NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl

Yanqing Liu; Li Tang; Can Wang; Jinting Li

doi:10.1371/journal.pone.0229490

. 2020 Feb 27;15(2):e0229490. doi: 10.1371/journal.pone.0229490

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl

Yanqing Liu ¹, Li Tang ¹, Can Wang ¹, Jinting Li ^1,^2,^*

Editor: Marie-Joelle Virolle³

PMCID: PMC7046271 PMID: 32107496

Abstract

Application of plant growth regulators has become one of the most important means of improving yield and quality of medicinal plants. To understand the molecular basis of phytohormone-regulated oleanolic acid metabolism, RNA-seq was used to analyze global gene expression in Achyranthes bidentata treated with 2.0 mg/L 1-naphthaleneacetic acid (NAA) and 1.0 mg/L 6-benzyladenine (6-BA). Compared with untreated controls, the expression levels of 20,896 genes were significantly altered with phytohormone treatment. We found that 13071 (62.5%) unigenes were up-regulated, and a lot of differentially expressed genes involved in hormone or terpenoid biosynthesis, or transcription factors were significantly up-regulated. These results suggest that oleanolic acid biosynthesis induced by NAA and 6-BA occurs due to the expression of key genes involved in jasmonic acid signal transduction. This study is the first to analyze the production and hormonal regulation of medicinal A. bidentata metabolites at the molecular level. The results herein contribute to a better understanding of the regulation of oleanane-type triterpenoid saponins accumulation and define strategies to improve the yield of these useful metabolites.

Introduction

Achyranthes bidentata Blume, a member of the family Amaranthaceae, is a well-known and widely prescribed traditional Chinese herb that has been listed in the Chinese Pharmacopoeia [1]. In China, this species is primarily distributed in the Guhuaiqingfu area, located in Jiaozuo in Henan Province. A. bidentata is also called ‘Huainiuxi’. Its dried root is an important herbal medicine that is used to maintain liver and kidney function, strengthen the muscles and bones, promote blood flow, remove blood stasis, and increase longevity [2–4]. Oleanane-type triterpenoid saponins belong to the major active phytochemical compounds in A. bidentata, and possess hepatoprotective effects as well as anti-inflammatory, antioxidant, and anticancer activities [5, 6].

Oleanolic acid, a pentacyclic triterpenoid, is an isoprenoid-derived compound. The triterpenoid biosynthetic pathway has been found in many plants, such as Platycodon grandiflorum [7], Panax japonicus [8, 9], Astragalus membranaceus Bge. [10], Anemone flaccida [11], Phyllanthus amarus [12], Gynostemma pentaphyllum [13], Panax ginseng [14], Eleutherococcus senticosus [15], and A. bidentata [16]. In general, the triterpenoid biosynthetic pathway can be divided into three steps: synthesis of universal terpenoid precursors, formation of carbon skeletons, and modification of triterpenoid skeletons. Triterpenoid precursors are synthesized mainly through the cytoplasmic mevalonate (MVA) pathway. The plastidic 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway is believed to be a minor pathway for triterpenoid biosynthesis [17]. Triterpenoid skeleton synthesis genes include FPS (farnesyl diphosphate synthase), SS (squalene synthase), SE (squalene epoxidase), and OSCs (oxidosqualene cyclases). The cyclization of 2,3-oxidosqualene catalyzed by OSCs is a key step in the biosynthesis of triterpenoid saponins and sterols. β-amyrin synthase (β-AS) and cycloartenol synthase (CAS) catalyze the conversion of 2,3-oxidosqualene to β-amyrin and cycloartenol, which are precursors for oleanolic acid and phytosterols, respectively. A lot is known about several genes involved in modification of the triterpenoid skeleton downstream of the cyclization step are known. Some cytochrome P450 monooxygenases (CYP450s) and glycosyl transferases (GTs) were shown to catalyze modifications of triterpenoid skeletons, including hydroxylation and glycosidation. An increasing number of studies have indicated that the CYP716 family belongs to the CYP85 clan of CYP450s, and is involved in biosynthesis of various triterpenoids including oleanane backbones [18, 19]. For example, CYP716A12 is involved in oxidation of the β-amyrin skeleton, which modifies the oleanane backbone [20]. CYP716A52v2 catalyzes the conversion of β-amyrin to oleanolic acid in P. ginseng [21]. However, the CYP450 genes involved in the synthesis of oleanolic acid in A. bidentata are unknown.

Application of plant growth regulators has become one of the most important means of improving yield and quality of medicinal plants. Numerous studies have shown that plant growth regulators not only regulate the growth of medicinal plants, but also secondary metabolism, affecting the synthesis of phenols, terpenoids, nitrogen compounds, and other medicinally active ingredients [22]. For example, in P. quinquefolium adventitious root, treatment with methyl jasmonate (MeJA) results in an increase in ginsenoside content (43.66 mg/g compared to 8.32 mg/g in control group) [23]. In P. ginseng hairy root cultures, the three phytohormones, 2,4-dichlorophenoxyacetic acid (2,4-D), NAA, and indolebutyric acid (IBA), improved the growth of the hairy roots and promoted saponin accumulation to different degrees, and 0.5 mg/L IBA significantly promoted the accumulation of total saponins and ginsenoside Rb1 [24]. In Calendula officinalis in vitro hairy root culture, jasmonic acid (JA) treatment increased both the accumulation of oleanolic acid saponins in the hairy root tissue (up to 20-fold) and, in particular, the secretion of these compounds to the medium (up to 113-fold) [25]. In addition, in previous studies we found that application of 1.0 mg/L IBA alone and in combination with 1.0 mg/L 6-BA was advantageous to growth and increased total oleanolic acid and ecdysterone contents in A. bidentata roots. Soaking the seeds of A. bidentata with 0.15 mmol/L MeJA can increase the accumulation of oleanolic acid in its roots and leaves, which are significantly increased by 114.3% and 60% respectively compared with the control group [26, 27]. Although multiple studies have shown that combining cytokinin and auxin promotes secondary metabolism in medicinal plants, the underlying molecular signaling mechanisms are largely unknown, except for JA. In the absence of JA-Ile, the JAZ repressor proteins bind to the MYC2 transcription factor, thereby blocking downstream JA response. In the presence of JA-Ile, COI1 binds to the JAZ proteins, which leads to their ubiquination by the SCF^COI1 complex and their subsequent degradation by the 26S proteasome. Degradation of the JAZ proteins releases MYC2, leading to transcriptional activation of the JA-responsive genes. The JAZ proteins contain a conserved TIFY motif within the ZIM domain that mediates homo- and hetero-dimeric interactions between different JAZ proteins. The ZIM domain also functions to recruit transcriptional corepressors through the novel interactor of JAZ (NINJA) protein. The JAZ proteins contain a Jas domain that is required for the interaction of both COI1 and a broad array of TFs. In the presence of JA or its bioactive derivatives, JAZ proteins are degraded and freeing TFs for expression of specific sets of JA-responsive genes, regulate enzymes involved in the biosynthesis of secondary metabolites, such as ginsenoside, artemisinin, and vinblastine [28].

We have previously conducted extensive studies on the cultivation, phytochemistry, and pharmacology of A. bidentata and the relationship between plant structure and accumulation of active components. However, the molecular mechanisms of phytohormone-elicited plant growth and secondary metabolism are still under investigation, largely due to the lack of genomic information [29, 30]. In the present study, RNA-seq was used to sequence the transcriptomes of young A. bidentata leaves treated with a combination of 2.0 mg/L NAA and 1.0 mg/L 6-BA and compared to untreated controls with the aim of uncovering the molecular mechanisms of enhanced oleanolic acid production following the application of exogenous hormones.

Materials and methods

Plant materials and treatment

Seeds of A. bidentata were planted and grown in the natural environment at the Wenxian Agricultural Science Institute of Henan Province, China. 20 days after emerging, the leaves were sprayed with a mixture of different concentrations of NAA and 6-BA until there was liquid dripping at the edge of the blade. The mixture included: 1.0 mg/L NAA + 0.5 mg/L 6-BA (T1); 2.0 mg/L NAA + 1.0 mg/L 6-BA (T2); 4.0 mg/L NAA + 2.0 mg/L 6-BA (T3) and 8.0 mg/L NAA + 4.0 mg/L 6-BA (T4). Experimental controls were treated at the same time with equal volumes of distilled water. At various times after treatment, A. bidentata plants were collected to measure growth parameters. Roots, which are usually used for medicinal purposes, were collected to determine oleanolic acid content, and leaves were sampled to measure chlorophyll (Chl. a, Chl. b) and carotenoid contents, endogenous JA content, and to extract RNA for RNA-seq and quantitative real-time PCR (qRT-PCR) as described below. All experiments were performed independently in triplicate.

Determination of growth indexes and oleanolic acid content

Twenty days after treatment, A. bidentata plants from all groups were collected to measure growth parameters, including root length, fresh weight of root, dry weight of root, plant height, fresh weight of plant, and dry weight of plant. In addition, the dried roots from all groups were ground into powder to measure oleanolic acid content. Extraction and quantification of oleanolic acid by high-performance liquid chromatography (HPLC) were conducted according to previously published methods [30]. We weighed 0.5 g dry weight of A. bidentata roots powder into a triangle bottle (repeated 3 times for each sample group), added 10 mL of methanol to it, sonicated for 40 minutes, and then concentrated using a rotary vacuum evaporator to completely dry the sample. Then 10 mL of 4 mol / L hydrochloric acid was added, and the extract was hydrolyzed at 85°C for 1 h. After cooling, 10 mL of chloroform was added to the sample, and then two reflux extractions were performed at 60°C for 15 minutes each. The lower liquid was collected and concentrated to dryness under reduced pressure. The residue was dissolved in 3 mL of methanol, filtered through a filter (0.22 μm), and measured by HPLC. The assay conditions were mobile phase: methanol-water-glacial acetic acid (90:10:0.1); flow rate: 0.9 mL/min; column temperature: 25°C; detection wavelength: 210 nm.

Determination of chlorophyll (Chl. a, Chl. b) and carotenoid contents

The second pair of leaves from the top of plants (Control and T2 group) was harvested to measure the contents of chlorophyll a, b, and carotenoid. Fresh chopped leaves (0.1 g) added in 10 mL of 80% acetone (v/v) at room temperature in a dark environment for 24 h, and then measured the absorbance at 645 nm, 663 nm and 470 nm, respectively, for detecting chlorophyll a, b and carotenoids contents [31].

