharide, and starch pathways for qRT-PCR analysis. Our findings that the overall correlation coefficient of the linear regression analysis between FPKM and qRT-PCR was 0.959 (R = 0.959) indicated a good correlation between the transcript abundance detected by RT-PCR and the transcription spectrum of RNA sequence data analysis (S2 Fig). Data obtained from RNA-seq and qRT-PCR analyses revealed that those genes associated with terpene (LPS, CDS/LIS, ISPS, and TPS06), curcumin (CHS2, CHS1, and TT4), and polysaccharide (TSTA, GALE, and GH) pathways were significantly up-regulated in the rhizomes of C. wenyujin cultivated in Wenzhou, whereas genes associated with polysaccharide (LOC103985749) and starch (GAE, PME, BGLU, and SUS) pathways were significantly up-regulated in the rhizomes of C. wenyujin obtained from Haikou. These differences in gene expression patterns were consistent with those obtained based on transcriptome FPKM analysis (Fig 10).

Fig 10 — Using c121677 as a control/reference gene, the relative expression of genes was calculated using the 2-^ΔΔCt method. Asterisks denote significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars represent the standard deviations of mean values (n = 6). (CHS2: chalcone synthase2; CHS1: chalcone synthase1; TT4: chalcone and stilbene synthase family protein; LPS: levopimaradiene synthase; CDS/LIS: 3S-linalool synthase; ISPS: isoprene synthase; TPS06: dolabella-3,7-dien-18-ol synthase/sesquiterpene synthase 6; GAE: UDP-glucuronate 4-epimerase; PME: pectin methylesterase; BGLU: beta-glucosidase; SUS: sucrose synthase; TSTA: GDP-l-fucose synthase; GALE: UDP-glucose 4-epimerase; GH: glycoside hydrolase; LOC103985749: uncharacterized protein LOC103985749).

Transcription factor analysis

Transcription factor analysis revealed that 57 classes of transcription factors showed dynamic changes in Haikou rhizomes compared with those from Wenzhou. Compared with the former, 52 types of transcription factor were up-regulated in Wenzhou-derived rhizomes, of which the expression levels of genes in the RLK, C2H2, Zn-clus, TKL, bZIP, CAMK, AP2/ERF, AGC, STE, C2C2, CMGC, C3H, SNF2, WRKY, bHLH, GNAT, MYB, HSF, and NAC families were significantly up-regulated. Among these, the gene expression of certain types of transcription factor only showed up-regulation, whereas a few genes in the RLK, bZIP, AP2/ERF, WRKY, bHLH, MYB, HSF, and NAC classes also showed down-regulation in C. wenyujin rhizomes from WZ compared with those from HK (S3 Fig, S11 Table).

Discussion

Environmental factors play a prominent role in determining the growth of plants, and environmental quality and climatic conditions in different growing areas are the most important factors contributing to variations in plant primary and secondary metabolites [18, 19].

Previous studies that have conducted chemical component and transcriptome analyses of Curcuma species have reported large variations in the content and composition of volatile oils and the content of curcumin and polysaccharide among different Curcuma species grown in different areas, as well as in the corresponding Rhizoma Curcumae [58–61]. Consistently, the findings of the present revealed large differences in the content and composition of volatile oils and the content of curcumin, polysaccharide, and starch in the rhizomes of C. wenyujin sourced from the traditional production areas in Wenzhou and the more recently established production areas in Haikou. Notably in this regard, we detected significantly higher content of the volatile oil terpene, curcumin, and polysaccharides in C. wenyujin rhizomes from Wenzhou compared with that in the rhizomes of plants cultivated in the Haikou area, whereas the content of starch was significantly higher in the Haikou rhizomes. Additionally, we detected certain differences between Wenzhou and Haikou rhizomes with respect to the composition of the volatile oil, which is consistent with the observations of Huang et al., who found that the volatile oil content of C. wenyujin rhizomes from Haikou (2.9%) was slightly lower than that of rhizomes from Zhejiang (3.0%), and also reported considerable differences in the volatile oil constituents of plants growing in different habitats [39]. Further studies have also shown differences in the chemical constituents of volatile oils in C. wenyujin from different planting areas, which were found to be associated with differences in the quality of the corresponding medicinal materials [17, 62, 63]. It has similarly been demonstrated that volatile oil content may vary depending upon the origin of the variety, cultivation area, cultivation techniques, and extraction method [8], and Cao et al. have reported that the content of volatile oil and curcumin differed substantially in different experimental areas, with the curcumin content varying from 1.5% to 5% and volatile oil content ranging from 0.4% to 0.7% [63]. In the present study, RT-PCR and transcriptome RNA-seq analyses revealed that genes involved in terpene, curcumin, and polysaccharide production were significantly up-regulated in C. wenyujin rhizomes from Wenzhou, thereby indicating that these genes are closely associated with observed differences in the profiles of these metabolites in C. wenyujin cultivated in different areas. In contrast, we observed that starch-related pathways were significantly up-regulated in the rhizomes of C. wenyujin cultivated in Haikou. These results are consistent with the findings of previous studies that have indicated that C. wenyujin rhizomes from Wenzhou have higher content of the volatile oil, terpene, curcumin, polysaccharide but lower starch content than those in C. wenyujin rhizomes from Haikou. We also found that the gene expression patterns were consistent with those determined based on transcriptome FPKM analysis (Figs 3 and 10). Huang et al. have previously suggested that whereas differences in the starch content of C. wenyujin rhizomes from different producing areas are not appreciably influenced by latitude and longitude, they may be related to local soil, temperature, and light conditions, as well cultivation measures [64]. Starch formation is a photosynthesis-related dark reaction involving a series of enzymatic reactions within the chloroplast matrix, which is influenced to varying degrees by temperature, CO₂ concentration, and pH. It can therefore be speculated that differences in these environmental factors between Wenzhou and Haikou contribute to the observed differences in the content of starch and/or other substances in C. wenyujin rhizomes. Similarly, gene expression analysis has revealed that the curcumin content of C. wenyujin rhizomes is markedly affected by environmental factors and changes in nutritional conditions [19].

