Citation: Pan Y, Zhao X, Wang Y, Tan J, Chen D-x (2021) Metabolomics integrated with transcriptomics reveals the distribution of iridoid and crocin metabolic flux in Gardenia jasminoides Ellis. PLoS ONE 16(9):
Editor: Dinesh Nagegowda, CSIR-Central Institute of Medicinal and Aromatic Plants, INDIA
Received: February 3, 2021; Accepted: August 16, 2021; Published: September 10, 2021
Copyright: Â© 2021 Pan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The whole set of transcript data can be found in the National Center for Biotechnology Information (NCBI) SRA database (PRJNA688705; SRR13360533 – SRR13360544).
Funding: This work was supported by Natural Science Foundation of Chongqing province of China, grant number– cstc2018jcyjAX0553 (Yuan Pan).
Competing interests: The authors have declared that no competing interests exist.
10-hydroxygeraniol oxidoreductase; ALDH,
acetaldehyde dehydrogenase; CCD,
carotenoid cleavage dioxygenase; CRTISO,
carotenoid isomerase; DAM,
differentially accumulated metabolite; DEG,
differentially expression gene; DXR,
2-C-methyl-D-erythritol-4-phosphate reductoisomerase; DXS,
1-deoxy-D-xylulose-5-phosphate synthase; FP,
the peel of red fruit; FPPS,
farnesyl diphosphate synthase; FS,
the sarcocarp of red fruit; G10H,
geraniol 10-hydroxylase; G8O,
8-hydroxygeraniol dehydrogenase; GES,
geraniol synthase; GFP,
the peel of green fruit; GFS,
the sarcocarp of green fruit; GGPPS,
geranylgeranyl diphosphate synthase; GPPS,
geranyl diphosphate synthase; HDR,
4-hydroxy-3-methyl-2-(E)-butenlyl diphosphate reductase; HMGR,
3-hydroxy-3-methyl glutaryl CoA reductase; HMGS,
3-hydroxy-3-methylglutaryl CoA synthase; IS,
iridoid synthase; LCYB,
lycopene cyclase; MK,
mevalonate kinase; NCED,
9-cis-epoxycarotenoid dioxygenase; PDS,
phytoene desaturase; PSY,
phytoene synthase; SS,
squalene synthase; UGTs,
zeta-carotene desaturase; Z-ISO,
Gardenia jasminoides Ellis (G. jasminoides), belonging to the Rubiaceae family, is an evergreen shrub that is cultivated in multiple areas in China and is adapted to many temperate regions . It has been used for many years to obtain a natural yellow dye [2, 3], and itâ€™s mature and dried fruits are employed in traditional Chinese medicine. Its fruits exhibit a cold and bitter taste, present anti-inflammatory , antidiabetic , antidepression , hepatocyte-protective , antioxidant properties  and improve the quality of sleep [1, 9]. Iridoids, organic acids, flavonoids and triterpenes have been identified in the fruit of G. jasminoides . Many chemicals have been isolated and characterized in the fruit, such as geniposide, genipin, gardenoside, crocin and other iridiods. Geniposide is recorded as the quality control standard for G. jasminoides in the Chinese Pharmacopoeia (2000â€“2015 edition) [10, 11]. Because of the pharmacological benefits of crocins and their abundance, G. jasminoides crocins have attracted increasing attention . Research on bioactive constituent synthesis with a focus on G. jasminoides has gradually increased. Iridoid are cyclopentapyranoid monoterpenoids that exist in many medicinal plants and are still expanding. Iridoid was derived from terpenoids, which was synthesized upstream of the cytosolic mevalonate (MVA) and plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, providing precursors of isoprene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) for the biosynthesis of iridoid glycosides . Iridoid biosynthesis is initiated from gernyl pyrophosphate, which is converted to secologanin by a series of reactions that include oxidation, reduction, glycosylatios and methylation steps . The pathway is further modified to produce various subclasses of iridoid by different classes of enzymes. The functional analysis of medicinal plants has identified a set of genes that encode key components of the terpenoid and iridoid biosynthesis pathway and its constituent enzymes, such as 1-deoxy-D-xylulose-5-phosphate synthase (DXS); 2-C-methyl-D-erythritol-4-phosphate reductoisomerase (DXR); isopentenyl diphosphate isomerase (IPPI); 4-(cytidyl-5-diphospho)-2-C-methyl-D-erythritol transferase (CMS); 2-phospho-4-(cytidine 5â€™-diphospho)-2-C-methyl-D-erythritol kinase (CMK); 4-hydroxy-3-methyl-2-enbutenyl-4-diphosphate synthase (HDS); 4-hydroxy-3-methyl-2-(E)-butenlyl diphosphate reductase (HDR); geranyl diphosphate synthase (GPPS); and geranylgeranyl pyrophosphate synthase (GGPPS) [15â€“19]. Transcriptome analysis of G. jasminoides tissues has successfully identified genes that are involved in terpenoid and iridoid biosynthesis [10, 11, 20]. In recent years, studies on the biosynthetic pathway of crocin have attracted increasing attention because of their high medicinal and commercial value. The crocin biosynthesis pathway in Crocus sativus L. includes the upstream methylerythritol phosphate (MEP) pathway from pyruvate/glyceraldehyde 3-phosphate to geranylgeranyl pyrophosphate (GGPP), the midstream carotenoid pathway from GGPP to zeaxanthin, and the downstream crocin pathway from zeaxanthin to crocin [12, 21]. A variety of catalytic enzymes and coding genes are involved in the whole pathway, including phytoene desaturase (PDS), zeta-carotene isomerase (Z-ISO), zeta-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), lycopene-cyclase (LCYB), carotenoid cleavage dioxygenase (CCD), aldehyde dehydrogenase (ALDH) and glucosyltransferases (UGTs) [22â€“27]. Previous studies showed that the biosynthesis of iridoids and crocins originated from the same precursor pathway. In G. jasminoides, relevant studies have been carried out on the above substances, but few studies have investigated the integration of the biosynthesis pathways of the two kinds of substances. Additionally, the distribution of bioactive substances in different organs of G. jasminoides fruit and the expression of related genes are not clear. Recent technical advancements in transcriptome and metabolome analyses have provided effective ways to identify new genes and metabolites and to elucidate complex secondary metabolic bioprocesses in plants . In this study, based on the widely targeted metabolomics approach and RNA-Seq sequencing, we profiled the transcriptome and metabolome changes in the peel and sarcocarp of G. jasminoides fruit during various ripening stages to investigate the biosynthesis mechanism of iridoid and crocin. This study analyzes the molecular mechanisms of the synthesis and distribution of the active components of G. jasminoides fruit from a new perspective, which provides a theoretical basis for the subsequent selection of dominant provenances and variety improvement.