Assay of endogenous JA content

The leaves of the Control and T2 groups were collected at 0, 6, 12, 24, 48, and 72 h to measure endogenous JA content using HPLC. Extractions of endogenous JA were done as described previously [32].

Library construction and high-throughput RNA sequencing

Young and healthy A. bidentata leaves (2nd and 3rd leaves from the top) were treated with 2 mg/L NAA and 1 mg/L 6-BA or distilled water (as Control) and harvested separately at 0, 3, 6, and 9 days after treatment. Total RNA was extracted from each leaf sample for RNA-seq. Libraries were constructed from total RNA isolated from the Control and treated leaves. Equal amounts of total RNA extracted from samples taken at different time points were pooled together. The methods for total RNA extraction and cDNA library construction were described previously [16]. Three biological replicates were used for RNA extraction and two replicates were used for leaf transcriptome sequencing. The libraries were sequenced on the Illumina HiSeq 2500 platform. All reads generated in this study are available from the NCBI Sequence Read Archive database (http://www.ncbi.nlm.nih.gov/sra/) under the project accession number PRJNA350183.

RNA sequencing data analysis

The raw RNA-seq reads were processed to remove sequencing adapters and low quality bases using Trimmomatic [33], and clean reads shorter than 80 bp were discarded. Then, the high-quality reads were assembled into unigenes with previously published data [16] using Trinity [34] with “min_kmer_cov” set to 10. The unigenes were compared to the ribosome RNA database [35] and GenBank Nucleotide (nt) database using BLAST+ [36] to remove ribosome RNA, viral and bacterial DNA, and other contaminants. To remove redundancies in the cleaned unigenes, they were further de novo assembled using iAssembler [37] with 97% minimum percent identify. The high-quality clean reads were aligned back to non-redundant unigenes using Bowtie [38], allowing up to 2 mismatches. Reads per kilobase of exon model per million mapped reads (RPKM) was calculated to determine the expression levels of unigenes. The unigenes with extremely low expression (RPKM < 0.001) in all samples were discarded in downstream analysis.

The functional descriptions of unigenes were predicted using automated assignment of human readable descriptions (AHRD: https://github.com/groupschoof/AHRD). The unigenes were queried against the UniProt database using BLAST+ [36], and the Gene ontology (GO) terms were assigned to the unigenes based on the GO terms annotated to their corresponding homologs in the UniProt database. Biochemical pathways were predicted based on the AHRD and GO annotation of unigenes using the Pathway Tools program [39].

Statistical analysis was performed using DESeq [40] and those unigenes with fold changes ≥2 or ≤0.5 and with adjusted P < 0.05 were categorized as differentially expressed genes (DEGs). GO and pathway enrichment analysis was done to gain insight into the biological function of DEGs. GO enrichment analysis of DEGs was implemented in GO::TermFinder [41]. The pathway enrichment analysis was performed using the in-house Perl scripts. The raw P values generated by GO and pathway enrichment analysis were corrected using Benjamini-Hochberg methods [42] and the corrected P < 0.05 were considered significantly enriched in DEGs.

Phylogenetic analysis

Phylogenetic analysis was performed based on deduced amino acid sequences of CYP450 from A. bidentata and other plants. All of the deduced amino acid sequences were aligned with Clustal W with a delay divergent cutoff of 30%, and the other parameters set to defaults as described previously [43]. The evolutionary distances were computed using the Jones-Taylor-Thornton (JTT) method. For the phylogenetic analysis, a neighbor-joining tree was constructed with bootstrap values obtained after 1000 replications using MEGA7.0 [44].

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation and expression analysis

Six DEGs involved in triterpenoid saponin metabolic pathways were chosen for real-time qRCR verification of the Illumina RNA-seq results. The gene-specific primers, designed using Primer Premier 5.0 software, are listed in S1 Table. All reactions were performed in 96-well plates in a LightCycler 96 (Roche, Switzerland) using SYBR^®Green Master Mix (Vazyme, China) according to the manufacturer’s instructions. All reactions were performed for three biological replicates with three technical replicates per experiment, and for each sample the results were expressed relative to the expression levels of an internal reference gene, Actin (UN011760) using the 2^–ΔΔCt method.

Data analysis

Results are presented as means ± standard deviations. All data were subjected to one-way analysis of variance (ANOVA), and means were compared with Student’s t-test at a 5% or 1% level of probability using SPSS 20.0.

Results

Effects of NAA and 6-BA on the growth of A. bidentata

The growth parameters of the plants 20 days after treatment with various combinations of NAA and 6-BA are presented in Fig 1. Lower concentrations of NAA and 6-BA (treatment T1 and T2) improved these parameters, while higher concentrations (particularly T4) exerted adverse effect. Under T2 treatment, all morphological parameters were at the highest values, and root length, fresh weight of root, dry weight of root, plant height, fresh weight of plant, and dry weight of plant were significantly increased by 19.9%, 39.5%, 35.6%, 15.9%, 42.4%, and 19.2%, respectively, compared to the Control (P < 0.05). Chlorophyll a, chlorophyll b and carotenoid contents in T2 group leaves were measured, and compared with the Control group, all were significantly decreased (S2 Table). The results indicated that 2.0 mg/L NAA and 1.0 mg/L 6-BA treatment significantly reduced biosynthesis of photosynthetic pigments.

Effects of NAA and 6-BA on oleanolic acid content in A. bidentata roots

In this study, the roots of A. bidentata were collected 20 days after treatment to measure the levels of oleanolic acid. The oleanolic acid content in all treatment groups except T4 was significantly higher than in Control roots; the content in T1 was the highest, followed by T2, and oleanolic acid contents in T1 and T2 were not significantly different (Fig 2). These results indicated that low concentrations of NAA and 6-BA combinations significantly promoted the accumulation of oleanolic acid, while high concentrations inhibited accumulation. Therefore, the T2 group was taken as the focus of our investigation and analysis in this study. Furthermore, to determine the molecular mechanism of auxin and cytokinin-induced changes in oleanolic acid accumulation, we used Control and T2 (2.0 mg/L NAA and 1.0 mg/L 6-BA treatment) A. bidentata leaves as experimental materials for constructing Control and treated (T) RNA-seq libraries, respectively.

Fig 2 — T1: 1.0 mg/L NAA + 0.5 mg/L 6-BA; T2: 2.0 mg/L NAA + 1.0 mg/L 6-BA; T3: 4.0 mg/L NAA + 2.0 mg/L 6-BA; T4: 8.0 mg/L NAA + 4.0 mg/L 6-BA. Each value represents the mean (±SD) of three experiments. Statistical analysis was performed with one-way ANOVA, and Student’s t-tests were performed to compare the differences in mean oleanolic acid content among different treatments. P < 0.05 was considered statistically significant. Different letters indicate significant differences between groups.

RNA-seq analysis of A. bidentata leaves

The transcriptomes of Control and T leaves were sequenced in order to gain a comprehensive overview of the transcriptional response of A. bidentata to NAA and 6-BA. To enhance data reliability, cDNA libraries were prepared for two biological repeats of each sequencing sample. Combined with clean reads generated by our previous study [16], about 164 million clean read pairs (16.4 Gbp) were used for de novo transcriptome assembly. A total of 102,126 unigenes were assembled. More than 80% of clean reads could be mapped back to these assembled unigenes, indicating that the assemblies were good quality and suitable for downstream analysis (Table 1).

Table 1. RNA-seq reads for four RNA-seq libraries.

Summary	Control-rep1	Control-rep2	T-rep1	T-rep2
No. of raw reads	35,359,331	27,630,495	32,028,032	33,307,916
No. of clean reads	29,637,493	22,759,674	26,819,403	27,707,163
Total % of clean reads	83.82	82.37	83.74	83.18
No. of mapped reads	23,895,374	18,503,286	21,457,401	22,285,936
Total % of mapped reads	80.63	81.30	80.01	80.43

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Changes in gene expression profiles in leaves treated with NAA and 6-BA

Among A. bidentata unigenes, 53.8% and 59.4%, respectively, expressed in the Control and T samples, had RPKM values in the range of 1–100, while 44.9% and 39.3%, respectively, had RPKM values in the range of 0–1 (Fig 3). Differentially expressed genes (DEGs) between the Control and T samples were identified. Compared with Control, a total of 20,896 genes were significantly differentially expressed in the T group, among which 13,071 (62.55%) were up-regulated and 7,825 (37.45%) were down-regulated. These results are consistent with the higher number of unigenes in the T group than the Control group with RPKM values in the range of 1–100. These results suggest that NAA and 6-BA induced the expression of a large number of genes.

Fig 3 — The different colors indicate the percentage of genes with the range of RPKM values shown in the legend.

To investigate the functions of all 20,896 DEGs, GO and pathway enrichment analysis was applied. DEGs annotated in GO were grouped into 47 groups based on GO level2 classification (S1 Fig). The assigned GO terms belonged to three main ontologies: biological process, cell component, and molecular function with 22, 14, and 11 groups, respectively. In the biological process group, the most common categories were “cellular process” (11,362, 54.4%), “single-organism process” (10,434, 49.9%) and “metabolic process” (9,793, 46.9%). In the cell component group, “cell”, “cell part” and “organelle” were the top three categories. Most DEGs categorized in molecular function were involved in “binding” and “catalytic activity”.

To further identify metabolic or signal transduction pathways in which the DEGs are likely to be involved in promoting accumulation of oleanolic acid by JA regulation by NAA and 6-BA treatment in A. bidentata, pathway enrichment analysis was performed using KEGG database. For the pathway enrichment analysis, a total of 28 pathways were enriched (adjusted P < 0.05) with DEGs (Table 2). These pathways were divided into five categories, including metabolite degradation, photosynthesis, carbohydrate metabolism, terpenoid biosynthesis, and biosynthesis of other compounds. In the metabolite degradation pathways, 58 DEGs were predicted to be involved in processes that detoxify hydrogen peroxide, including baicalein, betanidin, and luteolin triglucuronide degradation processes. For instance, the expression levels of 43 DEGs encoding peroxidase were significantly increased in the T group compared with Control. Photosynthesis metabolic pathways, such as oxygenic photosynthesis, NAD-ME-type and PEPCK-type photosynthetic carbon assimilation cycle, the Calvin-Benson-Bassham cycle, and the chlorophyll cycle were enriched in DEGs. In treated leaves, most DEGs involved in oxygenic photosynthesis, the Calvin-Benson-Bassham cycle and the chlorophyll cycle were down-regulated except for 3 transketolases. However, 12 malic enzyme genes, 6 out of 10 aspartate aminotransferase genes (AST), and 8 phosphoenolpyruvate carboxykinase genes (PEPCK) were up-regulated in the T group, and are involved in NAD-ME-type and PEPCK-type photosynthetic carbon assimilation cycle metabolic pathways. These results and the reduction in the levels of the photosynthetic pigments chlorophyll a and b and carotenoid (S2 Table) suggest that photosynthesis was inhibited by hormone treatment.