Transcriptomics is concerned with analysis of the gene expression levels and diversity profiles of different tissues in animals and plants [65–67], one of the important focuses of which is the study of the biosynthesis of metabolites in non-model medicinal plants [68, 69]. In the present study, we adopted hierarchical clustering to obtain complete transcriptional profiles of C. wenyujin cultivated in the Wenzhou and Haikou production areas, and accordingly found that the rhizomes of C. wenyujin grown in Wenzhou showed significant differences with respect to the transcriptional profiles of the co-expressed transcripts when compared with the rhizomes of plants grown in Haikou. Among these co-expressed transcripts, we found that when compared with those in Haikou rhizomes, the number of genes up-regulated in Wenzhou rhizomes was greater than those down-regulated. Functional analysis indicated that the enriched KEGG pathways appeared to be primarily associated with endocytosis and phagosome among cellular processes; ribosome, endoplasmic reticulum protein processing, RNA transport and spliceosome in genetic information processing; and carbon metabolism and biosynthesis of amino acids with respect to metabolism, thereby implying that the associated regulatory genes are significantly differentially expressed between the C. wenyujin rhizomes obtained from Wenzhou and Haikou.

Overall, we found that 980 DEGs were allocated to KEGG pathways, among which those associated with spliceosome, endoplasmic reticulum protein processing, ribosome, ubiquitin-mediated proteolysis, RNA transport, phagosome, glyoxylate and dicarboxylate metabolism, and glutathione metabolism were predominantly enriched. With respect to up-regulated DEGs, pathways associated with spliceosome, carbon metabolism, ubiquitin-mediated proteolysis, proteasome, citrate cycle (TCA cycle), aminoacyl-tRNA biosynthesis, arginine and proline metabolism, and glutathione metabolism were predominantly enriched, whereas for the down-regulated DEGs, pathways relating to ribosome, starch and sucrose metabolism, endoplasmic reticulum protein processing, plant hormone signal transduction, protein export, and riboflavin metabolism were significantly enriched. These findings are thus indicative of the active biosynthesis of not only secondary but also primary metabolites. Conversely, we noted that plant hormone signal transduction and starch and sucrose metabolism were inhibited and down-regulated in the rhizomes of C. wenyujin cultivated in Wenzhou. These DEGs annotations will provide a valuable resource for characterizing the relative importance of different signal transduction pathways and specific genes in the rhizomes of C. wenyujin grown in both the traditional and recently established production areas [52], which in turn could provide useful information for examining biosynthesis mechanisms of interest in C. wenyujin (S4–S7 Figs).

In the present study, we found that among all genes that were differentially expressed between Wenzhou and Haikou rhizomes, the GO terms of plastid and chloroplast (CC); methylerythritol 4-phosphate (MEP) pathway, isopentenyl diphosphate biosynthetic process, and thylakoid membrane organization (BP), and protein kinase activity and protein serine/threonine kinase activity (MF) were predominantly enriched in the rhizomes from Wenzhou compared with those from Haikou. Among up-regulated DEGs, the GO terms of pollen tube tip (CC), leaf morphogenesis (BP), and polyamine oxidase activity, isomerase activity, ion channel activity, and phospholipase activity (MF) were significantly enriched in Wenzhou rhizomes, whereas for down-regulated DEGs, the GO terms cell wall (CC), defense response to fungus (BP), and peroxidase activity, copper ion binding, sucrose synthase activity, heat shock protein binding, and catalytic activity (MF) were significantly enriched in Wenzhou rhizomes. We assume that these differences reflect the fact that when grown under different environmental conditions, C. wenyujin requires different metabolites and levels of energy to respond to the different stimuli associated with the prevailing conditions.

When exposed to abiotic stresses, such as drought and elevated temperatures, plants respond by expressing a range transcription factors, induced via a series of signaling pathways. The stimulated transcription factors bind to corresponding cis-acting elements to initiate specific patterns of gene expression to counter the adverse effects of abiotic stress. Plant transcription factors known to respond to abiotic stress mainly include those in the AP2/EREBP, MYB, WRKY, bZIP, and HSFs [70–75]. AP2/EREBP, MYB, WRKY, bZIP, HSFs, RLK, bHLH, NAC, and CAMK families, the regulation of which is determined by a diverse range of biotic and abiotic pressures. plant growth and material metabolic processes, including trichome and seed coat development, embryogenesis leaf senescence, biosynthesis pathways, and the regulation of hormonal signals [76–80]. C2H2 and C2HC zinc finger structures are characteristic features of the WRKY family of transcription factor from which the WRKY family can be explained [73]. In the present study, we detected significantly up-regulated expression of transcription factors in the RLK, C2H2, Zn-clus, TKL, bZIP, CAMK, AP2/ERF, AGC, STE, C2C2, CMGC, C3H, SNF2, WRKY, bHLH, GNAT, MYB, HSF, and NAC families. Thus, on the basis of our findings that the genes of transcription factors known to respond to abiotic environmental factors were significantly up-regulated, we can speculate that observed differences in the compositions and gene regulation of C. wenyujin rhizomes derived from the traditional (Wenzhou) and recently established (Haikou) production areas, are probably attributable to differences in environmental factors specific to these two regions.

Conclusions

In summary, our findings indicate that the content of volatile oil, curcumin, and polysaccharides in the rhizomes of C. wenyujin cultivated in the Wenzhou production region is higher than those in the rhizomes of C. wenyujin grown in Haikou, whereas the content of starch is lower. We also detected significant differences in the constituents of the volatile oils of rhizomes obtained from the two production areas. Transcriptome analysis revealed notable differences in the gene expression patterns of rhizomes sourced from the Wenzhou and Haikou regions. We found that the expression of genes in pathways related to volatile oil, curcumin, and polysaccharide was significantly up-regulated in Wenzhou rhizomes, whereas that of starch-associated genes was significantly down-regulated, compared with those in Haikou rhizomes. Moreover, we identified the significantly up-regulated expression of transcription factors (including those in the RLK, C2H2, bZIP, CAMK, AP2/ERF, WRKY, bHLH, MYB, HSF, and NAC families) that are typically associated with responses to abiotic environmental stress. Collectively, the findings of this study provide important insights into the molecular mechanisms underlying differences in the metabolite profiles of C. wenyujin, which are assumed to reflect differences in environmental factor characterizing the two examined production regions. We also describe new methodologies that may prove beneficial with respect to evaluating the authenticity of Chinese herbal medicines.

