Accumulation mechanism of indigo and indirubin in Polygonum tinctorium revealed by metabolite and transcriptome analysis

Industrial Crops & Products 141 (2019) 111783

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Accumulation mechanism of indigo and indirubin in Polygonum tinctorium revealed by metabolite and transcriptome analysis

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Wenjing Wanga, Yuan Wua, Huihui Xua, Yuan Shangb,c, Yichen Chena, Miaoer Yana, Zhaohui Lib, , ⁎ David R. Waltd, a

Institute of Chemical Biology and Nanomedicine, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China c Super computer center, Smart City Institute, Zhengzhou University, Zhengzhou 450001, China d Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, 60 Fenwood Road, Boston, MA 02115, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Polygonum tinctorium Indigo Indirubin High-performance liquid chromatography Transcriptome analysis

The harvest time of a plant usually affects the yield of its metabolites. Polygonum tinctorium (Polygonum tinctorium Ait., P. tinctorium) is an important cultivated plant that produces the natural blue dye indigo and the medicinal metabolite indirubin. To explore the mechanism of indigo and indirubin accumulation, the leaves of P. tinctorium at different harvest times, namely July, August, and October, were investigated by metabolite and de novo transcriptome analyses. The results revealed that the indigo and indirubin content varied at different harvest times. Moreover, their contents were significantly higher (p < 0.05) in August than in either July or October. Transcriptome analysis showed different gene expression patterns of P. tinctorium leaves in August. Almost all the identified differentially expressed genes (DEGs) that were responsible for the biosynthesis of indigo and indirubin, including four anthranilate synthase (AS) genes, two indole-3-glycerolphosphate synthase (IGPS) genes, three tryptophan synthase (TS) genes and seven cytochrome P450 (CYP450) genes were expressed at significantly higher levels in August (p < 0.05), which correlated with the increase of the two compounds. Overall, the results of this study provide useful information for understanding the accumulation mechanism of indigo and indirubin in P. tinctorium, which may promote the development of agricultural breeding and industrial production of P. tinctorium. Moreover, sequence data obtained by de novo transcriptome sequencing of P. tinctorium provides a platform for future molecular biological studies.

1. Introduction Polygonum tinctorium (Polygonum tinctorium Ait., P. tinctorium) is an annual herbal medicine plant, which has been reported to possess a wide range of pharmacological activities such as anti-neuroinflammatory (Lee et al., 2018), anticancer (Heo et al., 2014), and antinociception (Ishihara et al., 2000). In addition to its medicinal properties, P. tinctorium also has been widely used as a coloring agent for thousands of years because of its secondary metabolite indigo (Shin et al., 2017), the most important natural blue dye for textile and dyeing industry (Minami et al., 2015). Natural indigo is considered to be the oldest dye (Gillam et al., 2000). It has been reported that indigo dyeing in China can date back 3400 years ago (Li et al., 2019a). In ancient times, indigo was a commodity with large economic impact and socalled ‘indigoferous’ plants were the only source of the blue dye (Warzecha et al., 2007). Baden Aniline and Soda Factory (BASF) began

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to market synthetic indigo in 1897 and it is now synthesized on a large scale (Gilbert and Cooke, 2001). However, in recent years, awareness and concern for environmental pollution of synthetic dyes has increased, because the syntheses of many dyes introduces toxins and carcinogens, such as benzidine and other aromatic compounds (Ali, 2010). Moreover, naturally dyed textiles exhibit anti-psoriatic, antifungal, and antibacterial properties (Shahid ul et al., 2013). With increased demand for natural dyes, plant-derived indigo is growing in popularity (Kim et al., 2010; Kukula-Koch et al., 2015; Minami et al., 1999). Indirubin, an isomer of indigo, is also a metabolite of P. tinctorium. Indirubin’s importance as a natural compound stems from its recent use for the treatment of chronic myelogenous leukemia (Xiao et al., 2002). It has also been reported that indirubin exhibits other significant pharmacological activities, such as anti-HIV-1 (Zhong et al., 2005), antiproliferative (Kim et al., 2012), and anti-inflammatory (Kunikata

Corresponding authors. E-mail addresses: [email protected] (Z. Li), [email protected] (D.R. Walt).

https://doi.org/10.1016/j.indcrop.2019.111783 Received 29 May 2019; Received in revised form 12 September 2019; Accepted 13 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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the late Ming Dynasty (hundreds of years ago) (Ou et al., 2017). The leaves were washed thoroughly with sterile water, frozen immediately in liquid nitrogen and stored at -80℃ for further use.

et al., 2000) properties. Due to the important economic and medicinal value of natural indigo and indirubin, a growing number of researchers are exploring their biosynthesis pathways in order to improve their yields. Both indigo and indirubin are indole alkaloids derived from indole (Celik et al., 2005). Their biosyntheses in P. tinctorium are influenced by many factors. Previous studies reported that environmental conditions can greatly affect both the growth of the plant and the production of indigo, and also indicated that P. tinctorium can be harvested three times a year from July to October (Angelini et al., 2004). The biomass of P. tinctorium varies at each harvest time, and so do the yields of indigo and indirubin (Campeol et al., 2006). Little is known about the accumulation mechanism of indigo and indirubin. RNA sequencing (RNA-seq) provides a powerful tool for acquiring transcriptomic information and identifying genes involved in the biosynthesis and metabolism of natural products in plants (Huang et al., 2018; Suo et al., 2019; Wu et al., 2018; Sahoo et al., 2019). To better understand the molecular mechanism underlying the accumulation of indigo and indirubin in P. tinctorium at different harvest times, the yields of indigo, indirubin, and their precursor indole, as well as the transcriptomes of the leaves were analyzed comprehensively. The fresh leaves of P. tinctorium were collected in July, August, and October. High-performance liquid chromatography (HPLC) was performed to measure the contents of indigo, indirubin, and indole at different harvest times. Simultaneously, the gene expression patterns of the leaves were also studied by de novo transcriptome analysis using RNA-seq and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between the metabolite contents of indigo and indirubin and the gene expression patterns were also explored. By combining the chemical analysis and gene expression with bioinformatic analysis, the candidate genes related to the biosynthesis of indigo and indirubin were identified (Fig. 1).