Table 2. Pathway enrichment analysis of genes differentially expressed in phytohormone-treated A. bidentata leaves.

Pathway ID	Pathway Name	Nd	Bn	p value	adj P
Degradation
PWY-7214	baicalein degradation (hydrogen peroxide detoxification)	58	106	2.24E-06	1.51E-04
PWY-5461	betanidin degradation	58	106	2.24E-06	1.51E-04
PWY-7445	Luteolin triglucuronide degradation	58	106	2.24E-06	1.51E-04
PWY-1081	homogalacturonan degradation	30	49	4.03E-05	1.26E-03
MANNCAT-PWY	D-mannose degradation	5	5	3.86E-03	4.82E-02
Photosynthesis
PHOTOALL-PWY	oxygenic photosynthesis	38	64	1.06E-05	5.96E-04
PWY-7115	photosynthetic carbon assimilation cycle, NAD-ME type	33	60	3.15E-04	6.64E-03
PWY-7117	photosynthetic carbon assimilation cycle, PEPCK type	30	52	1.89E-04	4.26E-03
CALVIN-PWY	Calvin-Benson-Bassham cycle	28	49	3.86E-04	7.64E-03
PWY-5068	chlorophyll cycle	9	9	4.48E-05	1.26E-03
Carbohydrate metabolism
GLUCONEO-PWY	gluconeogenesis I	39	80	2.17E-03	3.18E-02
PWY-4821	UDP-D-xylose biosynthesis	17	18	7.28E-08	1.23E-05
PWY-7346	UDP-alpha-glucuronate biosynthesis (from UDP-glucose)	5	5	3.86E-03	4.82E-02
Terpenoid biosynthesis
PWY-5910	superpathway of geranylgeranyl diphosphate biosynthesis I (via mevalonate)	36	61	2.19E-05	9.23E-04
PWY2OL-4	superpathway of linalool biosynthesis	29	45	1.32E-05	6.34E-04
PWY-7182	linalool biosynthesis I	28	44	2.64E-05	9.87E-04
PWY-7141	linalool biosynthesis II	21	37	2.32E-03	3.25E-02
PWY-5122	geranyl diphosphate biosynthesis	20	36	4.11E-03	4.95E-02
PWY-6475	trans-lycopene biosynthesis II (plants)	19	33	2.98E-03	4.02E-02
PWY-922	mevalonate pathway I	15	22	7.36E-04	1.31E-02
PWY-7186	superpathway of scopolin and esculin biosynthesis	10	13	1.46E-03	2.34E-02
PWY-5725	farnesene biosynthesis	8	8	1.36E-04	3.29E-03
PWY-6275	beta-caryophyllene biosynthesis	8	8	1.36E-04	3.29E-03
PWY-5670	epoxysqualene biosynthesis	6	6	1.27E-03	2.14E-02
Others
PWY0-541	cyclopropane fatty acid (CFA) biosynthesis	26	27	4.42E-12	1.49E-09
PWY-6163	chorismate biosynthesis from 3-dehydroquinate	17	26	6.79E-04	1.27E-02
PWY-7498	phenylpropanoids methylation (ice plant)	13	19	1.61E-03	2.47E-02
PWY-2181	free phenylpropanoid acid biosynthesis	9	9	4.48E-05	1.26E-03

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Bn (Background number) indicates the total number of unigenes present in each pathway. Nd (Number of DEGs) indicates the number of differentially expressed unigenes in each pathway.

For carbohydrate metabolism, there were three categories enriched in DEGs: gluconeogenesis I, UDP-D-xylose biosynthesis, and UDP-alpha-D-glucuronate biosynthesis. Genes encoding malic enzyme and phosphoenolpyruvate carboxylase, which participate in the gluconeogenesis I pathway, were up-regulated in treated leaves. All DEGs involved in UDP-D-xylose biosynthesis and UDP-alpha-D-glucuronate biosynthesis were up-regulated. For terpenoid metabolism, the top three pathways enriched in DEGs were the geranylgeranyl diphosphate biosynthesis I super pathway, the linalool biosynthesis super pathway, and linalool biosynthesis I. It is worth noting that mevalonate pathway I, farnesene biosynthesis, and epoxysqualene biosynthesis pathway were enriched in DEGs, and the expression levels of 26 out of 29 DEGs in these pathways were significantly increased in treated leaves. For instance, the expression level of HMGR, which encodes the first rate-limiting enzyme involved in the MVA pathway, was 501.66 times higher in T than in Control leaves. These results indicate that triterpenoid biosynthesis is induced by NAA and 6-BA treatment. For biosynthesis of other compounds, the cyclopropane fatty acid, chorismate, and free phenylpropanoid acid biosynthesis pathways were enriched in DEGs. All DEGs involved in these pathways were down-regulated except for the cyclopropane fatty acid synthase gene. The results of pathway enrichment analysis suggest that the expression of A. bidentata genes involved in photosynthesis metabolic pathways and terpenoid metabolic processes are affected by the combination of 2.0 mg/L NAA and 1.0 mg/L 6-BA. In addition, this is the first report of C4 type photosynthetic carbon assimilation cycle in A. bidentata suggesting that A. bidentata may be a C4 plant. About 28% of the 900 species in the Amaranthaceae estimated to occur in the family are C4 species, such as Gomphrena meyeniana, Amaranthus hypochondriacus [45].

DEGs involved in JA signaling pathways

Seventeen candidate DEGs were predicted to be involved in JA signaling pathways (Table 3), which regulate the synthesis of secondary metabolites [46]. Most DEGs involved in JA signaling were up-regulated. Our results suggest that the expression of JA-responsive genes was affected by treatment with 2.0 mg/L NAA + 1.0 mg/L 6-BA.

Table 3. Differentially expressed genes in response to NAA and 6-BA treatment in A. bidentata.

JA Signaling	Number of DEGs	Up-regulated genes	Down-regulated genes
Jasmonate ZIM domain protein	7	7	0
Jasmonate-induced protein	2	2	0
Methyl esterase 1	4	1	3
Ninja-family protein	2	2	0
Topless-related protein	2	2	0
Total	17	14	3

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Transcription factors differentially expressed in response to auxin and cytokinin treatment

Several TF families such as MYC, MYB, WRKY, and AP2/ERF are involved in regulating secondary metabolism in different medicinal plants [28]. Therefore, we asked whether DEGs included TFs. We found that 308 TFs were differently expressed in the T group compared with the Control group, including 17 MYBs, 21 bHLHs, 14 WRKYs, and 21 bZIPs (Table 4 and S3 Table). Many more TFs were up-regulated than down-regulated. WRKYs and bZIPs were mainly down-regulated, while AP2-ERFs, ERFs, bHLH, MADS, heat stress transcription factors, GATAs, and Nuclear transcription factor Y were mainly up-regulated.

Table 4. Transcription factors differentially expressed in response to auxin and cytokinin treatment in A. bidentata.

TF family	Number of DEGs	Up-regulated TF genes	Down-regulated TF genes
MADS box transcription factor	34	27	7
Ethylene-responsive transcription factor	31	20	11
AP2-like ethylene-responsive transcription factor	21	19	2
bHLH transcription factor	21	16	5
bZIP transcription factor	21	7	14
Heat stress transcription factor	19	12	7
Nuclear transcription factor Y	17	16	1
MYB transcription factor	17	10	7
WRKY transcription factor	14	4	10
GATA transcription factor	7	5	2
Others	106	77	29
Total	308	213	95

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DEGs involved in the oleanolic acid biosynthesis pathway

In the present study we identified a total of 395 unigenes (S4 Table) encoding almost all the enzymes known to be involved in oleanolic acid biosynthesis via the MVA and MEP pathways, including 200 DEGs. The DEGs related to oleanolic acid metabolism are schematically represented in Fig 4. In the MVA pathway, the transcription level of unigenes involved in terpenoid precursor biosynthesis, such as HMGS (HMG-CoA synthase), HMGR (HMG-CoA reductase), PMK (phosphomevalonate kinase), MDD (mevalonate-5-diphosphate decarboxylase), and IDI (isopentenyl diphosphate isomerase), were up-regulated by 2.0 mg/L NAA and 1.0 mg/L 6-BA. In contrast, most terpenoid precursor synthesis genes in the MEP pathway, such as DXS (1-deoxy-D-xylulose5-phosphate synthase), CMS (2-C-methyl-D-erythritol 4-phosphate cytidylyl-transferase), HDS (4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase), and HDR (4-hydroxy-3-methylbut-2-enyl diphosphate reductase), were down-regulated by NAA and 6-BA treatment. Triterpenoid skeleton synthesis genes, such as FPS, SS, SE and β-AS were also up-regulated in the T group. In the stage when triterpenoid skeletons are modified, the hydroxylation and glycosylation processes, which are catalyzed by cytochrome P450 monooxygenases and glycosyl transferases in turn, are important for the production of triterpenoid saponins. In this study, 186 unigenes were annotated as CYP450s, including 41 up-regulated and 48 down-regulated unigenes in the T group. In order to further narrow the potential range of CYP450s involved in biosynthesis of oleanolic acid saponin, 22 up-regulated CYP450s (with amino acid sequence lengths > 450) were selected for phylogenetic analysis from A. bidentata treated leaves. As a template, 245 CYP450 annotated genes in the Arabidopsis genome were obtained from http://www.p450.kvl.dk/p450.shtml. Twenty-two unigenes (S6 Table) from A. bidentata were clustered in the CYP71 (10 unigenes), CYP711 (5 unigenes), CYP85 (3 unigenes), CYP72 (2 unigenes), and CYP86 (2 unigenes) clans (S2 Fig). After phylogenetic analysis of five unigenes and fifty-five CYP450s (S5 Table) related to triterpene synthesis (S4 Fig), and the CYP450s with similar functions were further analyzed with five unigenes. Among them, unigene UN082587 was annotated to P. ginseng CYP716A52v2 (Fig 5). This result indicated that the UN082587 gene might encode an enzyme that converts β-amyrin to oleanolic acid. Surprisingly, UN082587 was 117.8 times higher in T than Control leaves. Three unigenes (UN046523, UN046524 and UN085763) were homologous to Glycyrrhiza uralensis CYP88D6 (β-amyrin 11-oxidase). A total of 77 DEGs encoding glycosyl transferases were identified, 35 of which were up-regulated in treated leaves. These results indicate that the expression level of most genes involved in oleanolic acid biosynthesis were up-regulated by NAA and 6-BA treatment.