Supporting information

S1 Fig. Curcuma unigene length distribution.

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S2 Fig. Comparison of RNA-seq and quantitative reverse-transcription PCR (qRT-PCR) analyses of 15 selected genes.

log2Fold Change [Wenzhou (WZ)/Haikou (HK)].

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S3 Fig. Transcription factor expression for Wenzhou (WZ) versus Haikou (HK).

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S4 Fig. Terpenoid backbone synthesis: KEGG enrichment structure for Wenzhou (WZ) versus Haikou (HK).

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S5 Fig. Flavonoid biosynthesis: KEGG enrichment structure for Wenzhou (WZ) versus Haikou (HK).

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S6 Fig. Amino sugar and nucleotide sugar metabolism: KEGG enrichment structure for Wenzhou (WZ) versus Haikou (HK).

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S7 Fig. Starch and sucrose metabolism: KEGG enrichment structure for Wenzhou (WZ) versus Haikou (HK).

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S1 Table. Environmental and weather conditions in Wenzhou and Haikou.

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S2 Table. RNA-seq verification using RT-PCR primers in Curcuma wenyujin.

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S3 Table. GC-MS detection results of the main volatile oil constituents in Curcuma wenyujin from Wenzhou (WZ) and Haikou (HK).

(XLS)

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S4 Table. Sample sequencing data evaluation statistics.

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S5 Table. Assembly result statistics.

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S6 Table. Comparison of sequencing data and assembly results.

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S7 Table. Unigene annotation statistics.

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S8 Table. Expression data of enriched key DEGs in different assemblies for Wenzhou (WZ) versus Haikou (HK).

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S9 Table. Details of topGO enrichment for Wenzhou (WZ) versus Haikou (HK).

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S10 Table. Details of KEGG enrichment for Wenzhou (WZ) versus Haikou (HK).

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S11 Table. Transcription factor expression for Wenzhou (WZ) versus Haikou (HK).

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S1 File. Enrichment analysis of responsive genes and transcripts using GO terms for Wenzhou (WZ) versus Haikou (HK).

The node size is proportional to the number of targets in the GO category. Node color represents enriched significance; a deeper color represents a higher significance [a1, a2, and a3: total (topGO_BP, topGO_CC, and topGO_MF); b1, b2, and b3: down-regulated (topGO_BP, topGO_CC, and topGO_MF); c1, c2, and c3: up-regulated (topGO_BP, topGO_CC, and topGO_MF)].

(ZIP)

Click here for additional data file.^{(748.8KB, zip)}

Acknowledgments

We would like to thank Editage (www.editage.cn) for editing the English language in this manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

YES, Hainan province Key Scientific and Technological Research and Development Special Project (Grant No: ZDKJ2016006), The funders had a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0242776.r001

Decision Letter 0

Christian Schönbach

4 Dec 2019

PONE-D-19-24237

Transcriptome analysis of Curcumawenyujin from Haikou and Wenzhou and a comparison of the main substances and related genes of Rhizoma Curcumae

PLOS ONE

Dear Dr. Lilan,

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PLOS ONE

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Comments to the Author

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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: Partly

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: 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: Yes

Reviewer #2: No

**********

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: Curcumae Rhizoma, known as Ezhu (Chinese), and Curcumae Radix, known as Yujin (Chinese), are different plant parts coming from three same species of Curcuma according to China Pharmacopoeia. The Chinese Pharmacopoeia recorded that Curcumae Radix should be the dry radix of Curcuma wenyujin Y. H. Chen and C. Ling, C. longa L., C. kwangsiensis S. G. Lee, and C. phaeocaulis Valeton. And Curcumae Rhizoma should be the dry rhizomes derived from the above-mentioned species except C. longa L. They are similar in source but different in medicinal parts. Curcumae Rhizoma and Curcumae Radix are confused in variety and source, even in clinical trials by some nonprofessional workers. So, it is important for us to make them clear. Also, genotype and environment interaction is high for secondary metabolites in Curcuma sp. The main objective of the present study was to analyze the constituents of essential oils such as terpene, curcumin, polysaccharide, starch, and other important substances from C. Wenyujin rhizomes from the traditional (Wenzhou) and introduced (Haikou) production areas. In this study authors also attempted comparative RNAseq profiles of C. Wenyujin rhizomes produced in traditional (Wenzhou) and introduced (Haikou) areas. The study has some interesting findings in the poorly understood crop species of Curcuma.

However, authors need to clarify following points.

The introduction part seems to be too lengthy – cab be shortened.

L.71: The terminology 'Species variety' is not botanically correct. Authors may use the term ‘genotype’ instead.

L.86: There is nothing called “lime soil”. Is it alkaline soil?

L.120-122: Give a suitable reference for this statement.

L.146: What do you mean by treatment conditions?

L.146-153: Authors silent on experimental conditions. Similar sampling and growing conditions are important for studying secondary metabolites and comparative transcriptomics.

The environmental and weather conditions of the two provinces need to be given as supplementary table to understand the differences in secondary metabolites and related genes.

L.154: Protocol for RNA isolation is not mentioned. Also specify the tissue from which RNA was isolated.

In case of RNASeq, number of biological replicates sequences is not clear. Also, replicates need to be compared at sequence level and prove that they are not significantly different.

L.209: Sufficient experimental details viz., temperature, pressure and slice thickness need to be given on boiling and drying of samples?

L.234: What is the sample? rhizome or leaf, or at what stage?

L. 248: Is it a curcumin or curcuminoids

L.268: Polysaccharide and starch content methodology are too elaborate and need to shorten with suitable references.

L 361………..

In many literatures, the C. weeyujin is referred as synonymous to C. aromatica. In that case can authors compare the present transcritomes with already available C. aromatica transcriptome to draw the meaningful conclusions.