2.2. Extraction and measurement of indigo and indirubin For extraction of indigo and indirubin from P. tinctorium leaves, several frozen leaves collected at the same harvest time were mixed and dried in an oven at 105℃ for about two hours. Then the oven-dried leaves were ground finely to a powder with a mortar and pestle. A total of 90 mg powder was immersed in 30 mL N, N-dimethylformamide (DMF). After ultrasonic treatment for 3 h, the mixture was filtered through a 0.45 μm Millipore filter. Finally, the clear filtrate was collected for HPLC analysis. For each harvest time, three extracts were used for HPLC detection for replication and each extract contained multiple leaves. The measurements of indigo and indirubin were performed according to a previously described method (Xie et al., 2011) using a Waters Breeze2 HPLC system with a PDA detector (2298), autosampler (2707) and fluorescence detector (2475). The sample was separated on a C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase of water: methanol (70:30, v/v) and a flow rate of 1 ml min−1. The column temperature was set as 30℃. Standards of indigo and indirubin were purchased from Shioko Biotechnology Co. Ltd (Beijing, China). The samples were monitored at 260–290 nm for indigo and indirubin detection. Each measurement was repeated three times to obtain the mean values and standard deviations (SD). Indole, the precursor of indigo and indirubin, was also quantified by HPLC according to a previously described method (Rius and GarciaRegueiro, 2001) with slight modification. To extract indole from P. tinctorium leaves, the frozen leaves collected at the same harvest time were mixed and ground to a powder in liquid nitrogen. The powders were freeze-dried and then immersed in acetonitrile. After ultrasonic treatment for 3 h in an ice-water bath, the extracts were collected. Before HPLC analysis, the extracts were filtered through a 0.45 μm Millipore filter. The HPLC detection was carried out by measuring the fluorescence using an excitation wavelength of 280 nm and emission wavelength of 360 nm with a mobile phase of water: acetonitrile (60:40, v/v). The other conditions were the same with the detection of indigo and indirubin. Standards of indole were purchased from Aladdin Biochemical Technology Co. Ltd (Shanghai, China). Each measurement

2. Materials and methods 2.1. Plant material The first leaves, that were young, fresh, and healthy, were collected randomly from the top of different P. tinctorium plants on 18 July, 20 August, and 15 October, 2016, in a rural farmland in Nantong, Jiangsu province, China, where the planting of natural indigo can be dated to

Fig. 1. Schematic diagram of indigo and indirubin accumulation investigation. Abbreviations: ASA, anthranilate synthase subunit alpha; ASB, anthranilate synthase subunit beta; PAT, phosphoribosylanthranilate transferase; PAI, phosphoribosylanthranilate isomerase; TSA, tryptophan synthase subunit alpha; TSB, tryptophan synthase subunit beta.

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the RNA-seq results. Specific primer pairs were designed and targeted for 200–300 bp regions of different genes (Supplementary Table S1). Total RNA was extracted using the above described method. For each gene analyzed by qRT-PCR, three RNA samples from the same harvest times were used for replication. In each qRT-qPCR run, PU1 (Polyubiquitin gene) was selected as an endogenous standard gene (Tsugama et al., 2017). All qRT-PCR reactions were carried out using a SYBR Green Quant one-step qRT-PCR Kit (Tiangen Biotech co., Ltd, China) according to the manufacturer’s instructions. The qRT-PCR analysis was performed using the QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The conditions were adjusted as follows: maintain at 50℃ for 30 min, denaturation at 95℃ for 2 min, followed by 40 cycles of 94℃ for 20 s, 55℃ for 20 s, 68℃ for 20 s. The rate of temperature change was 1.6℃/s. Transcript abundance was calculated based on the normalized relative quantitation 2−△△Ct method.

was also repeated three times to obtain the mean values and SD. 2.3. RNA extraction and de novo transcriptome sequencing For each RNA extraction, three leaves that were collected at the same harvest time were mixed and processed with an RNAprep Pure Plant Kit (Tiangen Biotech co., Ltd, China). The integrity of total RNA was checked using an Agilent Bioanalyzer 2100 system. Only when the RNA Integrity Number (RIN) > 7.0, was the total RNA used further. The mRNA was isolated from the total RNA by the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, USA) according to the manufacturer’s specification. Immediately, the mRNA was used to construct cDNA libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendations. The fragment lengths and concentrations of the cDNA libraries were detected by an Agilent Bioanalyzer 2100 system and Qubit 3.0 (Life Technologies, USA), respectively. Finally, a total of six cDNA libraries (two replicates for each harvest time and each replicate contained three leaves) were sequenced on an Illumina NextSeq 500 platform and a 150-bp pair-end sequencing protocol was employed.

2.7. Statistical analysis For HPLC and qRT-PCR analysis, each measurement was repeated three times. All data are expressed as the mean ± standard deviation (SD). Statistical significance was tested by one-way ANOVA with Duncan's multiple range test (p < 0.05). The DEGs were selected with the threshold values of log-fold expression change (logFC) > 1 or < −1 and adjusted p-value < 0.05.

2.4. Comparative transcriptome and bioinformatics analysis After removing adaptor sequences and low-quality reads by Trimmomatic (Bolger et al., 2014), the clean reads were used for de novo transcriptome assembly by Trinity (Grabherr et al., 2011) with a default k-mer size of 25. The assembled transcriptome was then annotated by standard Trinotate (v2.0.0) (Bryant et al., 2017) against Swissprot (Boeckmann et al., 2003), Pfam (Finn et al., 2016), eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) (Powell et al., 2014), GO (Gene Ontology) (Harris et al., 2004), and KEGG (Kyoto Encyclopedia of Genes and Genomes) (Kanehisa et al., 2012) databases with a cut-off E-value of 1e−5. The GO functional enrichment for all the unigenes was analyzed by GOseq (a perl script in Trinity software) and visualized by the online drawing tool WEGO (Ye et al., 2006). To evaluate the expression level of each unigene, the clean reads from each of the samples were mapped back to the assembled unigenes by Bowtie2 software (Langmead and Salzberg, 2012). The unigene expression was normalized by TPM (Transcripts per million reads) method. In order to identify the difference of gene expression at different harvest times, the edgeR package in R program was used to filter the DEGs.