Fig 4 — For each gene, the top row of squares represents the Control and the bottom row represents the NAA and 6-BA treatment. The number of squares indicates the number of differentially expressed genes with fold changes ≥ 2 or fold change ≤ 0.5 at adjusted P < 0.05. Different colors indicate genes that were down-regulated (green) or up-regulated (red) in the T group compared with the Control group. Abbreviations are as follows: AACT, acetyl-CoA acetyltransferase; MK, mevalonate kinase; DXR, 1-deoxy-D-xylulose5-phosphate reductoisomerase; CMK, 4-(cytidine-diphospho)-2-C-methyl-D-erythritol kinase; MCS, 2-C-methyl-D-erythritol-2,4- cyclodiphosphate synthase; GPPS, geranyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; DDS, dammarenediol synthase; LS, lanosterol synthase; LUS, lupeol synthesis; and UGT, UDP-glycosyltransferase.

Fig 5 — The phylogenetic tree was constructed based on the deduced amino acid sequences for the A. *bidentata* CYP450s (red circle) and other plant CYP450s involved in triterpenoid biosynthesis. The protein sequence ID was retrieved from the NCBI GenBank using the accession numbers marked in red provided in S5 Table.

RNA-seq accuracy determined by real-time PCR

To confirm the reliability of the RNA-seq data, the expression levels of six DEGs involved in the oleanolic acid biosynthesis pathways (HMGR, PMK, FPS, SS, SE, and β-AS) were validated using qRT-PCR. The expression patterns of these genes in the T and Control leaves were consistent with the expression patterns based on RNA-seq, and there was a close correlation (r = 0.94) between the expression changes determined by RNA-seq and by qRT-PCR (Fig 6).

Discussion

JA-mediated transcriptional regulation of oleanolic acid metabolism

In the present study, the levels of JA, stress hormones that regulate the synthesis of secondary metabolites, increased in A. bidentata leaves treated with 2.0 mg/L NAA + 1.0 mg/L 6-BA (S3 Fig). This is consistent with a previous finding that endogenous JA concentration was significantly increased by 2.65-fold after NAA treatment in Chlorella vulgaris [46]. The increase in JA may be due to direct activation of the JA signaling pathway or may be indirectly caused by the accumulation of IAA due to the collaborative relationship between IAA and JA [47].

JA ZIM domain (JAZ) is a key molecule that serves as the on/off switch for JA signaling. In the presence of JA or its bioactive derivatives, JAZ proteins are degraded, freeing TFs for expression of specific sets of JA-respective genes, thereby promoting physiological activity, including producing specific sets of secondary metabolites [48]. In addition to the F-box protein COI1, some transcription factors were JAZ interactors including bHLH TFs, R2R3 MYB TFs, and TFs of other hormone signaling pathways [49]. Multiple experiments have demonstrated that MYC, MYB, WRKY and AP2/ERF TFs are involved in the regulation of terpenoid metabolism in different medicinal plants [28, 50]. For example, AaWRKY1, TSAR1 (triterpene saponin activating regulator), and TSAR2 regulate HMGR expression involved in the MVA pathway as a rate-limiting enzyme in Artemisia annua and Medicago truncatula [50, 51]. The bHLH transcription factors TSAR1 and TSAR2 are two homologous jasmonate-inducible transcription factors [51]. In M. truncatula hairy roots, overexpression of TSAR1 and TSAR2 resulted in significantly elevated transcript levels of the MVA pathway genes and all consecutive genes involved in the generation of the β-amyrin backbone, such as HMGS, HMGR1, SS, and β-AS [51, 52]. GbWRKY1 regulates GbMVD expression involved in terpene trilactone biosynthesis by binding to the W-box in Ginkgo biloba [53]. In the present study, most AP2/ERF TFs, bHLH TFs, and MYB TFs were up-regulated in the T group, with significantly increased transcript levels of terpenoid biosynthesis genes, especially HMGR, SS, SE, and β-AS in A. bidentata (Fig 7). Therefore, JA regulates oleanolic acid accumulation by freeing TFs and increasing expression of terpenoid biosynthesis genes in A. bidentata. These results were consistent with overexpression of TSARs to promote oleanane-type triterpene saponin in M. truncatula.

NAA and 6-BA promote terpenoid biosynthesis and photosynthesis processes

For the pathway enrichment analysis of DEGs, most metabolic pathways were classified as terpenoid biosynthesis or photosynthesis. For terpenoid biosynthesis, three linalool biosynthesis pathways and geranyl diphosphate biosynthesis pathway were identified, and most DEGs involved in their biosynthesis pathway were down-regulated, especially 1, 4-dihydroxy-2-naphthoate (DHNA) polyprenyltransferase. Previous studies demonstrated that DHNA polyprenyltransferase belongs to the prenyltransferases family of aromatic prenyltransferases and catalyzes the transfer of prenyl moieties to aromatic acceptor molecules, forming C–C bonds between C-1 or C-3 of the isoprenoid substrate and one of the aromatic carbons of the acceptor substrate [54, 55]. The prenyl moiety is derived from allylic isoprenyl diphosphates including dimethylallyl diphosphate (DMAPP; C5), geranyl diphosphate (GPP; C10), and farnesyl diphosphate (FPP; C15). These allylic isoprenyl diphosphates were substrates of triterpenoid biosynthesis. Therefore, more and more allylic isoprenyl diphosphates were involved in triterpenoid biosynthesis and promoted accumulation of oleanolic acid due to low expression levels of 1, 4-dihydroxy-2-naphthoate polyprenyltransferase in the T group. For photosynthesis, most genes of malic enzyme, AST and PEPCK were up-regulated and involved in NAD-ME-type and PEPCK-type photosynthetic carbon assimilation cycle metabolic pathways in treated leaves. The use of C4 pathway is conducive to the use of low concentrations of carbon dioxide, enhancing plant stress resistance. Therefore, hormones affect the level of endogenous JA and promote oleanolic acid biosynthesis.

Conclusions

Transcriptomes of A. bidentata leaves sprayed with 2.0 mg/L NAA + 1.0 mg/L 6-BA allowed us to gain insight into the molecular mechanisms of the response to phytohormone treatment. A total of 20,896 DEGs were identified, and 62.55% of these DEGs were up-regulated by phytohormone treatment. The pathway enrichment analysis demonstrated that photosynthesis metabolic pathways and terpenoid biosynthesis were overrepresented by genes differentially expressed in the treatment group, suggesting that photosynthesis and terpenoid metabolic processes are affected by treatment with 2.0 mg/L NAA and 1.0 mg/L 6-BA. Furthermore, the expression levels of genes involved in JA signaling were differentially expressed between Control and T. The endogenous JA content of treated leaves also significantly increased over time, suggesting that endogenous JA levels were regulated by exogenous phytohormones. In conclusion, exogenous phytohormones work in conjunction with endogenous phytohormones to promote the accumulation of oleanolic acid in A. bidentata.

Supporting information

S1 Fig. GO enrichment analysis for the differentially expressed genes in A. bidentata.

(TIF)

Click here for additional data file.^{(3.2MB, tif)}

S2 Fig. Phylogenetic tree constructed based on the deduced amino acid sequences of the A. bidentata CYP450s and all Arabidopsis CYP450s.

The phylogenetic tree was generated using the neighbor-joining (NJ) method in MEGA6.

(TIF)

Click here for additional data file.^{(8.1MB, tif)}

S3 Fig. Effects of exogenous NAA and 6-BA on JA contents in A. bidentata leaves.

(TIF)

Click here for additional data file.^{(902.3KB, tif)}

S4 Fig. Phylogenetic tree constructed based on the deduced amino acid sequences of the five A. bidentata CYP450s and 55 CYP450s related to triterpene synthesis.

The phylogenetic tree was generated using the neighbor-joining (NJ) method in MEGA6.

(TIF)

Click here for additional data file.^{(951.3KB, tif)}

S1 Table. Primers used for qRT-PCR analysis.

(DOCX)

Click here for additional data file.^{(14.3KB, docx)}

S2 Table. Photosynthetic pigment content in leaves.

(DOCX)

Click here for additional data file.^{(13.8KB, docx)}

S3 Table. List of transcription factors.

(DOCX)

Click here for additional data file.^{(68.7KB, docx)}

S4 Table. List of candidate genes encoding enzymes in the oleanolic acid biosynthesis pathways.

(DOCX)

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

S5 Table. List of fifty-five CYP450s related to triterpene synthesis in constructing phylogenetic tree.

The CYP450s marked in red represent those used in the construction of Fig 5.

(DOCX)

Click here for additional data file.^{(17.5KB, docx)}

S6 Table. List of twenty-two unigenes of A. bidentata used to build phylogenetic tree.

The unigenes marked in red represent those used in the construction of Fig 5.

(DOCX)

Click here for additional data file.^{(15.1KB, docx)}

Data Availability

All the readings obtained in this study have been uploaded to the NCBI Sequence Read Archive, and the accession number is PRJNA350183. All other relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The National Nature Science Foundation of China (81274076) and the Key Projects of Henan Province Colleges and Universities (17A180026).