Hope above suggestions help authors to improve the manuscript.

Reviewer #2: Language correction is essential before acceptance. Method used for RNA isolation is not mentioned (what mentioned as RNAse free DNAse, Takara; is just an enzyme and is not the method used for RNA extraction).

Minor Corrections Suggested

1) Line no. 38, 39: better replace ‘was’ with were.

2) Species name should always be in small letters, throughout the manuscript its written as CurcumaWenyujin , few line numbers are mentioned ( Line no.119, 134, 148, 342, 350, 347, 355, 506, 509, 522, 528, 538, 541, 544, 546, 549, 558, 562, 563, 574, 582, 583, 594, 609, 611, 625, 627, 644, 650).

3) Make sure that Curcuma wenyujin is in italics (not followed).

4) Line no. 265: Rephrase the sentence in a better conveyable form.

5) Line no. 297: spelling correction; ‘using’ is written as uing.

6) Line no. 329: better replace ‘was’ with were.

7) Line no. 561 is contradictory to Line no. 564 (about polysaccharide).

**********

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: PONE-D-19-24237_reviewer.pdf

Click here for additional data file.^{(2.6MB, pdf)}

PLoS One. 2020 Nov 30;15(11):e0242776. doi: 10.1371/journal.pone.0242776.r002

Author response to Decision Letter 0

29 Feb 2020

Dear Plos one editor and reviewers,

We thank you for all of your comments and suggestions about ways to improve our manuscript, now retitled “Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou and a comparison of the main constituents and related genes of Rhizoma Curcumae”. The following are our response to your questions and suggestions.

Journal requirements:

1. When submitting your revision, we need you to address these additional requirements. 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

Revision: Thanks for the suggestion, We have revised the manuscript to meet PLOS ONE's style requirements by consulting The PLOS ONE style templates.

Revision: Thanks for the suggestion, We have created a new ORCID：0000-0003-4317-0029

3. Both reviewers pointed out deficiencies in description of methodological details, conditions and sample details and conditions. According to Publication Criterion #3 You are required to describe your methods and samples in sufficient detail to ensure replicability. This extends also to purchased products that may not be sufficiently described or characterized, or whose purchase locations were not provided.

Revision: Thanks for the suggestion, I read it carefully and have made changes in the text. The purchased products was sufficiently described or characterized, purchase locations were provided in the paper. We have described methods and samples in sufficient detail to ensure replicability in the revised manuscript.

4. You are required to state how the Illumina RNASeq was done (details and instrument) and submit the reads to NCBI SRA Archive (PloS ONE publication criterion #7 Data Availability (see also https://journals.plos.org/plosone/s/data-availability).

Revision: Thanks for the suggestion, the data reads of Illumina sequencing have been deposited in the National Center for Biotechnology Information Sequence Read Archive (SRA accession: PRJNA598542; Temporary Submission ID: SUB6764411; SAMPLE: Curcuma wenyujin (SAMN13707351); Release date: 2020-01-08. https://www.ncbi.nlm.nih.gov/sra/PRJNA598542).

Additional Editor Comments:

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: Partly

Reviewer #2: Yes

Revision: Thanks for the suggestion, According to this suggestion “Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. ” We have Modified this article based on detailed recommendations from experts in red highlight place.

2. 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

Reviewer #2: No

Revision: Thanks for the suggestion, This language polish has been corrected by the company(Editage) polish, to ensure clear, correct, and unambiguous.

Review Comments to the Author

Reviewer #1”: The introduction part seems to be too lengthy – cab be shortened.

Revision: Thanks for the suggestion, We have shortened the introduction in the revised manuscript.

Reviewer #1: L.71: The terminology 'Species variety' is not botanically correct. Authors may use the term ‘genotype’ instead.

Revision: Line 71: “Species variety” was replaced with “genotype”. Shown in line 69 in the revised manuscript.

Reviewer #1: L.86: There is nothing called “lime soil”. Is it alkaline soil?

Revision: Line 86: “lime soil” was replaced with “alkaline soil”. Shown in line83 in the revised manuscript.

Reviewer #1: L.120-122: Give a suitable reference for this statement.

Revision: Thanks for the suggestion, we have given a suitable reference for this statement in line 104 in the revised manuscript.

Reviewer #1: L.146: What do you mean by treatment conditions?

Revision: Thanks for the suggestion, L.146: “Plant materials and treatment conditions? ” was replaced with “Plant materials”. Shown in line 131in the revised manuscript.

Reviewer #1: L.146-153: Authors silent on experimental conditions. Similar sampling and growing conditions are important for studying secondary metabolites and comparative transcriptomics. The environmental and weather conditions of the two provinces need to be given as supplementary table to understand the differences in secondary metabolites and related genes.

Revision: Thank you for the recommends. The environmental and weather conditions of the two provinces was given in S1 Table (shown in L141 in the revised manuscript).

Reviewer #1: L.154: Protocol for RNA isolation is not mentioned. Also specify the tissue from which RNA was isolated. In case of RNASeq, number of biological replicates sequences is not clear. Also, replicates need to be compared at sequence level and prove that they are not significantly different.

Revision: L.154: “Protocol for RNA isolation and specify the tissue from which RNA was isolated ”is added in L143-145.Three biological replicates samples in sequences were mentioned, and were shown in line 145 in the revised manuscript. “replicates need to be compared at sequence level and prove that they are not significantly different” were mentioned in line 324-326 in the revised manuscript.

Reviewer #1: L.209: Sufficient experimental details viz., temperature, pressure and slice thickness need to be given on boiling and drying of samples?

Revision: L.209: “ Sufficient experimental details viz., temperature, pressure and slice thickness need to be given on boiling and drying of samples? ” were revised . Shown in line 205-207in the revised manuscript.

Reviewer #1: L.234: What is the sample? rhizome or leaf, or at what stage?

Revision: L.234: “What is the sample? rhizome or leaf, or at what stage? ” were revised with “C. wenyujin rhizomes ” . Shown in line 230 in the revised manuscript.