2.8. Data availability The sequencing reads were deposited in the NCBI Bioproject database with the accession number of PRJNA515386. Other data supporting the findings of the study are available in this article and its Supplementary Information files, or from the authors upon request. 3. Results 3.1. Content change of indigo, indirubin, and indole in P. tinctorium leaves at different harvest times In order to elucidate the effect of harvest times on the biosynthesis of indigo and indirubin in P. tinctorium leaves, the leaves were extracted and HPLC was used to quantify the content of indigo and indirubin in July, August and October. The results reveal that the content of indigo and indirubin were different at the three harvest times (Fig. 2). Moreover, the content of both indigo and indirubin in August are significantly higher compared with the other two months (p < 0.05). Specifically, indigo in the leaves in August was 2.14 times the content in July (p < 0.05) and 1.36 times the content in October (p < 0.05) (Fig. 2A). Likewise, indirubin in P. tinctorium leaves in August was 1.89 times the content in July (p < 0.05) and 1.63 times the content in October (p < 0.05) (Fig. 2B). To evaluate the effect of intermediate content on the yields of indigo and indirubin, the precursor indole was also measured by HPLC. As shown in Supplementary Fig. S1, the content of indole in August was also significantly higher compared with the other two months (p < 0.05). Specifically, indole in the leaves in August was 1.66 times the content in July (p < 0.05) and 1.36 times the content in October (p < 0.05).

2.5. Identification of related unigenes involved in the metabolic pathway of indigo and indirubin Based on the differences in content of indigo and indirubin at different harvest times, it was speculated that the expression levels of the related unigenes would exhibit similar or opposite trends as the concentrations of indigo and indirubin change. Therefore, expression trends of DEGs at different harvest times were analyzed using the short time-series expression miner (STEM) software program (Ernst and BarJoseph, 2006). The target trends were selected for further study to identify the potential unigenes involved in the biosynthesis of indigo and indirubin. The GO enrichment analysis and visualization for the selected DEGs were performed by the Bingo plugin in Cytoscape software (Maere et al., 2005). To better understand the biological processes at different harvest times and identify the genes involved in the biosynthesis and accumulation of indigo and indirubin, the selected DEGs were further assigned to the reference pathways in the KEGG database.

3.2. Transcriptome sequencing, assembly and annotation To explore the mechanism underlying the accumulation of indigo and indirubin in P. tinctorium leaves in August, the transcriptomes at different harvest times were compared by RNA-seq. After filtering the adaptor sequences, primer sequences and low-quality reads, a total of 148,390,328 clean reads were obtained with an average of 24,731,721 reads per library (Supplementary Table S2). These clean reads were de

2.6. qRT-PCR analysis A total of nine candidate genes related to indigo and indirubin biosynthesis were selected randomly for qRT-PCR analysis to confirm 3

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Fig. 2. The content of indigo (A) and indirubin (B) in the leaves of P. tinctorium in July, August, and October. The data represent the mean from three replicates with three biological repeats. Error bars indicate standard deviation (SD). Asterisks indicate significant differences in August compared with July and October (p < 0.05).

novo assembled into 267,093 unigenes with an average length of 704 bp and N50 length of 1061 bp (Supplementary Table S3). The comprehensive annotation of the obtained unigenes are shown in Supplementary Table S4. To further understand the functional classification of the unigenes, WEGO was used to display the GO functional groups. In Supplementary Fig. S2, a total of 69,204 unigenes are categorized into 61 functional groups. Most of the GO terms fall into the category “molecular function” (61,794 terms), followed by “cellular component” (58,128 terms) and “biological process” (57,728 terms). In addition, under the “molecular function” category, the sub classifications “binding” (GO: 0005488, 45,975 unigenes) and “catalytic activity” (GO:0003824, 38,596 unigenes) are the dominant terms. Under the “cellular component” category, the sub classifications “cell” (GO:0005623, 54,118 unigenes) and “cell part” (GO:0044464, 54,032 unigenes) are the most assigned terms. Moreover, under the “biological process” category, “cellular process” (GO:0009987, 48,826 unigenes) and “metabolic process” (GO:0008152, 42,913 unigenes) are the most abundant terms.

Fig. 3. The differentially expressed unigenes in July vs. August and October vs. August. Venn diagram reveals the unigenes that were consistently differentially expressed in August compared with July and October.

3.3. Comparison of transcriptional profiles at different harvest times 3.4. Expression trends analysis of the potential DEGs

To evaluate the repeatability of samples, correlation analysis was performed among all six transcriptomes. A high correlation was observed between the samples collected at the same harvest time (Supplementary Table S5). The correlation coefficient ranged from 0.95 to 0.98. It is particularly noteworthy that samples taken on consecutive days had very good correlation. As expected, the correlation of the samples from different harvest times was much lower with correlation coefficients less than 0.78. In summary, correlation analysis demonstrated good repeatability among the samples from the same harvest time and considerable differences between the samples from different harvest times. The contents of indigo and indirubin in August were significantly higher than in July and October (p < 0.05); Therefore, it was hypothesized that the DEGs involved in the biosynthesis of indigo and indirubin would be also expressed differentially in August when compared with both July and October. The DEGs were filtered by edgeR with a |logFC| > 1 and an adjusted p < 0.05. Compared with the unigenes in August, a total of 9526 unigenes expressed differentially in July; specifically, 5184 up-regulated unigenes and 4342 down-regulated unigenes. In addition, 9132 unigenes were up-regulated and 6988 unigenes were down-regulated in October compared with August (histogram in Fig. 3). When compared with July and October simultaneously, 3364 unigenes were consistently differentially expressed in August (Venn diagram in Fig. 3), some of which are likely to be genes related to the higher accumulation of indigo and indirubin in August. These genes were selected for further analysis.