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PLoS One. doi: 10.1371/journal.pone.0229490.r001

Decision Letter 0

Marie-Joelle Virolle

4 Sep 2019

PONE-D-19-21410

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl. through de novo transcriptomics

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Reviewer #1: The manuscript by Liu and co-workers describes the investigation of the oleanolic acid biosynthesis in the Chinese medicinal plant Achyranthes bidentata. Compared to control plants, A. bidentata plants exogenously treated with NAA and 6BA accumulated higher amounts of oleanolic acid, a major phytochemical in the roots of the plant. RNA-Seq analysis of control and treated plants revealed that almost all genes encoding enzymes involved in oleanolic acid biosynthesis were upregulated upon NAA and 6BA treatment. Furthermore, also genes involved in JA signaling and a large amount of transcription factors were NAA/6BA-responsive. As also an increased JA accumulation was observed, the authors conclude that combined auxin and cytokinin treatment leads to increased oleanolic acid accumulation via a JA-mediated signaling cascade. To my opinion this conclusion is not supported by the provided evidence (for more details, see my specific comments below).

Remarks:

1/ Lines 47-50 needs a reference.

2/ Lines 52-54: OSCs are not generally known as rate-limiting enzymes in the biosynthesis of sterols and/or triterpenoids. Rather, HMGR and SQE are. To be either corrected or supported by references that support the notion of OSCs being rate-limiting enzymes.

3/ Line 56: Cholesterol? Apart from Solanaceae, cholesterol is only a minor plant sterol.

4/ Lines 87-89: explain better the JA signaling mechanism; it is a bit too summarily described here.

5/ Lines 104-107: how exactly were the plants treated? Spraying? If so, how much (volume) was sprayed on the plants?

6/ Lines 126-128: this sentence is not clear to me.

7/ Lines 252-254: this is not clear to me, how can you have such high percentages for the different processes, unless the genes are part of different processes?

8/ Lines 297-299: nice observation, it could be supported by the fact that other amaranthaceae are known to be C4 plants.

9/ Lines 313-314: Use bZIP and bHLH as abbreviation.

10/ Lines 346-347: What is the reason the CYP88D6 orthologs were pointed out? Likely the encountered CYP88 genes are involved in ent-kaurene biosynthesis rather than triterpene biosynthesis. Furthermore, oxidation at the C-11 position is not required for oleanolic acid biosynthesis.

11/ Lines 347-349: What is the reason the differentially expressed UGTs are indicated? No UGTs are required for the oleanolic acid biosynthesis.

12/ Remark Figure 4. I don’t entirely agree with the downstream MVA pathway presented in Figure 4. There is a differentiation between the cytosolic FPP production, which is done by a single FPS enzyme that condenses two units of IPP and one unit of DMAPP and the plastidial FPP production, which is catalyzed by two distinct enzymes, GPPS and FPS. The cytosolic pathway leads to sesquiterpenes and triterpenes, the plastidial pathway leads to mono-, di- and tetraterpenes. In this respect it seems to me that all plastid-derived metabolite biosynthesis pathways (linalool = monoterpene) are down, whereas all cytosolic (triterpene and farnesene = sesquiterpene) are upregulated by the NAA/6BA treatment. Another remark on figure 4 is the inclusion of the ginsenoside biosynthesis. Why is this included here? Are ginsenosides reported in A. bidentata?

13/ Remark Figure 5: Why were these specific P450s chosen? Many more have been shown to be involved in triterpene biosynthesis. Either provide an explanation for the limited choice of P450s or be more complete and include all P450s involved in triterpene or oleanane biosynthesis.

14/ Line 374: A link is made between JAZ repressors and transcription factors (like bHLH) that bind these JAZ proteins. However, the TSAR transcription factors, for instance, do not have a JAZ interaction domain (JID) and as such they don’t interact with JAZ. Yet they are involved in the regulation of triterpene biosynthesis.

15/ Lines 389-391: The conclusion that JA regulates oleanolic acid accumulation is not supported by the provided evidence. You show that JA levels and JA response are upregulated upon NAA/6BA treatment and that triterpene levels are upregulated upon NAA/6BA treatment. This is only a correlation, not necessarily a causation. To point to a possible involvement of JA signaling, it would be more convincing to carry out a time-dependent transcript profiling of some JAZ/TF/JA genes, and some triterpene biosynthesis genes. Only if the JAZ/TF/JA genes are upregulated before the triterpene biosynthesis genes you can point to a possible role of the JA signaling in the increased biosynthesis of oleanolic acid. Another option is to carry out a JA-treatment of your plants and measure the effect of it on oleanolic acid accumulation or transcription of OA biosynthesis genes. In a perfect world you could also work in JA biosynthesis or signaling mutants, but this is clearly impossible in your plant species…

16/ Lines 410-411: tertripenoid?

Reviewer #2: The research subject of this manuscript is interesting and valuable because data on regulation of triterpenoid biosynthesis in medicinal plants are still scarce. However, I have several doubts about the experimental model, moreover, some obtained results are difficult to understand.

Line 40. „Oleanolic acid, a pentacyclic triterpenoid saponin”. Oleanolic acid is not a saponin! It is an aglycon of numerous saponins, but it can also occur in plants in a free form. The Authors are not explaing if Achyrantes bidentata contains oleanolic acid in a free form or in the form of glycosides (saponins). It is rare that there is only one saponin in the plant, usually there are several, with different sugar chains. Moreover, the Authors are not describing the details of extraction and HPLC metods which they used for oleanolic acid determination (the reference 29 does not contain a suitable description of these procedures!). It is not clear if extracts were purified, if they were hydrolyzed (to release oleanolic acid from its glycosides), what conditions were used for HPLC etc.

Line 103. „Seeds of A. bidentata were planted and grown in the natural environment at the

Wenxian Agricultural Science Institute of Henan Province, China.” Why these conditions („natural”) have been chosen? Plants were then growing during quite a long time (e.g., 20 days after treatment), they could be exposed to the influence of various abiotic and biotic stresses (drought, temperature, light, herbivore attack, pathogen infection), which can change the biosynthesis and accumulation of oleanolic acid as a result of the response of secondary plant metabolism to stress conditions. So it cannot be concluded that the changes in oleanolic acid content are only due to the hormonal treatment. It is true that Authors are comparing the treated samples with the controls, but it cannot be excluded that the controls had slightly different growth conditions (the spraying with hormones could for example act as an attractant or repelent for some insects or microorganisms). Authors should explain why they have chosen „natural” conditions for their experiments, instead of the controlled conditions in a green house.

Line 202. „After 20 days of treatment, the root length, fresh weight of root, dry weight of root, plant height, fresh weight of plant, and dry weight of plant had the same trend of first increasing then decreasing”. This is very difficult to understand and hard to believe. The plants were suddenly shrinking? How the root length and plant height can be at first bigger, and than smaller?

Line 204. „Lower concentrations of NAA and 6-BA improved these indexes while higher concentrations inhibited these indexes”. This is an awkward expression. Indexes cannot be inhibited. Phenomenon or processes can, but not indexes which are just the parameters.

Line 257. For the pathway enrichment analysis, a total of 28 pathways were enriched. What do it mean?

Line 318.” …almost all the enzymes known to be involved in oleanolic acid biosynthesis via the MVA and MEP pathways..”. The participation of MEP pathway in oleanolic acid biosynthesis – in normal conditions - is very doubful. The possibility of the cross-talk between these two pathway cannot be ruled out but it usually occurs in special circumstances, particularly when the MVA pathway is blocked or inhibited. Do the Authors expect such conditions after applied treatment?

The style of writing is sometimes a little chaotic and full of repetitions.

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Reviewer #1: Yes: Jacob Pollier

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PLoS One. 2020 Feb 27;15(2):e0229490. doi: 10.1371/journal.pone.0229490.r002

Author response to Decision Letter 0

29 Sep 2019

Responds to academic editor:

1.I have carefully checked the manuscript, and the manuscript has met PLOS ONE's style requirements, including those for file naming.

2.I have modified the manuscript for language usage, spelling, and grammar and sincerely hope that it can meet the requirements of the magazine.

My colleague, Li Tang, and I participated in the revision of the manuscript,and my name is Yanqing Liu.

For the resubmission, I have provide the following files:

“Response to Reviewers”;

“Revised Manuscript with Track Changes” (uploaded as a *supporting information* file) and a clean copy of the edited manuscript (uploaded as the new *manuscript* file).

3.In my Methods section, I have provided additional details regarding the A. bidentata seeds used in the study.

4.In the revised cover letter, I have provided the relevant accession numbers that may be used to access data. All the readings obtained in this study have been uploaded to the NCBI Sequence Read Archive, and the accession number is PRJNA350183.

Responds to reviewers:

Reviewer #1

1/ Lines 47-50

I have added the following references:

Miettinen K, Iñigo S, Kreft L, Pollier J, De Bo C, Botzki A, et al. The TriForC database: a comprehensive up-to-date resource of plant triterpene biosynthesis. Nucleic Acids Res. 2018; 46(D1): D586-D594. https://doi.org/10.1093/nar/gkx925 PMID:PMC5753214

2/ Lines 52-54

The previous paper was incorrectly stated. I have changed the content of the paper that “The cyclization of 2,3-oxidosqualene catalyzed by OSCs is a key step in the biosynthesis of triterpenoid saponins and sterols”.

3/ Line 56

I have mistakenly spelled before, and have already changed the word “Cholesterol” to “phytosterol”.

4/ Lines 87-89 (changed to lines 87-98)

A more detailed statement about the JA signaling mechanism is :

In the presence of JA-isoleucine, the SCFCOI1 complex forms, and JAZ proteins are degraded by the 26S proteasome. The ubiquitination of JAZ protein binds to substrate-specific SCFCOI1, and the degradation of JAZ protein by the 26S proteasome abolishes this interaction. The JAZ proteins contain a conserved TIFY motif within the ZIM domain that mediates homoand hetero-dimeric interactions between different JAZ proteins. The ZIM domain also functions to recruit transcriptional corepressors through the novel interactor of JAZ protein. The JAZ proteins contain Jas domain that is required for the interaction of both COI1 and a broad array of TFs. In the presence of JA or its bioactive derivatives, JAZ proteins are degraded and freeing TFs for expression of specific sets of JA-responsive genes, regulate enzymes involved in the biosynthesis of secondary metabolites, such as ginsenoside, artemisinin, and vinblastine.

5/ Lines 104-107 (changed to lines 112-115)

The detailed treatment of plants is described below.