Reviewer #1: L. 248: Is it a curcumin or curcuminoids

Revision: Curcuminoids mainly cantain curcumin , demethoxycurcumin, bisdemethoxycurcumin and other curcumin substances, and in this paper, we analyzed a curcumin in rhizome of Curcuma wenyujin, so, "It is a curcumin in this paper" was confirmed, shown in line L244 in the revised manuscript.

Reviewer #1: L.268: Polysaccharide and starch content methodology are too elaborate and need to shorten with suitable references.

Revision: Thank you for the recommends. L.268: “Polysaccharide and starch content methodology are too elaborate and need to shorten with suitable references” was revised and shorten with suitable references[53-54]. Shown in line L264-279 in the revised manuscript.

Reviewer #1: In many literatures, the C. weeyujin is referred as synonymous to C. aromatica. In that case can authors compare the present transcritomes with already available C. aromatica transcriptome to draw the meaningful conclusions. Hope above suggestions help authors to improve the manuscript.

Revision: Thank you for the recommends. In discussions, We cited the literature and discussed and compared C. wenyujin between HK and WZ areas. Shown in line L486-489 in the revised manuscript.

Revision: This language polish has been corrected by the company (Editage) polish, and RNA extraction methods have been mentioned in the article L43-145.

Reviewer #2: Line no. 38, 39: better replace ‘was’ with were.

Revision: Line 38, 39: “was” was replaced with “were”. Shown in line 39 in the revised manuscript.

Reviewer #2: Species name should always be in small letters, throughout the manuscript its written as Curcuma Wenyujin, few line numbers are mentioned ( Line no.119, 134, 148, 342, 350, 347, 355, 506, 509, 522, 528, 538, 541, 544, 546, 549, 558, 562, 563, 574, 582, 583, 594, 609, 611, 625, 627, 644, 650).

Revision: Line (119, 134, 148, 342, 350, 347, 355, 506, 509, 522, 528, 538, 541, 544, 546, 549, 558, 562, 563, 574, 582, 583, 594, 609, 611, 625, 627, 644, 650) “Species name should always be in small letters, throughout the manuscript its written as Curcuma Wenyujin ” were revised. Shown in Line (100, 111, 134, 289, 295, 297, 302, 457, 461, 474, 480, 491, 493, 497, 498, 501, 510, 512, 513, 515, 524, 532, 533, 544, 559, 561, 574, 577, 594) in the revised manuscript.

Reviewer #2: Make sure that Curcuma wenyujin is in italics (not followed).

Revision: “Curcuma wenyujin is in italics ” were revised . Shown in the revised manuscript.

Reviewer #2: Line no. 265: Rephrase the sentence in a better conveyable form.

Revision: Line no. 265“Rephrase the sentence in a better conveyable form”were revised . Shown in line 260-265 in the revised manuscript.

Reviewer #2: Line no. 297: spelling correction; ‘using’ is written as uing.

Revision: Line no. 297 “uing” was replaced with “using”. Shown in line 267 in the revised manuscript.

Reviewer #2: Line no. 329: better replace ‘was’ with were.

Revision: Line no. 329 was revised. Shown in line 274-279 in the revised manuscript.

Reviewer #2: Line no. 561 is contradictory to Line no. 564 (about polysaccharide).

Revision: Line no561-564 “561 is contradictory to Line no. 564 (about polysaccharide) in Line no.561-564” were revised . Shown in line 511-515 in the revised manuscript.

Other revisions:

1. Line 469 “15 of the transcriptomes of differentially expressed genes- How did you select these genes? What was the criteria followed to shortlist 15 genes? ”

Revision: These 15 genes are genes related to terpenoids, curcumin, polysaccharides, and starch metabolism pathways of Rhizoma Curcuma, and the selected genes are expressed in both samples. The differential expression of genes in the two regions is high, and the p value and FDR value are within the range. These genes are designed with appropriate primers and quantified.

2. Line 376“A total of six samples-what are they? ”

Revision: Thank you for the recommends. they are HK1, HK2, HK3, WZ1, WZ2, WZ3. Shown in line 326-327 in the revised manuscript.

Best regards,

Lulilan,

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(27.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0242776.r003

Decision Letter 1

Christian Schönbach

8 Apr 2020

PONE-D-19-24237R1

Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou and a comparison of the main constituents and related genes of Rhizoma Curcumae

PLOS ONE

Dear Dr. Lu Lilan,

While some of the major issuses raised by reviewers in the previous round were addressed more work is required improve descriptions of methods and language to meet publicaiton criteria #3 and #5. Especially, add parameters and thresholds to methods applied, explain rational for chosing a particular statistical test (here univariate ANOVA), and most importantly revisit and revise the DEG analysis part to ensure enrichment results are statically correctly supported. In additon your are required to improve lablelling and quality of figures.

We would appreciate receiving your revised manuscript by May 23 2020 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.

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

We look forward to receiving your revised manuscript.

Kind regards,

Christian Schönbach, Dr.rer.nat.

Academic Editor

PLOS ONE

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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 #2: All comments have been addressed

Reviewer #3: (No Response)

**********

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

Reviewer #2: Yes

Reviewer #3: Partly

**********

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

Reviewer #2: Yes

Reviewer #3: No

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #2: Authors incorporated the suggestions into their manuscript and as a result the MS quality has been improved. The revised MS can be accepted as it is.

Reviewer #3: The authors analyzed C. wenyujin rhizomes in two different locations and showed that the difference in metabolome and transcriptome were consistent.

The conclusion sounds OK but there are so many points to be improved in figures, statistical analysis and sentences.

The comparison along the metabolic pathway, and their conclusion would be OK. However, the distribution of transcriptome should be evaluated and normalized before detail statistical analysis such as DEG. Also, the detail about clustering analysis and enrichment analysis are not clear to evaluate them.

All of the figures were looks very low in resolution (converted from compressed JPEG?) and most of the labels are difficult to read, even I downloaded each tiff file.

Use vector format images or, at least, more high resolution and low compression images.

It is strongly recommended to submit the whole manuscript to an English editorial service.

There are detail comments below.

l85. ~ indicating that the oil content of Curcuma rhizomes can

85 be affected by factors such as soil quality and climate.