To further select the relevant genes, STEM was used to analyze the expression trends of the 3364 potential DEGs. Twenty trends were obtained including four colored trends with p-values less than 0.05 (Supplementary Fig. S3). Among the four significant trends, profile 18 and profile 1 showed highest and lowest expression levels in August, which exhibited similar or opposite trends to the content changes of indigo and indirubin. In detail, there were 1045 unigenes expressed significantly higher (p < 0.05) in August than in the other two months (Fig. 4A). Moreover, 484 unigenes expressed significantly lower (p < 0.05) when compared with July and October (Fig. 4B). It is likely that some of these significantly higher or lower expressed genes are related to the biosynthesis and accumulation of indigo and indirubin.

3.5. Candidate genes related to the biosynthesis of indigo and indirubin The biological functions of the unigenes that were significantly increased or decreased in expression levels in August were analyzed by the Bingo plugin in Cytoscape. Within the 1045 significantly highly expressed genes, 602 unigenes were assigned to the 33 GO enrichment terms, in which 20 sub-terms were significantly enriched with a corrected p < 0.05 (Fig. 5). Among the significantly enriched terms, the sub-terms “biosynthetic process” (238 genes, 39.53%), “cytosol” (114 genes, 18.94%), and “DNA binding” (83 genes, 13.78%) were the top assigned terms in the “biological process” category, “cellular component” category, and “molecular function” category, respectively. Importantly, it was noticed that in the “biological process” term, there 4

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Fig. 4. The expression trends of significantly different expression genes in August compared with July and October. The salient points are the TPM values of the (A) significantly highexpressed genes (profile 18) and (B) significantly low-expressed genes (profile 1) obtained by RNA-seq. Inserts: the expression trends of the two types of the DEGs, the color means p < 0.05 (p = 0 and p = 3.3×10−155, respectively).

were some terms related to stress and stimulus, such as “response to stress”, “response to external stimulus” “response to abiotic stimulus”, etc. In addition, within the 484 lower expressed genes, many genes were also assigned to the stress-response terms with p < 0.05 (Supplementary Fig. S4). The proposed DEGs were further assigned to biochemical pathways in the KEGG database. In total, 96 KEGG pathways were obtained, among which, seven pathways such as “phenylalanine, tyrosine, and tryptophan biosynthesis”, “alpha-Linolenic acid metabolism” etc. were significantly enriched (p < 0.05, Supplementary Table S6). It is known that the secondary metabolites indigo and indirubin are indole alkaloids that derive from indoles. Thus, the biosynthesis of indole plays a key role in the formation of indigo and indirubin. In the most significantly enriched pathway “phenylalanine, tyrosine, and tryptophan biosynthesis” (p < 0.001), nine DEGs were identified to be involved in the biosynthesis pathway of indole (Fig. 6, Supplementary Table S7). Moreover, all the nine genes including four AS genes, two IGPS genes and three TSB genes were up-regulated in August (Supplementary Fig. S5). In addition, seven up-regulated CYP450 genes, three up-regulated and three down-regulated uridine diphosphate glucoseglucosyltransferase (UGT) genes were also identified in the selected

DEGs (Supplementary Figs. S6 and S7, Supplementary Table S8), which have been reported to be involved in the downstream biosynthesis of indigo and indirubin (Minami et al., 1996, 1997; Song et al., 2011). 3.6. Candidate genes related to the biosynthesis of indigo and indirubin To confirm the expression level obtained by RNA-seq, nine DEGs related to the biosynthesis of indigo and indirubin were selected for qRT-PCR analysis. As expected, eight selected genes, including two ASA gene (gene86009 (ASA1), gene97347 (osASA2)), one TSB gene (gene88810 (TSB)), one IGPS gene (gene108971 (IGPS)), three CYP450 gene (gene85036 (CYP82C2), gene89942 (CYP71A9), gene100326 (CYP71A1)) and one UGT gene (gene82528 (UGT78D3)) exhibited highest expression levels in August (Fig. 7). Moreover, only one UGT gene (gene102174 (UGT76B1)) expressed lowest in the leaves harvested in August compared with July and October. 4. Discussion In this work, the chemical contents of indigo, indirubin, and indole as well as the transcriptome of P. tinctorium leaves at different harvest

Fig. 5. GO functional enrichment analysis of the highly expressed genes in August. The areas of the circles correspond to the number of enriched genes. The colored terms mean p < 0.05. 5

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Fig. 6. The simplified pathway and related genes involved in the biosynthesis of indigo and indirubin. The genes and numbers colored in red or green mean they exhibit higher or lower gene expression in August. The red molecules mean that they are more likely to accumulate at higher levels in August.

Fig. 7. The qRT-PCR verification of the expression profiles in July, August, and October in P. tinctorium leaves. The relative expression levels of gene86009 (ASA1), gene97347 (osASA2), gene88810 (TSB), gene108971 (IGPs), gene85036 (CYP82C2), gene89942 (CYP71A9), gene100326 (CYP71A1), gene82528 (UGT78D3), and gene102174 (UGT76B1) were calculated by 2−△△Ct method. Error bars indicate SD. 6

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between PtUGT1 and PtUGT2, and the amino acid sequence of PtIGS had 99% identity to PtUGT1 and PtUGT2. In this work, six genes encoding UGTs were identified. Among the six UGT genes, three genes were up-regulated, and three genes were down-regulated in August (Supplementary Fig. S7), which made the transformation from indoxyl to indican elusory at this harvest time. However, the transformation of indoxyl to indican is reversible. The glucose moiety of indican can be removed to release indoxyl by the catalysis of β-glucosidase, an enzyme in chloroplasts. When subjected to damage, such as physical wounds, disease, or attack by insects or fungi, the intracellular endomembranes of vacuoles and chloroplasts are lysed, resulting in the mixture of indican and β-glucosidase (Kim et al., 2018, 2010; Shahid ul et al., 2013; Inoue et al., 2018; Hsu et al., 2018; Minami et al., 1996). Therefore, indican can eventually convert to indoxyl. The enriched indoxyl can be further spontaneously oxidized to indigo and indirubin by a second branch. In the presence of oxygen, indoxyl can be oxidized rapidly and dimerized spontaneously to form indigo (Ensley et al., 1983; Murdock et al., 1993). At the same time, indirubin, a common byproduct of indigo synthesis, is also generated by the reaction of indoxyl with isatin (indole-2,3-dione), an oxidation product of indoxyl. The data suggest that the accumulated indoxyl promotes the production of indigo and indirubin in August. Overall, this work demonstrated that the contents of indigo, indirubin, and indole in P. tinctorium leaves varied at different harvest times and reached their highest concentrations in August relative to July and October. Transcriptome analysis of leaves from different harvest times revealed that all the DEGs involved in the biosynthesis of indole were significantly increased in August, which led to the enrichment of indole (Supplementary Fig. S1). The up-regulated CYP450 s in turn promote the transformation of accumulated indole to more indoxyl. Furthermore, more indigo and indirubin were generated by spontaneous oxidation of the increased indoxyl in the presence of oxygen. Moreover, the relative expression levels obtained by qRT-PCR were consistent with the results of RNA-seq, enabling qRT-PCR to potentially be used to routinely monitor the production of indigo and indirubin during harvest time to maximize their production (Fig. 7). In summary, it was found that almost all the unigenes involved in the biosynthesis of indigo and indirubin were highly expressed in August, providing a credible explanation for why indigo and indirubin accumulate at this harvest time.