After 20 days of emergence, the leaves of plants were spraied with a mixture of different concentrations of NAA and 6-BA until there was liquid dripping at the edge of the blade. The mixture included: 1.0 mg/L NAA + 0.5 mg/L 6-BA (T1); 2.0 mg/L NAA + 1.0 mg/L 6-BA (T2); 4.0 mg/L NAA + 2.0 mg/L 6-BA (T3) and 8.0 mg/L NAA + 4.0 mg/L 6-BA (T4).

6/ Lines 126-128 (changed to lines 136-139)

The method for determining the chlorophyll (Chl. a, Chl. b) and carotenoid contents is expressed as follows:

7/ Lines 252-254 (changed to lines 262-264)

Some genes indeed are part of different processes. Many of the genes in A. bidentata treated with NAA and 6-BA were affected because the various biological processes are complex and some genes are involved in other biological reaction process. Therefore, the percentage of different processes in the GO analysis is very high.

8/ Lines 297-299 (changed to lines 312-314)

About 28% of the 900 species in the Amaranthaceae estimated to occur in the family are C4 species, such as Gomphrena meyeniana, Amaranthus hypochondriacus.

The references is:

Sage RF, Sage TL, Pearcy RW, Borsch T. The taxonomic distribution of C4 photosynthesis in Amaranthaceae sensu stricto. Am J Bot. 2007; 94(12): 1992-2003. https://doi.org/10.3732/ajb.94.12.1992 PMID: 21636394

9/ Lines 313-314 (changed to lines 328-329)

After the modification, I have used bZIP and bHLH as abbreviation.

10/ Lines 346-347 (changed to lines 361-362)

The results are the data analysis we have done. The production of glycyrrhizin is oxidized at the C-11 position while oleanolic acid biosynthesis is at C-28 position oxidized. It may involved in ent-kaurene biosynthesis, because we observed that the ent-laurene synthase gene is up-regulated in the biosynthesis of gibberellin.

11/ Lines 347-349 (changed to lines 362-364)

The saponins currently isolated and identified from A. bidentata are all oleanane triterpenoid saponins. UGT can add sugar chains to oleanolic acid to form oleanane-type triterpenoid saponins, and UGT gene expression is up-regulated, which proves that more oleanane-type triterpenoid saponins are produced. From the side, it is indicated that the content of oleanolic acid is increased. So we analyzed the UGT slightly here.

12/ Remark Figure 4.

Your comments are meaningful, but the data we analyze does have some differences from your point of view. We also selected some genes for qRT-PCR validation, and the results were consistent with the expression pattern of RNA-seq assay. This result may be due to some differences in the expression of some enzyme genes in different species in MVA and MEP pathways.

Ginsenoside biosynthesis is part of our data analysis. In addition, ginsenosides have been reported in the achyranthes. Ginsenosides belong to the triterpenoids, one is the oleanane type pentacyclic triterpenoid saponin Ro, and the other two are the ginsengdiol type saponins (such as Rb1, Rb2, Rc, Rd, F2, Rg3, Rh2, etc.) and Ginseng triol saponins (such as Re, Rg1, Rg2, Rf, Rh1, etc.), both of which belong to the dammarane type tetracyclic triterpenoid saponins.

13/ Remark Figure 5

We used DEGs to analyze the gene data between samples, and analyzed the key enzymes involved in the synthesis of triterpenoid saponins. It was found that there were more differential genes involved in the synthesis of triterpenoid carbon skeleton, and 38 were co-expressed in control roots and treated leaves up-regulated CYP450s gene sequence. In order to further narrow the potential range of CYP450s involved in biosynthesis of oleanolic acid saponin, 25 up-regulated CYP450s (with sequence lengths > 1500 bp) were selected for phylogenetic analysis from A. bidentata treated leaves.

14/ Line 374 (changed to lines 389)

Your comment makes a lot of sense, we also consulted a lot of literature: more experiments have shown that MYC, MYB, WRKY and AP2/ERF transcription factors are involved in the regulation of terpene metabolism in different medicinal plants [1, 2]. For example, AaWRKY1, TSAR1, and TSAR2 are found to regulate the expression of HMGR genes in the MVA pathway in ArtemisiaannuaL. and Medicago truncatula. TSAR1 and TSAR2 are two transcription factors that are induced by jasmonic acid in the bHLH family [3, 4]. In the Medicago truncatula hairy roots, overexpression of TSAR1 and TSAR2 significantly increased the transcriptional expression levels of the MVA pathway gene and a series of genes involved in the synthesis of β-amyrin, such as HMGS, HMGRI, SS and β-AS [4, 5]. In our study, most AP2/ERF TFs, BHLHTFS and MYB TFs were up-regulated in treated leaves, and significantly increased the expression of triterpenoid biosynthesis genes in A. bidentata, especially HMGR, SS, SE and β-AS gene. Therefore, JA regulates oleanolic acid accumulation by freeing TFs and increasing expression of terpenoid biosynthesis genes in A. bidentata. These results were consistent with overexpression of TSARs to promote oleanane-type triterpene saponin in M. truncatula.

References:

[1] Afrin S, Jing-Jia H, Zhi-Yong L. JA-mediated transcriptional regulation of secondary metabolism inmedicinal plants[J]. Science Bulletin. 2015, 60(12): 1062-1072.

[2] Wasternack C, and Hause B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. an update to the 2007 review in annals of botany[J]. Annals of Botany. 2013, 111(6): 1021.

[3] Patra B, Schluttenhofer C, Wu Y, et al. Transcriptional regulation of secondary metabolite biosynthesis in plants[J]. Biochimica Et Biophysica Acta. 2013, 1829(11): 1236-1247.

[4] Goossens A, Mertens J, Pollier J, et al. The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula[J]. Plant Physiology. 2015, 170(1): 194.

[5] Mertens J, Moerkercke A V, Bossche R V, et al. Clade IVa Basic Helix -Loop -Helix Transcription Factors Form Part of a Conserved Jasmonate Signaling Circuit for the Regulation of Bioactive Plant Terpenoid Boynesis[J]. Plant and Cell Physiology. 2016, 57(12): 2564.

15/ Lines 389-391 (changed to lines 404-406)

We have already done it. (Li Jinting. Effects of Methyl Jasmonate on the Growth and Distribution of Main Medicinal Components in Achyranthes Bidentata Blume.)

Spraying 1.0mg/L MeJA could increase the content of oleanolic acid in A. bidentata.

16/ Lines 410-411 (changed to lines 425-426)

Spelling mistakes, change tertripenoid to triterpenoid.

Reviewer #2

Line 40

The language expression is not accurate and should be “Oleanolic acid, a pentacyclic triterpenoid, is an isoprenoid-derived compound ”.

Oleanolic acid has been purified during the extraction process.

Preparation of oleanolic acid determination sample: 0.5 g sample was added with methanol 10 mL, ultrasonically extracted for 40 min, and then concentration using a rotary vacuum evaporator. To the concentrated samples 10 mL of 4 mol/L hydrochloric acid was added for hydrolyzing at 85°C for 1 h. After being allowed to cool, 10 mL of chloroform were added to the sample, followed by two countercurrent extractions at 60°C, each extraction being 15 min. The bottom liquid was harvested and evaporated under reduced pressure. Chromatography of pure methanol to a volume of 3 mL, mixed and filtered through a Millipore filter (0.22 μm), and determined by HPLC. The assay conditions were mobile phase: methanol‐water‐glacial acetic acid (90: 10: 0.05); flow rate: 0.9 mL/min; column temperature: 30°C; detection wavelength: 210 nm.

Line 103 (changed to lines 111)

Achyranthes bidentata is one of the famous Dao-di herbs in China, this species is primarily distributed in the Guhuaiqingfu area, located in Jiaozuo in Henan Province. So A. bidentata is also called ‘Huainiuxi’. Dao-di herbs mean that natural Chinese medicines that grow naturally under the influence of natural environment and social environment such as specific geography and climate, or are processed and cultivated with good quality and high curative effect and is synonymous with quality medicinal herbs in China. The quality of A. bidentata in Jiaozuo is better than in other regions, therefore, we choose to experiment in the natural environment.

Line 202 (changed to lines 212)

Here, it should be compared with the control, these characteristics showed a trend of increasing first and then decreasing. The reason we explore was lower concentrations of NAA and 6-BA promote the growth of A. bidentata while higher concentrations inhibited growth of A. bidentata. Therefor, it presenting this trend.

Line 204 (changed to lines 214-215)

We have replaced it with a more appropriate expression, using ‘growth parameters’ instead of ‘index’.

Line 257 (changed to lines 267-270)

I have improved the “For the pathway enrichment analysis, a total of 28 pathways were enriched (adjusted P < 0.05) with DEGs (Table 2)” to “To further identify metabolic or signal transduction pathways in which the DEGs are likely to be involved in promoting accumulation of oleanolic acid by JA regulation by NAA and 6-BA treatment in A. bidentata, and pathway enrichment analysis was performed using KEGG database. For the pathway enrichment analysis, a total of 28 pathways were enriched (adjusted P < 0.05) with DEGs (Table 2)”.

Line 318 (changed to lines 333)

We identified 22 key enzyme genes involved in the MVA pathway and 23 key enzyme genes involved in the MEP pathway. Comparing the expression in the roots and leaves of A. bidentata, it was found that the RPKM values of MCT, HDS and HDR genes were significantly higher than roots. The HMGS, HMGR, PMK and MDD genes in the MVA pathway are highly expressed in roots and low in leaves, which is completely opposite to the expression of key enzyme genes in the MEP pathway. These results are consistent with the expression of key enzyme genes in the MVA pathway and the MEP pathway in different vegetative organs of Astragalus membranaeus Bge. In addition, more documents revealed that protein of CMS, DXS, DXR and HDS enzymes are highly expressed in young roots and floral organs of Arabidopsis. These results clearly demonstrate that the MEP pathway synthesized in Plastid pathway prefers expressed in leaves, while the MVA pathway favors in roots. In root and leaf samples, it is clearly that chloroplasts are common in leaves and are relatively rare in roots. This difference may be due to the fact that the expression of key enzyme genes in the majority of MEP synthesis pathways is significantly higher in leaves than in roots and in the up-regulation of genes in roots in most MVA pathways. The expression patterns of key enzyme genes in different tissues in these two pathways indicated that the regulation of the biosynthetic pathways of IPP and DMAPP in different tissues of A. bidentata is parallel but separate.