> Add some reference if there are some preceding related studies.

l. 192

> It would be specify the name of the test and parameters since "DESeq" is a name of a function in the R package rather than a name of a method.

l. 282 Univariate analysis of variance was applied to determine the significance of the results among different treatments.

> The target of the analysis unclear. Does it means "different species", and the variables are compound components? Since they have only two groups, are there any rational reason to apply univariate ANOVA instead of t-test or U-test?

Through three replications, the average value of each parameter was calculated, and the standard error (SE) of the mean value was obtained. Univariate analysis of variance was applied to determine the significance of the results among different treatments. Multidimensional tests were considered significant at a P value < 0.05. SPSS V.13 was used for statistical analysis.

l 327 41.74 Gb

> If it means giga "byte", show the amount in giga "base pair" instead.

l. 328 Q30 percentages

> It should be more concretely and use general term.

For example, "Number of samples which Phred quality score is higher than 30."

l. 340 1.00e-05

> It would be better to use scientific format (superscripts) instead of computational format.

l 340

> In the DEG analysis the number of up regulated genes were much larger than that of down regulated. The authors should concern about experimental bias between samples or consider to apply some compensation such as quantile normalization.

Fig 4 shows the ratio of DEG unigenes in each ontology is highly correlated

with that of all annotated unigenes.

> It seems that DEG are not correlate with their functions but almost randomly sampled from the all unigenes, so that the significance of enrichment analysis is rather doughtful.

Fig. 7

> Make clear the number of genes shown in this figure, is it 3720? If it is so make clear how and why these genes were chosen. It is not clear what means "up/down regulated" in this figure, I mean compared with what values.

> And the labels in the columns should be replaced by their real labels (T01 = HK1, T02 = HK2, T03 = HK3, T04 = WZ1, T05 = 393 WZ2, T06 = WZ3).

l. 409 A comparison of DEGs in 26 COG classifications between HK and WZ is shown in Fig 8.

> It is not clear that the numbers of DEG in this figure means up regulated or down regulated. And for enrichment analysis, it is also important to show the number of annotated genes in each category and the ratio of DEG to the annotated genes.

l. 454

> It seems meaningless to show the sequence id such like c118842.grraph-co, show gene name or protein name instead. It would be much better to show them in the pathway map also.

Fig. 9

> Arrange the plots for FPKM and rt-PCR side by side when the comparison about the same gene is important.

**********

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 #2: No

Reviewer #3: Yes: Naoaki ONO

PLoS One. 2020 Nov 30;15(11):e0242776. doi: 10.1371/journal.pone.0242776.r004

Author response to Decision Letter 1

6 Sep 2020

Dear Plos one editor and reviewers,

We thank you for all of your comments and suggestions about ways to improve our manuscript, and give us a second chance to revise the paper, now retitled “Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou, and a comparison of the main constituents and related genes of Rhizoma Curcumae”. The following are our response to your questions and suggestions.

Journal requirements:

Revision: Thank you very much for the suggestion, and We have accepted the expert's suggestion that we improve the description of the method and language by polishing the company to meet the #3 and #5 public standards. In particular, Why are parameters and thresholds added to the applied method to explain the rationality of choosing a specific statistical test (here, univariate ANOVA), Curcuma rhizomes from different species, and they have only two groups, We had applied univariate ANOVA instead of t-test or U-test. Seen in lines 282 in the revised. and the DEG analysis part and enrichment results have been modified and explained. In addition, we also made adjustments to meaningful tags and improved image quality. We have Modified this article based on detailed recommendations from experts in red highlight place.

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #2: Yes

Reviewer #3: No

Revision: Thanks for the suggestion, According to this suggestion “the statistical analysis been performed appropriately and rigorously. ” and according to this suggestion We had applied univariate ANOVA instead of t-test or U-test. Seen in lines 282 in the revised manuscript. We have Modified this article based on detailed recommendations from experts in red highlight place.

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

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: No

Revision: Thanks for the suggestion, This language polish has been corrected by the company(Editage) polish, to ensure clear, correct, and unambiguous. Seen in red highlight place in the revised manuscript.

6. Review Comments to the Author

Reviewer #2: Authors incorporated the suggestions into their manuscript and as a result the MS quality has been improved. The revised MS can be accepted as it is.

Answer: Thank you for your comments

Reviewer #3:

The authors analyzed C. wenyujin rhizomes in two different locations and showed that the difference in metabolome and transcriptome were consistent. The conclusion sounds OK but there are so many points to be improved in figures, statistical analysis and sentences. The comparison along the metabolic pathway, and their conclusion would be OK. However, the distribution of transcriptome should be evaluated and normalized before detail statistical analysis such as DEG. Also, the detail about clustering analysis and enrichment analysis are not clear to evaluate them.

Revision: Thank you for your comments, the distribution of transcriptome was evaluated and normalized before detail statistical analysis such as DEG and unigenes in S1-Fig, S4-S7 Table. Also, the detail about clustering analysis and enrichment analysis are evaluate them in S9, S10 Table and S1 File. In-depth research in the future, we will further analyze.

All of the figures were looks very low in resolution (converted from compressed JPEG?) and most of the labels are difficult to read, even I downloaded each tiff file.Use vector format images or, at least, more high resolution and low compression images.

Revision: Thank you for your comments, all of the figures were adjusted by using more high resolution and low compression images.

It is strongly recommended to submit the whole manuscript to an English editorial service.

Revision: Thank you for your comments, We had submited the whole manuscript to an English editorial service again (Editage) . Seen in red highlight place.

There are detail comments below.

L85. ~ indicating that the oil content of Curcuma rhizomes can be affected by factors such as soil quality and climate. Add some reference if there are some preceding related studies.

Revision: Thank you for your comments. We have added a reference about preceding related studies, However, now, There is still not much research on related studies about the soil quality and climate, in lines L84-88 in the revised manuscript.

L192: It would be specify the name of the test and parameters since "DESeq" is a name of a function in the R package rather than a name of a method.

Revision: Thank you for your comments. "DESeq" is revised for a name of a function in the R package. The P value results were regulated using the Benjamin and Hochberg method (1995) for controlling the false discovery rate (FDR), and genes at a P value < 0.05 were classified as differentially expressed genes (DEGs), in lines L198-202 in the revised manuscript.