times were systemically investigated. The chemical analysis revealed that both indigo and indirubin were present at significantly higher levels in August than in the other two months (p < 0.05, Fig. 1), indicating that the accumulation of indigo and indirubin in P. tinctorium leaves is indeed affected by the harvest times. To explore the underlying mechanism for the accumulation of indigo and indirubin, transcriptome analysis was performed to identify the genes related to the biosynthesis of indigo and indirubin. In the bioinformatics analysis, the potential DEGs that showed the highest or lowest expression levels (p < 0.05) in August were selected for further analysis. In general, the yield of indigo and indirubin are influenced by environmental factors and the quality of the growing season (Angelini et al., 2004; Campeol et al., 2006). In the GO enrichment analysis, many DEGs were found to be associated with response to stress and stimulus, which may be caused by changes in the growth environment in August. These changes of environmental factors may be more conducive to the production of indigo and indirubin. It has been reported that the precursors of indigo and indirubin are probably produced in the cytosol (Inoue et al., 2018); in this work, 114 genes that were highly expressed in August were related to “cytosol” terms. Indoles are widely found in numerous compounds produced in plants or animals. Indigo and indirubin are both derived from indole (Celik et al., 2005). Therefore, the biosynthesis of indole can directly affect the accumulation of indigo and indirubin. Indole mainly originates from the shikimate pathway (Fig. 6). Briefly, shikimate is converted to chorismate by a series of reactions. Chorismate converts to anthranilate in the presence of AS (including ASA and ASB). Anthranilate is subsequently transformed to indole-3-glycerol phosphate (IGP) via the catalyzing of PAT, PAI, and IGPS (Jin et al., 2015). The next steps are the mutual transformation of IGP, tryptophan, and indole by the two catalytic subunits of TS, subunit alpha (TSA) and subunit beta (TSB); TSA catalyzes the release of indole, and TSB catalyzes the formation of tryptophan by combining indole with serine (Li et al., 2019b). Moreover, TSA catalyzes indole synthesis only in a TSB-dependent manner (Maeda and Dudareva, 2012; Zeng et al., 2016). Previous studies have reported that the up-regulation of the genes of AS, IGPS, and TS can result in an increase of tryptophan and indole (Gao et al., 2016; Jin et al., 2015; Chen et al., 2013). In this work, nine unigenes involved in indole biosynthesis were identified. Notably, these genes include four AS genes (gene86009 (ASA1), gene86713 (ASA2), gene97347 (OsASA2), and gene102890 (ASB2)), two IGPS genes (gene95714 (IGPS) and gene108971 (IGPS)), and three TS genes (gene88810 (TSB), gene101490 (TSB), and gene102007 (TSB)), all of which were significantly more highly expressed in August (Supplementary Fig. S5), which strongly suggests that P. tinctorium produce more indole at this harvest time. The increase of indole in August was confirmed by HPLC analysis (Supplementary Fig. S1). The higher expression of indole biosynthetic genes provided more precursors for downstream biosynthesis of indigo and indirubin. It has been reported that indole can be oxidized to form indoxyl in the presence of CYP450 (Song et al., 2011). In this work, seven DEGs encoding CYP450 s were identified. Moreover, all of the seven unigenes (gene39188 (CYP82A3), gene67221 (CYP94B1), gene81797 (CYP94C1), gene85036 (CYP82C2), gene89942 (CYP71A9), gene99655 (CYP71A1), and gene100326 (CYP71A1) exhibited significantly higher expression levels (p < 0.05) in August (Supplementary Fig. S6). The up-regulated CYP450s can promote the transformation of accumulated indole to indoxyl. In plants, indoxyl is unstable, and is converted into stable derivatives by two branches (Fig. 6). To avoid spontaneous oxidation, indoxyl is stabilized by a glucose moiety and converted to the colorless molecule indican (indoxyl β-D-glucoside), one of the indigo precursors that is mainly stored in vacuoles (Maier et al., 1990; Minami et al., 2000). Indoxyl is transformed to indican by UGTs (Song et al., 2011). In previous reports, three UGTs, including PtUGT1, PtUGT2, and PtIGS, were identified in P. tinctorium (Inoue et al., 2018; Hsu et al., 2018). In these previous studies, only four amino acid residues differed

5. Conclusion In this work, it was demonstrated that significant changes occur at harvest time in August in the transcriptome of P. tinctorium leaves and its content of indigo and indirubin. Collectively, to understand the underlying mechanisms of indigo and indirubin accumulation in P. tinctorium, the leaves at different harvest times were analyzed by HPLC and RNA-seq. The results showed that the significant increase of indigo and indirubin were caused by high expression of the associated biosynthetic genes during harvest time in August. These results provide insights into the understanding of indigo and indirubin accumulation and the molecular mechanism of their biosynthesis. Moreover, this work identified a series of candidate genes that can be potentially applied in the breeding of P. tinctorium cultivars.

Declaration of Competing Interest There are no conflicts to declare.