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PLoS One. doi: 10.1371/journal.pone.0229490.r003

Decision Letter 1

Marie-Joelle Virolle

16 Oct 2019

PONE-D-19-21410R1

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl. through de novo transcriptomics

PLOS ONE

Dear Mrs. Li,

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

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We look forward to receiving your revised manuscript.

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Academic Editor

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Reviewers' comments:

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Comments to the Author

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

Reviewer #1: (No Response)

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**********

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

Reviewer #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: (No Response)

**********

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

Reviewer #1: No

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: Several of my comments were addressed, however some points remain:

1/ I still don’t agree with the pathway presented in Figure 4. It is true that two distinct pathways, the cytosolic MVA pathway and the plastidial MEP pathway produce IPP and DMAPP. Exchange of IPP and DMAPP between the cytosol and the plastids can occur. However, downstream of IPP and DMAPP there is also a difference between the cytosol and the plastids. In the cytosol, 2 molecules of IPP and one molecule of DMAPP are used to make FPP by the FPPS enzyme. No GPPS enzyme is involved, and no GPP is produced, as is incorrectly shown in Figure 4. In the plastids, however, GPP is produced by GPPS, and that GPP can then be used for further downstream product formation (without FPP production). Please correct the pathway in Figure 4. For more information on the MVA-MEP pathways, see for instance: https://www.ncbi.nlm.nih.gov/pubmed/23451776

On the dammarene-type saponins (dammarenediol-derived) in this figure: if there are studies showing these compounds accumulate in Achyranthes spp then it is okay to include them in the figure; but in that case, refer to them in the paper. For now only oleanane-type (β-amyrin-derived) saponins are mentioned in the paper.

2/ Also, make sure to have a clear distinction between saponins containing an oleanolic acid aglycone and free oleanolic acid in your manuscript. In many plant species OA occurs as a free metabolite, whereas here it occurs as a saponin aglycone. When you measure OA, you carry out an acid hydrolysis to liberate the OA aglycones. In lines 37-39 you clearly state that OA occurs, not that OA occurs as a saponin aglycone. There is a big difference between the biological activity and in planta role of free OA (hydrophobic metabolite) and a saponin (amphipathic metabolite). To clarify throughout the manuscript.

3/ Line 54: correct typo to cycloartenol.

4/ Line 56: Little is known about genes involved in triterpenoid skeleton modification… This statement was true 10 years ago, but now plenty of genes are known to be involved, especially P450s.

5/ In the intro you point out that many phytohormones are used to increase the production of specialized metabolites. The most obvious treatment, jasmonates, is omitted in the first part, whereas later it is the one you focus on to explain the signaling mechanism. Also provide some examples of JA treatment, there are plenty of studies that show their effect on saponin metabolism.

6/ Line 113, typo: sprayed.

7/ The reads were submitted to the NCBI SRA database, but where can we find the sequence information of the assembled transcripts? The entire assembly – or at least the sequences that you use for phylogenetics or mention as candidate genes should be made publicly available.

8/ For the phylogenetic analysis, please provide the alignment file that was used to create the tree as SI. Why was a NJ tree opted for and not an ML tree?

9/ Figure 5. My comment was not addressed. In your figure, only 13 published P450s are included, whereas over 50 have been shown to be involved in triterpene (saponin) biosynthesis. Was there a reason to choose this specific set of P450s?

10/ Also my remark that there is not enough data to support the conclusion that JA regulates OA accumulation is not properly addressed. You provide a reference in your rebuttal, but this information was not used to update the manuscript.

Reviewer #2: I am not satisfied by the answer about methodology. I did not want to obtain a detailed description of extraction and quantification procedure as an answer to my remark. I consider it as an important part of a paper, and it should be inserted in a text. Particularly because the Authors menton in their manuscript free oleanolic acid and its saponins really in a very confusing way. For example:

Line 37 „Oleanolic acid is one of the major active phytochemical compounds in A. bidentata, and possesses hepatoprotective effects as well as anti-inflammatory, antioxidant, and anticancer activities”.

So finally –from this manuscript – the reader does not know if A. bidentata contains free oleanolic acid or its saponins? And it is important, because free oleanolic acid might have different bioactivity than its saponins.

Please precise:

Line 37. Oleanolic acid saponins are …

Line 130. Add the general description of extraction , ACID HYDROLYSIS and quantification procedure. It is important what you are measuring: saponins or free aglycon.

It should not be as detailed as in the answer to my question, but making the sequence of this procedure understandable for the reader.

Another problem not solved is the misunderstanding about the growth parameters of plants.

Line 211. After 20 days of treatment, the root length, fresh weight of root, dry weight of root, plant height, fresh weight of plant, and dry weight of plant had the same trend of first increasing then decreasing.

What do this description mean? „Fist increasing then decresing” could be understood as a tendency occurring in time, i.e. that the plant are first growing than are becoming smaller. And it is not true because this experiment is not made in time, it is a comparison of different treatments. So why „first increasing then decresing” if the Authors are mentioned independent experiments and NOT the process occurring in time? Only because they numbered the treatments as 1,2,3,4, and then what is occuring for the treatment 1 is „first” and for the treatment 4 – the last?

The figure illustrating these results is even more confusing because it is presented as a line of trend, and T can mean „time”. It would be better understandable if it would be a chart with separate points or bars.

The same problem is with the Figure 2.

According to the comment of the reviewer 1, the Authors changed „cholesterol” to „phytosterol”. It is better than previously, but still not really perfect solution, because „phytosterol” is not one compound, this name is used for a numerous group of plant sterols (e.g., sitosterol, stigmasterol, campesterol…etc.), and in every plant a mixture of at least several sterols is synthesized. So it should be writen „phytosterols” better than just „phytosterol”.

**********

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

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

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

Reviewer #1: Yes: Jacob Pollier

Reviewer #2: No

PLoS One. 2020 Feb 27;15(2):e0229490. doi: 10.1371/journal.pone.0229490.r004

Author response to Decision Letter 1

30 Nov 2019

We have tried our best to revise and improve the manuscript and made many changes in the manuscript according to the reviwers′good comments. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval.

Once again, thank you very much for your comments and suggestions.

We look forward to your information about my revised papers and thank you for your good comments.

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PLoS One. doi: 10.1371/journal.pone.0229490.r005

Decision Letter 2

Marie-Joelle Virolle

18 Dec 2019

PONE-D-19-21410R2

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl. through de novo transcriptomics

PLOS ONE

Dear Dr Jinting Li,

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

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

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

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Marie-Joelle Virolle, PhD

Academic Editor

PLOS ONE

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors now have addressed most of my comments, however, when they changed the manuscript a lot of typographical errors were introduced that should be corrected prior to publication of the manuscript. Some examples are (line numbers from track changes upload) are given below:

- Line 38: saponins is now plural, adjust "is one of the"

- Line 55-56: CAS = cycloartenol synthase, this has been changed but is now incorrect.

- Line 73: For example is repeated.

- Line 74: Treatment "of with" MeJA, remove "of"

- Line 83: remove one of the repeated commas.

- Line 88: "singly", please correct.

- Line 91: Here you use mmol L-1, before you used mg/L, please be consistent.

- Line 100: correct "homoand"

- Line 141: Add space before "Sample"

- Line 141: Add space between "methanol" and "was" and do something about the large (tab?) between "to" and "0.5"

- Line 142-150: re-write in proper English.

- Line 377: remove the space before the comma.

- Line 441: Only keep the numbered references.

- Line 486: italicize A. bindentata.

Overall the use of "S6 Table" or "S4 Fig" instead of "Table S6" of Fig. S4" sounds weird, please correct the order throughout the entire paper.

Reviewer #2: I have still some comments to the Authors and I feel that they did not understand some of my requests.

Line 37. Oleanane-type triterpenoid saponins is one of the major active phytochemical compounds in A. bidentata. This sentence is not correct grammatically (saponins are plural). It would be better to write „(..) saponins belong to the major active …

The description in the lines 225-229 is still not corrected and I see that the Authors does not understand my remark. The experiment is composed of 4 independent treatments, and the results cannot be commented as „first incresing then decreasing” only because the results for treatments T1 and T2 appear first (it means on the left side of the chart). The description „first.. then...” suggests the phenomenon occurring in time, and it is not the case in the presented experiment.

It would be better to omit this problem, writing:

The growth parameters of the plants after 20 days of treatment with various combinations of NAA and 6-BA are presented in Fig. 1. Lower concentrations of NAA and 6-BA (treatment T1 and T2) improved these parameters, while higher concentrations (particularly T4) exerted adverse effect.

The same remark for the description in lines 239-240.

Please definitively delete the sentence: „Oleanolic acid content rose at first and then fell.” The explanation which appear in the following sentences is totally sufficient and understandable.

**********

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

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

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

Reviewer #1: Yes: Jacob Pollier

Reviewer #2: No

PLoS One. 2020 Feb 27;15(2):e0229490. doi: 10.1371/journal.pone.0229490.r006

Author response to Decision Letter 2

6 Jan 2020

Dear editor and reviewers:

We have tried our best to revise and improve the manuscript and made many changes in the manuscript according to the reviwers′good comments. We appreciate for editor and reviewers’ warm work earnestly, and hope that the corrections will meet with approval.

Once again, thank you very much for your comments and suggestions.

We look forward to your information about my revised papers and thank you for your good comments.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0229490.r007

Decision Letter 3

Marie-Joelle Virolle

15 Jan 2020

PONE-D-19-21410R3

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl. through de novo transcriptomics

PLOS ONE

Dear Jinting Li,

Thank you for submitting your revised manuscript to PLOS ONE. One of the two reviewers think that you did not fulfill his requests therefore I recommand again minor revision of your paper and invite you to submit a revised version of the manuscript that addresses the various points listed by this reviewer.

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

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

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Marie-Joelle Virolle, PhD

Academic Editor

PLOS ONE

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: In my previous review i indicated that there were still many typographical and grammatical errors that should be corrected in addition to the ones indicated in my review. It seems the authors only corrected the errors i pointed to, hence, underneath a list containing more errors i spotted after carefully reading the paper.

1/ The title is not in standard English. I suggest to remove "through de novo transcriptomics" or reformulate the title so it is clear that de novo transcriptomics revealed the effect of NAA and 6-BA on JA signaling and OA biosynthesis.

2/ In the abstract (line 28) you still mention OA accumulation, whereas the plant accumulates saponins with an oleanolic acid backbone.