L282 : Univariate analysis of variance was applied to determine the significance of the results among different treatments. The target of the analysis unclear. Does it means "different species", and the variables are compound components? Since they have only two groups, are there any rational reason to apply univariate ANOVA instead of t-test or U-test?

Revision: Thank you for your comments. Curcuma rhizomes from different species, and they have only two groups, We had applied univariate ANOVA instead of t-test or U-test. Seen in lines 288 in the revised manuscript.

L:327 41.74 Gb, If it means giga "byte", show the amount in giga "base pair" instead.

Revision: Thank you for your comments, it means giga "byte", 41.74 Gb was revised for 41.74 Gbp (base pair) in lines 335 in the revised manuscript.

L: 328 Q30 percentages, It should be more concretely and use general term.

For example, "Number of samples which Phred quality score is higher than 30."

Revision: Thank you for your comments, Q30 percentages was revised for general term that number of samples which Phred quality score is higher than 30) in lines 336-337 in the revised manuscript.

L 340: 1.00e-05, It would be better to use scientific format (superscripts) instead of computational format.

Revision: Thank you for your comments. It has been used scientific format (superscripts)( 1.00-5) instead of computational format in lines 342 in the revised manuscript.

L 340: In the DEG analysis the number of up regulated genes were much larger than that of down regulated. The authors should concern about experimental bias between samples or consider to apply some compensation such as quantile normalization.

Revision: Thank you for your comments. Yes, you are right about that “experimental bias between samples or consider to apply some compensation such as quantile normalization”. In the DEG analysis the number of up /down regulated genes, logarithm quantile normalization (Log2FC（WZ/HK）) were seen in S8 Table .

Fig 4 shows the ratio of DEG unigenes in each ontology is highly correlated with that of all annotated unigenes.It seems that DEG are not correlate with their functions but almost randomly sampled from the all unigenes, so that the significance of enrichment analysis is rather doughtful.

Revision: Thank you for your comments. Fig4 showed Gene ontology (GO) histogram of classification in annotated unigenes WZ (Wenzhou) versus HK (Haikou). The functions of the unique sequences extracted from C. wenyujin were classified based on the three GO terms: cellular component (CC), biological process (BP), and molecular function (MF), The DEGs were mainly assigned to ‘cell, go: 0005623’, ‘cell part, go: 0044464’, ‘organelle, go: 0043226’, ‘membrane, go: 0016020’,‘cellular process, go: 0009987’, ‘metabolic process, go: 0008152’, ‘single-organism process, go: 0044699’, ‘binding, go: 0005488’, and ‘catalytic activity, go: 0003824’ (Fig 4). and We had some KEEG and GO enrichment analysis seen in lines 405-418 and 446-458 in the revised manuscript and related enrichment information was seen in S9, S10 Table and S1 File. We will conduct more in-depth research in the future on the significance of enrichment analysis.

Fig. 7: Make clear the number of genes shown in this figure, is it 3720? If it is so make clear how and why these genes were chosen. It is not clear what means "up/down regulated" in this figure, I mean compared with what values.And the labels in the columns should be replaced by their real labels (T01 = HK1, T02 = HK2, T03 = HK3, T04 = WZ1, T05 = 393 WZ2, T06 = WZ3).

Revision: Thank you for your comments. Make clear the number of genes shown in this figure is 4620. These genes were chosen because of a significant difference in these genes between WZ and HK . the values of up/down regulated" in this figure were those of WZ compared HK. And the labels in the columns have been replaced by their real labels (HK1, HK2, HK3, WZ1, WZ2, WZ3) in Fig 7 in the revised manuscript.

L409: A comparison of DEGs in 26 COG classifications between HK and WZ is shown in Fig 8. It is not clear that the numbers of DEG in this figure means up regulated or down regulated. And for enrichment analysis, it is also important to show the number of annotated genes in each category and the ratio of DEG to the annotated genes.

Revision: Thank you for your comments, We mean that 26 COG functional classifications of consensus sequence of DEGs between HK and WZ are shown in Fig 8, not a comparison of DEGs in 26 COG classifications between HK and WZ is shown in Fig 8. We have revised it seen in Line 409. We have showed the number of annotated genes in main eight categories and the ratio of DEG to the annotated genes, seen in lines 424-432in the revised manuscript.

L 454:It seems meaningless to show the sequence id such like c118842.grraph-co, show gene name or protein name instead. It would be much better to show them in the pathway map also.

Revision: Thank you for your comments, We have showed gene name or protein name instead of the sequence id such like c118842.grraph-co seen in lines 475-478 in the revised manuscript.

Fig. 10Arrange the plots for FPKM and rt-PCR side by side when the comparison about the same gene is important.

Revision: Thank you for your comments, We have arranged the plots for FPKM (DGE) and RT-PCR side by side when the comparison, seen in Fig. 10 in the revised manuscript.

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.

Answer: Thank you, We choose “no”.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(18.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0242776.r005

Decision Letter 2

Christian Schönbach

10 Nov 2020

Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou and a comparison of the main constituents and related genes of Rhizoma Curcumae

PONE-D-19-24237R2

Dear Dr. Lilan,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Christian Schönbach, Dr.rer.nat.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #4: N/A

**********

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

Reviewer #1: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: No

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #1: All the comments have been addressed. Manuscript Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou and a comparison of the main constituents and related genes of Rhizoma Curcumae is now acceptable.

Reviewer #4: the revised manuscript has complied all the query of the reviewers and meets all the mandate of the journal.

**********

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: No

Reviewer #4: Yes: Enketeswara subudhi

PLoS One. doi: 10.1371/journal.pone.0242776.r006

Acceptance letter

Christian Schönbach

16 Nov 2020

PONE-D-19-24237R2

Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou, and a comparison of the main constituents and related genes of Rhizoma Curcumae

Dear Dr. Lu:

I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Curcuma unigene length distribution.

(TIF)

Click here for additional data file.^{(1MB, tif)}

S2 Fig. Comparison of RNA-seq and quantitative reverse-transcription PCR (qRT-PCR) analyses of 15 selected genes.

log2Fold Change [Wenzhou (WZ)/Haikou (HK)].