Acknowledgements This work was supported by the Fundamental Research Funds for the Central Universities from Hunan University. 7

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Appendix A. Supplementary data

Agric. Food Chem. 66, 9828–9838. https://doi.org/10.1021/acs.jafc.8b01995. Inoue, S., Morita, R., Kuwata, K., Kunieda, T., Ueda, H., Hara-Nishimura, I., Minami, Y., 2018. Tissue-specific and intracellular localization of indican synthase from Polygonum tinctorium. Plant Physiol. Biochem. 132, 138–144. https://doi.org/10. 1016/j.plaphy.2018.08.034. Ishihara, T., Okura, T., Kohno, K., Tanimoto, T., Ikegami, H., Kurimoto, M., 2000. Polygonum tinctorium extract suppresses nitric oxide production by activated macrophages through inhibiting inducible nitric oxide synthase expression. J. Ethnopharmacol. 72, 141–150. https://doi.org/10.1016/s0378-8741(00)00249-x. Jin, B., Zhou, X.R., Jiang, B.L., Gu, Z.M., Zhang, P.H., Qian, Q., Chen, X.F., Ma, B.J., 2015. Transcriptome profiling of the spl5 mutant reveals that SPL5 has a negative role in the biosynthesis of serotonin for rice disease resistance. Rice 8, 18. https://doi.org/10. 1186/s12284-015-0052-7. Kanehisa, M., Goto, S., Sato, Y., Furumichi, M., Tanabe, M., 2012. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 40, D109–D114. https://doi.org/10.1093/nar/gkr988. Kim, H.-A., Suh, H.-R., Kang, B., Choe, B.-H., Crohns, Colitis Association in, D, 2018. Acute pancreatitis associated with indigo naturalis in pediatric severe Crohn’s disease. Intest. Res. 17, 144–148. https://doi.org/10.5217/ir.2018.00104. Kim, J.Y., Lee, J.Y., Shin, Y.S., Kim, G.J., 2010. Characterization of an indican-hydrolyzing enzyme from Sinorhizobium meliloti. Process Biochem. 45, 892–896. https:// doi.org/10.1016/j.procbio.2010.02.017. Kim, K.S., Hwang, W.G., Jang, H.G., Heo, B.G., Suhaj, M., Leontowicz, H., Leontowicz, M., Jastrzebski, Z., Tashma, Z., Gorinstein, S., 2012. Assessment of Indigo (Polygonum tinctorium Ait.) water extracts’ bioactive compounds, and their antioxidant and antiproliferative activities. LWT-Food Sci. Technol. 46, 500–510. https://doi.org/10. 1016/j.lwt.2011.11.017. Kukula-Koch, W., Koch, W., Stasiak, N., Glowniak, K., Asakawa, Y., 2015. Quantitative standarization and CPC-based recovery of pharmacologically active components from Polygonum tinctorium Ait. leaf extracts. Ind. Crops Prod. 69, 324–328. https://doi.org/ 10.1016/j.indcrop.2015.02.048. Kunikata, T., Tatefuji, T., Aga, H., Iwaki, K., Ikeda, M., Kurimoto, M., 2000. Indirubin inhibits inflammatory reactions in delayed-type hypersensitivity. Eur. J. Pharmacol. 410, 93–100. https://doi.org/10.1016/s0014-2999(00)00879-7. Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359. https://doi.org/10.1038/nmeth.1923. Lee, S., Kim, D.C., Baek, H.Y., Lee, K.D., Kim, Y.C., Oh, H., 2018. Anti-neuroinflammatory effects of tryptanthrin from Polygonum tinctorium Lour. in lipopolysaccharide-stimulated BV2 microglial cells. Arch. Pharm. Res. 41, 419–430. https://doi.org/10.1007/ s12272-018-1020-8. Li, R., Jiang, J., Jia, S., Zhu, X., Su, H., Li, J., 2019b. Overexpressing broccoli tryptophan biosynthetic genes BoTSB1 and BoTSB2 promotes biosynthesis of IAA and indole glucosinolates. Physiol. Plant. https://doi.org/10.1111/ppl.12933. Li, S., Cunningham, A.B., Fan, R., Wang, Y., 2019a. Identity blues: the ethnobotany of the indigo dyeing by landian yao (iu mien) in yunnan, southwest china. J. Ethnobiol. Ethnomed. 15, 13. https://doi.org/10.1186/s13002-019-0289-0. Maeda, H., Dudareva, N., 2012. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu. Rev. Plant Biol. 63, 73–105. https://doi.org/10.1146/ annurev-arplant-042811-105439. Maere, S., Heymans, K., Kuiper, M., 2005. BiNGO: a cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21, 3448–3449. https://doi.org/10.1093/bioinformatics/bti551. Maier, W., Schumann, B., Gröger, D., 1990. Biosynthesis of indoxyl derivatives in Isatis tinctoria and Polygonum tinctorium. Phytochemistry 29, 817–819. https://doi.org/10. 1016/0031-9422(90)80025-C. Minami, Y., Kanafuji, T., Miura, K., 1996. Purification and characterization of a betaglucosidase from Polygonum tinctorium which catalyzes preferentially the hydrolysis of indican. Biosci. Biotech. Biochem. 60, 147–149. https://doi.org/10.1271/bbb.60. 147. Minami, Y., Nishimura, O., Hara-Nishimura, I., Nishimura, M., Matsubara, H., 2000. Tissue and intracellular localization of indican and the purification and characterization of indican synthase from indigo plants. Plant Cell Physiol. 41, 218–225. https://doi.org/10.1093/pcp/41.2.218. Minami, Y., Sarangi, B.K., Thul, S.T., 2015. Transcriptome analysis for identification of indigo biosynthesis pathway genes in Polygonum tinctorium. Biologia 70, 1026–1032. https://doi.org/10.1515/biolog-2015-0131. Minami, Y., Shigeta, Y., Tokumoto, U., Tanaka, Y., Yonekura-Sakakibara, K., Oh-oka, H., Matsubara, H., 1999. Cloning, sequencing, characterization, and expression of a βglucosidase cDNA from the indigo plant. Plant Sci. 142, 219–226. https://doi.org/10. 1016/S0168-9452(99)00015-1. Minami, Y., Takao, H., Kanafuji, T., Miura, K., Kondo, M., Hara-Nishimura, I., Nishimura, M., Matsubara, H., 1997. beta-Glucosidase in the indigo plant: intracellular localization and tissue specific expression in leaves. Plant Cell Physiol. 38, 1069–1074. https://doi.org/10.1093/oxfordjournals.pcp.a029273. Murdock, D., Ensley, B.D., Serdar, C., Thalen, M., 1993. Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Escherichia coli. Bio/technology (Nature Publishing Company) 11, 381–386. https://doi.org/10.1038/nbt0393-381. Ou, B.Y., Ye, H.G., Li, B., 2017. Study on the origin of Nantong Blue calico. In: Tseng, A.A., Ansari, S. (Eds.), Proceedings of the 2017 2nd International Conference on Humanities and Social Science. Atlantis Press, Paris. pp. 263–267. https://doi.org/ 10.2991/hss-17.2017.48. Powell, S., Forslund, K., Szklarczyk, D., Trachana, K., Roth, A., Huerta-Cepas, J., Gabaldon, T., Rattei, T., Creevey, C., Kuhn, M., Jensen, L.J., von Mering, C., Bork, P., 2014. eggNOG v4.0: nested orthology inference across 3686 organisms. Nucleic Acids Res. 42, D231–D239. https://doi.org/10.1093/nar/gkt1253. Rius, M.A., Garcia-Regueiro, J.A., 2001. Skatole and indole concentrations in Longissimus