3/ Line 39, possesses should be possess.

4/ Line 52, add a space between diphosphate and synthase.

5/ Line 57, correct "Many are known about" to e.g. "A lot is known about" or even better, change the sentence to something like "Several genes involved in modification of the triterpenoid skeleton downstream of the cyclization step are known."

6/ Line 60, change "are assumed" to "were shown to".

7/ Line 63-66: There is confusion about "oleanane". Oleanane points to triterpenes / saponins with a β-amyrin backbone. Hence, CYP716A12 is not per se involved in the biosynthesis of oleanane-type triterpene aglycones, it is just involved in the modification of the oleanane backbone.

8/ Line 86. Split into two sentences, add a dot after A. bidentata roots and start a new sentence with "Soaking the seeds..."

9/ Lines 92 and 94: COI1 in SCFCOI1 should be superscript.

10/ Line 92-95; the JA signaling and degradation of JAZ proteins is not clear here. Rephrase to for instance "In the absence of JA-Ile, the JAZ repressor proteins bind to the MYC2 transcription factor, thereby blocking downstream JA response. In the presence of JA-Ile, COI1 binds to the JAZ proteins, which leads to their ubiquination by the SCFCOI1 complex and their subsequent degradation by the 26S proteasome. Degradation of the JAZ proteins releases MYC2, leading to transcriptional activation of the JA-responsive genes."

11/ Line 98, add the abbreviation (NINJA) after novel interactor of JAZ.

12/ Line 99, add "a" between contain and Jas to "The JAZ proteins contain a Jas domain..."

13/ Line 117, correct to "20 days after emerging, the leaves were sprayed with a mixture..."

14/ Line 140, rephrase to: "... hydrochloric acid was added, and the extract was hydrolyzed at 85..."

15/ Line 226, correct to "... the plants 20 days after treatment with..."

16/ Legend Fig 2, add a space between 6-BA. and Each.

17/ In the abstract (line 21-23) it is claimed that 62% of the upregulated genes encode proteins involved in hormone or terpenoid biosynthesis or TFs. Lines 267-269 only claim that of the 20896 DEGs, 13071 were upregulated. I cannot imagine all upregulated genes encode proteins involved in hormone or terpenoid biosynthesis or TFs. This should be corrected in the abstract.

18/ Correct to bHLH and bZIP in Table 4.

19/ Legend Figure 4: check the CMK abbreviation, remove "50"

20/ Line 419, correct to bHLH.

21/ Legend Figure 7: "through de novo sequencing"?

22/ Legend Table S6: italicize "A. bidentata"

Reviewer #2: I am satisfied by the improvements made by the Authors. I consider the manuscript as acceptable for publication.

**********

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

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

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

Reviewer #1: Yes: Jacob Pollier

Reviewer #2: No

PLoS One. 2020 Feb 27;15(2):e0229490. doi: 10.1371/journal.pone.0229490.r008

Author response to Decision Letter 3

6 Feb 2020

Dear editor and reviewers:

Once again, thank you very much for your comments and suggestions.

We look forward to your information about my revised papers and thank you for your good comments.

Responds to reviewers:

Reviewer #1

We have modified the title according to your comments. We have removed“through de novo transcriptomics”.

2/ (line 28, changed to line 27)

We have modified the manuscript according to your comments. We have changed “oleanolic acid” as “oleanane-type triterpenoid saponins”.

3/ ( line 39)

We have corrected as“possess”. 4/ (Line 52)

We have added a space between “diphosphate” and “synthase”.

5/ (line 57)

We have modified the manuscript according to your comments.

6/ (line 60)

We have changed "are assumed" to "were shown to".

7/ ( line 63-66, changed to line 65)

We have modified the manuscript according to your comments, and changed as “oleanane backbone”.

8/ (line 86, changed to line 85)

We have modified the manuscript according to your comments.

9/ (line 92-95, changed to line 90-96)

We have modified the manuscript according to your comments.

10/ (line 98, changed to line 99)

We have added the abbreviation “NINJA” after “novel interactor of JAZ”.

11/ (line 99)

We have added "a" between “contain” and “Jas”.

12/(line 117, changed to line 118-119)

We have modified the manuscript according to your comments.

13/ (line 140)

We have modified the manuscript according to your comments.

14/ (line 226)

We have corrected to "... the plants 20 days after treatment with..."according to your comments.

15/ ( Legend Fig 2)

We have added a space between “6-BA.” and “Each”.

16/ (line 21-23, changed to line 20-23)

We have corrected it in the abstract.

17/ (Table 4)

We have corrected to “bHLH” and “bZIP”.

18/ ( Legend Figure 4)

We have rechecked the “CMK’’ abbreviation, and no errors found; and removed “50”.

19/ ( Line 419)

We have corrected to “bHLH”.

20/ ( Legend Figure 7)

We have deleted "through de novo sequencing", and changed to “Fig 7. NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in A. bidentata.”

21/ (Legend Table S6)

We have changed to "A. bidentata".

Reviewer #2

Thank you very much for your comments on the manuscript. Your comments have made the manuscript more clear and reasonable. Thank you again!

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PLoS One. doi: 10.1371/journal.pone.0229490.r009

Decision Letter 4

Marie-Joelle Virolle

10 Feb 2020

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl.

PONE-D-19-21410R4

Dear Dr. Li,

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

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

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

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

With kind regards,

Marie-Joelle Virolle, PhD

Academic 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 #2: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: Please correct the position 25 in the list of cited publications. It was added during the revision process and it is written with mistakes. The name of the first author is Alsoufi, the other Authors' names are Pączkowski, Szakiel, Długosz. It should be also added officinalis after Calendula.

**********

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

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

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

Reviewer #1: Yes: Jacob Pollier

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0229490.r010

Acceptance letter

Marie-Joelle Virolle

13 Feb 2020

PONE-D-19-21410R4

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl.

Dear Dr. Li:

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

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

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

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Marie-Joelle Virolle

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Fig. GO enrichment analysis for the differentially expressed genes in A. bidentata.

(TIF)

Click here for additional data file.^{(3.2MB, tif)}

S2 Fig. Phylogenetic tree constructed based on the deduced amino acid sequences of the A. bidentata CYP450s and all Arabidopsis CYP450s.

The phylogenetic tree was generated using the neighbor-joining (NJ) method in MEGA6.

(TIF)

Click here for additional data file.^{(8.1MB, tif)}

S3 Fig. Effects of exogenous NAA and 6-BA on JA contents in A. bidentata leaves.

(TIF)

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S4 Fig. Phylogenetic tree constructed based on the deduced amino acid sequences of the five A. bidentata CYP450s and 55 CYP450s related to triterpene synthesis.

The phylogenetic tree was generated using the neighbor-joining (NJ) method in MEGA6.

(TIF)

Click here for additional data file.^{(951.3KB, tif)}

S1 Table. Primers used for qRT-PCR analysis.

(DOCX)

Click here for additional data file.^{(14.3KB, docx)}

S2 Table. Photosynthetic pigment content in leaves.

(DOCX)

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S3 Table. List of transcription factors.

(DOCX)

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S4 Table. List of candidate genes encoding enzymes in the oleanolic acid biosynthesis pathways.

(DOCX)

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

S5 Table. List of fifty-five CYP450s related to triterpene synthesis in constructing phylogenetic tree.

The CYP450s marked in red represent those used in the construction of Fig 5.

(DOCX)

Click here for additional data file.^{(17.5KB, docx)}

S6 Table. List of twenty-two unigenes of A. bidentata used to build phylogenetic tree.

The unigenes marked in red represent those used in the construction of Fig 5.

(DOCX)

Click here for additional data file.^{(15.1KB, docx)}

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Data Availability Statement

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PERMALINK

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl

Yanqing Liu

Li Tang

Can Wang

Jinting Li

Roles

Abstract

Introduction

Materials and methods

Plant materials and treatment

Determination of growth indexes and oleanolic acid content

Determination of chlorophyll (Chl. a, Chl. b) and carotenoid contents

Assay of endogenous JA content

Library construction and high-throughput RNA sequencing

RNA sequencing data analysis

Phylogenetic analysis

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation and expression analysis

Data analysis

Results

Effects of NAA and 6-BA on the growth of A. bidentata

Fig 1. Effects of NAA and 6-BA on the growth of A. bidentata leaves.

Effects of NAA and 6-BA on oleanolic acid content in A. bidentata roots

Fig 2. Effects of NAA and 6-BA on oleanolic acid content in A. bidentata roots.

RNA-seq analysis of A. bidentata leaves

Table 1. RNA-seq reads for four RNA-seq libraries.

Changes in gene expression profiles in leaves treated with NAA and 6-BA

Fig 3. Percentage of genes expressed in control and 2.0 mg/L NAA + 1.0 mg/L 6-BA-treated (T) leaves.

Table 2. Pathway enrichment analysis of genes differentially expressed in phytohormone-treated A. bidentata leaves.

DEGs involved in JA signaling pathways

Table 3. Differentially expressed genes in response to NAA and 6-BA treatment in A. bidentata.

Transcription factors differentially expressed in response to auxin and cytokinin treatment

Table 4. Transcription factors differentially expressed in response to auxin and cytokinin treatment in A. bidentata.

DEGs involved in the oleanolic acid biosynthesis pathway

Fig 4. Overview of the triterpenoid biosynthetic pathway.

Fig 5. Phylogenetic tree of the A. bidentata CYP450s.

RNA-seq accuracy determined by real-time PCR

Fig 6. Expression patterns of genes involved in the triterpenoid saponin and phytosterols biosynthesis pathways determined by RNA-seq and qRT-PCR.

Discussion

JA-mediated transcriptional regulation of oleanolic acid metabolism

Fig 7. NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in A. bidentata.

NAA and 6-BA promote terpenoid biosynthesis and photosynthesis processes

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Marie-Joelle Virolle

Roles

Author response to Decision Letter 0

Decision Letter 1

Marie-Joelle Virolle

Roles

Author response to Decision Letter 1

Decision Letter 2

Marie-Joelle Virolle

Roles

Author response to Decision Letter 2

Decision Letter 3

Marie-Joelle Virolle

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Author response to Decision Letter 3

Decision Letter 4

Marie-Joelle Virolle

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Acceptance letter

Marie-Joelle Virolle

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Associated Data

Supplementary Materials

Data Availability Statement

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