(TIF)

Click here for additional data file.^{(756KB, tif)}

S3 Fig. Transcription factor expression for Wenzhou (WZ) versus Haikou (HK).

(TIF)

Click here for additional data file.^{(333KB, tif)}

S4 Fig. Terpenoid backbone synthesis: KEGG enrichment structure for Wenzhou (WZ) versus Haikou (HK).

(TIF)

Click here for additional data file.^{(974.9KB, tif)}

S5 Fig. Flavonoid biosynthesis: KEGG enrichment structure for Wenzhou (WZ) versus Haikou (HK).

(TIF)

Click here for additional data file.^{(156.7KB, tif)}

S6 Fig. Amino sugar and nucleotide sugar metabolism: KEGG enrichment structure for Wenzhou (WZ) versus Haikou (HK).

(TIF)

Click here for additional data file.^{(277.8KB, tif)}

S7 Fig. Starch and sucrose metabolism: KEGG enrichment structure for Wenzhou (WZ) versus Haikou (HK).

(TIF)

Click here for additional data file.^{(1MB, tif)}

S1 Table. Environmental and weather conditions in Wenzhou and Haikou.

(XLS)

Click here for additional data file.^{(18KB, xls)}

S2 Table. RNA-seq verification using RT-PCR primers in Curcuma wenyujin.

(XLS)

Click here for additional data file.^{(21.5KB, xls)}

S3 Table. GC-MS detection results of the main volatile oil constituents in Curcuma wenyujin from Wenzhou (WZ) and Haikou (HK).

(XLS)

Click here for additional data file.^{(35KB, xls)}

S4 Table. Sample sequencing data evaluation statistics.

(XLS)

Click here for additional data file.^{(26.5KB, xls)}

S5 Table. Assembly result statistics.

(XLS)

Click here for additional data file.^{(25.5KB, xls)}

S6 Table. Comparison of sequencing data and assembly results.

(XLS)

Click here for additional data file.^{(25KB, xls)}

S7 Table. Unigene annotation statistics.

(XLS)

Click here for additional data file.^{(26KB, xls)}

S8 Table. Expression data of enriched key DEGs in different assemblies for Wenzhou (WZ) versus Haikou (HK).

(XLS)

Click here for additional data file.^{(825KB, xls)}

S9 Table. Details of topGO enrichment for Wenzhou (WZ) versus Haikou (HK).

(XLS)

Click here for additional data file.^{(46KB, xls)}

S10 Table. Details of KEGG enrichment for Wenzhou (WZ) versus Haikou (HK).

(XLS)

Click here for additional data file.^{(93KB, xls)}

S11 Table. Transcription factor expression for Wenzhou (WZ) versus Haikou (HK).

(XLS)

Click here for additional data file.^{(20.5KB, xls)}

S1 File. Enrichment analysis of responsive genes and transcripts using GO terms for Wenzhou (WZ) versus Haikou (HK).

(ZIP)

Click here for additional data file.^{(748.8KB, zip)}

Attachment

Submitted filename: PONE-D-19-24237_reviewer.pdf

Click here for additional data file.^{(2.6MB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(27.2KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(18.8KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou, and a comparison of the main constituents and related genes of Rhizoma Curcumae

Lilan Lu

Peiwei Liu

Yanfang Yang

Yuxiu Zhang

Caixia Wang

Jian Feng

Jianhe Wei

Roles

Abstract

Introduction

Materials and methods

Plant materials

RNA isolation, quantification, and qualification

Library preparation and sequencing

Data analysis and assembly

Sequence annotation and functional classification

Quantification of gene expression

GO enrichment analysis

KEGG pathway enrichment analysis

Sample preparation and Gas Chromatography-Mass Spectrometry (GC-MS) analysis

Quantitative reverse-transcription PCR (qRT-PCR) analysis

Determination of curcumin content

Determination of polysaccharide content

Determination of starch content

Statistical analysis

Results

Analysis of rhizome constituent and volatile oil compositions

Fig 1. The rhizomes and volatile oils of Curcuma wenyujin from Wenzhou and Haikou.

Fig 2. Paraffin sections of Curcuma wenyujin rhizomes showing starch (potassium iodide staining) (a, b) and oil cells (PAS reaction) (c, d) in rhizomes from Wenzhou (b, d) and Haikou (a, c) observed under a biological microscope (a, b scale = 50 μm; c, d scale = 100 μm).

Fig 3. The content of oil, curcumin, polysaccharide, and starch in Curcuma wenyujin rhizomes produced in Wenzhou and Haikou.

mRNA sequencing, assembly, functional annotation, and classification

Fig 4. A Gene Ontology (GO) histogram of the classification of annotated unigenes that were differentially expressed between Wenzhou (WZ) and Haikou (HK) rhizomes.

Fig 5. Annotation of unique sequences (1,597) based on KEGG classification.

Differential gene expression

Fig 6. Differential expression of the unique genes in Curcuma wenyujin rhizomes obtained from Wenzhou (WZ) and Haikou (HK).

Fig 7. Cluster analysis of genes that were differentially expressed (4,620) between Wenzhou (WZ) and HK (HK) rhizomes.

Functional enrichment analysis of DEGs

Fig 8. Clusters of Orthologous Group (COG) functional classification of the consensus sequences of genes that are differentially expressed between Wenzhou (WZ) and Haikou (HK) rhizomes.

Fig 9. Enrichment of KEGG pathways with genes that were differentially expressed between Wenzhou (WZ) and Haikou (HK) rhizomes.

Verification of DEGs

Fig 10. qRT-PCR verification of genes that were differentially expressed between Wenzhou (WZ) and Haikou (HK) rhizomes (a) and the corresponding verification based on RNA-seq (b).

Transcription factor analysis

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Christian Schönbach

Roles

Author response to Decision Letter 0

Decision Letter 1

Christian Schönbach

Roles

Author response to Decision Letter 1

Decision Letter 2

Christian Schönbach

Roles

Acceptance letter

Christian Schönbach

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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