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111783. References Ali, H., 2010. Biodegradation of synthetic dyes—a review. Water Air Soil Pollut. 213, 251–273. https://doi.org/10.1007/s11270-010-0382-4. Angelini, L.G., Tozzi, S., Di Nasso, N.N.O., 2004. Environmental factors affecting productivity, indican content, and indigo yield in Polygonum tinctorium Ait., a subtropical crop grown under temperate conditions. J. Agric. Food Chem. 52, 7541–7547. https://doi.org/10.1021/jf040312b. Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M.C., Estreicher, A., Gasteiger, E., Martin, M.J., Michoud, K., O’Donovan, C., Phan, I., Pilbout, S., Schneider, M., 2003. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res. 31, 365–370. https://doi.org/10.1093/nar/gkg095. Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. https://doi.org/10.1093/ bioinformatics/btu170. Bryant, D.M., Johnson, K., DiTommaso, T., Tickle, T., Couger, M.B., Payzin-Dogru, D., Lee, T.J., Leigh, N.D., Kuo, T.H., Davis, F.G., Bateman, J., Bryant, S., Guzikowski, A.R., Tsai, S.L., Coyne, S., Ye, W.W., Freeman, R.M., Peshkin, L., Tabin, C.J., Regev, A., Haas, B.J., Whited, J.L., 2017. A tissue-mapped axolotl de novo transcriptome enables identification of limb regeneration factors. Cell Rep. 18, 762–776. https:// doi.org/10.1016/j.celrep.2016.12.063. Campeol, E., Angelini, L.G., Tozzi, S., Bertolacci, M., 2006. Seasonal variation of indigo precursors in Isatis tinctoria L. and Polygonum tinctorium ait. as affected by water deficit. Environ. Exp. Bot. 58, 223–233. https://doi.org/10.1016/j.envexpbot.2005. 09.006. Celik, A., Speight, R.E., Turner, N.J., 2005. Identification of broad specificity P450CAM variants by primary screening against indole as substrate. Chem. Commun. 3652–3654. https://doi.org/10.1039/b506156c. Chen, J.F., Dong, X., Li, Q., Zhou, X., Gao, S.H., Chen, R.B., Sun, L.N., Zhang, L., Chen, W.S., 2013. Biosynthesis of the active compounds of Isatis indigotica based on transcriptome sequencing and metabolites profiling. BMC Genomics 14, 857. https://doi. org/10.1186/1471-2164-14-857. Ensley, B.D., Ratzkin, B.J., Osslund, T.D., Simon, M.J., Wackett, L.P., Gibson, D.T., 1983. Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science (New York, N.Y.) 222, 167–169. https://doi.org/10. 1126/science.6353574. Ernst, J., Bar-Joseph, Z., 2006. STEM: a tool for the analysis of short time series gene expression data. BMC Bioinformatics 7, 191. https://doi.org/10.1186/1471-2105-7191. Finn, R.D., Coggill, P., Eberhardt, R.Y., Eddy, S.R., Mistry, J., Mitchell, A.L., Potter, S.C., Punta, M., Qureshi, M., Sangrador-Vegas, A., Salazar, G.A., Tate, J., Bateman, A., 2016. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279–D285. https://doi.org/10.1093/nar/gkv1344. Gao, C., Yang, B., Zhang, D., Chen, M., Tian, J., 2016. Enhanced metabolic process to indole alkaloids in Clematis terniflora DC. after exposure to high level of UV-B irradiation followed by the dark. BMC Plant Biol. 16, 231. https://doi.org/10.1186/ s12870-016-0920-3. Gilbert, K.G., Cooke, D.T., 2001. Dyes from plants: past usage, present understanding and potential. Plant Growth Regul. 34, 57–69. https://doi.org/10.1023/ a:1013374618870. Gillam, E.M.J., Notley, L.M., Cai, H.L., De Voss, J.J., Guengerich, F.P., 2000. Oxidation of indole by cytochrome P450 enzymes. Biochemistry 39, 13817–13824. https://doi. org/10.1021/bi001229u. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q.D., Chen, Z.H., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B.W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., Regev, A., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652. https://doi.org/10. 1038/nbt.1883. Harris, M.A., Clark, J., Ireland, A., Lomax, J., Ashburner, M., Foulger, R., Eilbeck, K., Lewis, S., Marshall, B., Mungall, C., Richter, J., Rubin, G.M., Blake, J.A., Bult, C., Dolan, M., Drabkin, H., Eppig, J.T., Hill, D.P., Ni, L., Ringwald, M., Balakrishnan, R., Cherry, J.M., Christie, K.R., Costanzo, M.C., Dwight, S.S., Engel, S., Fisk, D.G., Hirschman, J.E., Hong, E.L., Nash, R.S., Sethuraman, A., Theesfeld, C.L., Botstein, D., Dolinski, K., Feierbach, B., Berardini, T., Mundodi, S., Rhee, S.Y., Apweiler, R., Barrell, D., Camon, E., Dimmer, E., Lee, V., Chisholm, R., Gaudet, P., Kibbe, W., Kishore, R., Schwarz, E.M., Sternberg, P., Gwinn, M., Hannick, L., Wortman, J., Berriman, M., Wood, V., de la Cruz, N., Tonellato, P., Jaiswal, P., Seigfried, T., White, R., Gene Ontology, C, 2004. The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 32, D258–D261. https://doi.org/10.1093/nar/gkh036. Heo, B.G., Park, Y.J., Park, Y.S., Bae, J.H., Cho, J.Y., Park, K., Jastrzebski, Z., Gorinstein, S., 2014. Anticancer and antioxidant effects of extracts from different parts of indigo plant. Ind. Crops Prod. 56, 9–16. https://doi.org/10.1016/j.indcrop.2014.02.023. Hsu, T.M., Welner, D.H., Russ, Z.N., Cervantes, B., Prathuri, R.L., Adams, P.D., Dueber, J.E., 2018. Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nat. Chem. Biol. 14, 256–261. https://doi.org/10.1038/nchembio.2552. Huang, H., Yao, Q.Y., Xia, E.H., Gao, L.Z., 2018. Metabolomics and transcriptomics analyses reveal nitrogen influences on the accumulation of flavonoids and amino acids in young shoots of tea plant (Camellia sinensis L.) associated with tea flavor. J.

8

Industrial Crops & Products 141 (2019) 111783

W. Wang, et al.

and cell cultures. Plant Biotechnol. J. 5, 185–191. https://doi.org/10.1111/j.14677652.2006.00231.x. Wu, Y.Q., Guo, J., Zhou, Q., Xin, Y., Wang, G.B., Xu, L.A., 2018. De novo transcriptome analysis revealed genes involved in flavonoid biosynthesis, transport and regulation in Ginkgo biloba. Ind. Crops Prod. 124, 226–235. https://doi.org/10.1016/j.indcrop. 2018.07.060. Xiao, Z.J., Hao, Y.S., Liu, B.C., Qian, L.S., 2002. Indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in China. Leuk. Lymphoma 43, 1763–1768. https://doi.org/10.1080/1042819021000006295. Xie, Y., Baiyin, H.E., Yuanbin, L.I., Xuelan, Y.E., Liu, C., Chen, J., Lai, X., 2011. Simultaneous determination of indigo and indirubin in medicinal material of indigo naturalis. Tradit. Chin. Drug Res. Clin. Pharmacol. 22, 452–455. https://doi.org/10. 19378/j.issn.1003-9783.2011.04.026. Ye, J., Fang, L., Zheng, H.K., Zhang, Y., Chen, J., Zhang, Z.J., Wang, J., Li, S.T., Li, R.Q., Bolund, L., Wang, J., 2006. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 34, W293–W297. https://doi.org/10.1093/nar/gkl031. Zeng, L., Zhou, Y., Gui, J., Fu, X., Mei, X., Zhen, Y., Ye, T., Du, B., Dong, F., Watanabe, N., Yang, Z., 2016. Formation of volatile tea constituent indole during the oolong tea manufacturing process. J. Agric. Food Chem. 64, 5011–5019. https://doi.org/10. 1021/acs.jafc.6b01742. Zhong, Y., Yoshinaka, Y., Takeda, T., Shimizu, N., Yoshizaki, S., Inagaki, Y., Matsuda, S., Honda, G., Fujii, N., Yamamoto, N., 2005. Highly potent anti-HIV-1 activity isolated from fermented Polygonum tinctorium Aiton. Antiviral Res. 66, 119–128. https://doi. org/10.1016/j.antiviral.2005.02.003.

dorsi and fat samples of pigs. Meat Sci. 59, 285–291. https://doi.org/10.1016/s03091740(01)00081-x. Sahoo, A., Kar, B., Sahoo, S., Jena, S., Kuanar, A., Parameswaran, S., Patnaik, J., Nayak, S., 2019. De Novo transcriptome sequencing explored cultivar specific sequence variation and differential expression of pigment synthesis genes in turmeric (Curcuma longa L.). Ind. Crops Prod. 134, 388–402. https://doi.org/10.1016/j.indcrop.2019. 04.021. Shahid ul, I., Shahid, M., Mohammad, F., 2013. Perspectives for natural product based agents derived from industrial plants in textile applications - a review. J. Clean. Prod. 57, 2–18. https://doi.org/10.1016/j.jclepro.2013.06.004. Shin, Y., Son, K., Yoo, D., 2017. Evaluation of aroma functionality imparted on natural indigo-dyed cotton fabrics. Fiber. Polym. 18, 1146–1153. https://doi.org/10.1007/ s12221-017-6942-0. Song, K.S., Shim, J.Y., Jung, D.S., Kim, S.U., 2011. Origin of oxygen in indoxyl-derivatives of Polygonum tinctorium L. as probed by 18O2 feeding. J. Korean Soc. Appl. Biol. Chem. 54, 340–344. https://doi.org/10.3839/jksabc.2011.054. Suo, J.W., Tong, K., Wu, J.S., Ding, M.Z., Chen, W.C., Yang, Y., Lou, H.Q., Hu, Y.Y., Yu, W.W., Song, L.L., 2019. Comparative transcriptome analysis reveals key genes in the regulation of squalene and beta-sitosterol biosynthesis in Torreya grandis. Ind. Crops Prod. 131, 182–193. https://doi.org/10.1016/j.indcrop.2019.01.035. Tsugama, D., Matsuyama, K., Ide, M., Hayashi, M., Fujino, K., Masuda, K., 2017. A putative MYB35 ortholog is a candidate for the sex-determining genes in Asparagus officinalis. Sci. Rep. 7, 41497. https://doi.org/10.1038/srep41497. Warzecha, H., Frank, A., Peer, M., Gillam, E.M.J., Guengerich, F.P., Unger, M., 2007. Formation of the indigo precursor indican in genetically engineered tobacco plants

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