Materials and methods
My study did not involve human participants, specimens or tissue samples, or vertebrate animals, embryos or tissues:
- We state clearly that no specific permissions were required for these locations/activities, and provide details on why this is the case. The planting place is a Medicinal herb planting base of Chongqing Academy of Chinese Materia Medica, where we have carried out related research on the cultivation of Gardenia jasminoides Ellis.
- We confirm that the field studies did not involve endangered or protected species.
- We confirm that the anthors had received approval from Chongqing Academy of Chinese Materia Medica to collect samples from the plants.
The widely cultivated Gardenia jasminoides Ellis were used for the present experiment, which were presented and identified as Gardenia jasminoides Ellis by Professor Daxia Chen. The trees were grown in a Medicinal herb planting base located at the southern hilly region of Banan District, Chongqing, China (N29.34, E106.92). A total of three trees were chosen to collect different fruit samples. All the trees were planted in parallel and were 7 years old. In order to ensure the consistency of sampling, we mark the buds at the same time during the flowering period. The peel of green fruit (GFP), the sarcocarp (the part within the peel of a G. jasmonoides) of green fruit (GFS), the peel of red fruit (FP) and the sarcocarp of red fruit (FS) were harvested from three plants, respectively (Fig 1). After washing, the samples were immediately frozen in liquid nitrogen and stored at -80Â°C until further use in metabolite, RNA sequencing (RNA-Seq), and qPCR analyses.
The freezedried samples were ground with zirconia beads for 1.5 min at a 30 Hz stirring mill (MM 400, Retsch, Germany). A total of 100 mg of each powder sample was dissolved in 1.0 mL of the extraction solution (70% methanol solution), which was extracted overnight by a wheel at 4Â°C. Three vortexes were carried out in this process to ensure complete extraction. Following centrifugation at 10 000Ã—g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 mL; ANPEL, Shanghai, China, www.anpel.com.cn/cnw) and filtered (SCAA-104, 0.22 Î¼m pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) before LC-MS analysis. In addition, quality control samples (mixing) are prepared by mixing all samples in equal amounts. Quality control samples are performed every 10 needles to monitor the stability of the analytical conditions during analysis. The sample preparation, extract analysis, metabolite identification and quantification were performed at Wuhan MetWare Biotechnology Co., Ltd. (www.metware.cn) following their standard procedures and previously fully described by Yuan et al. (2018) and Wang et al. (2017) [29, 30].
Metabolite analysis by Liquid Chromatographyâ€“Electrospray Ionizationâ€“Tandem Mass Spectrometry (LC-ESI-MS/MS)
LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupolelinear ion trap mass spectrometer (Q TRAP), API 4500 Q TRAP LC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550Â°C; ion spray voltage (IS) 5500 V; ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; and the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 Î¼mol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions were performed with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the metabolites eluted within this period.
Qualitative and quantitative analysis
According to previous research results [31, 32], we identified standard substances in self-built databases (MetWare, Wuhan, China)  and public databases by comparing fragmentation pattern, retention time and accurate M/Z value. The supervised multivariate method, partial least squares discriminant analysis (PLS-DA), was used to maximize the metabolome differences between a pair of samples. The relative importance of each metabolite to the PLS-DA model was checked using the parameter called variable importance in projection (VIP). Metabolites with VIP â‰¥ 1 and fold change â‰¥2 or fold change â‰¤0.5 were considered differential metabolites for group discrimination .
RNA extraction and transcriptome sequencing
Total RNA was obtained from three different biological replicates for each group using TRIzol reagent (Invitrogen) following the manufacturerâ€™s protocol. Total RNA purity and concentration were determined using the NanoPhotometerÂ® spectrophotometer (IMPLEN, CA, USA) and the QubitÂ® RNA Assay Kit in QubitÂ® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1.5 Î¼g RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNextÂ® Ultraâ„¢ RNA Library Prep Kit for IlluminaÂ® (NEB, USA) following manufacturerâ€™s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturerâ€™s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 4000 platform and paired-end reads were generated. In the quality control step, raw reads of fastq format were firstly processed through in house perl scripts. In this step, clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw reads. At the same time, Q20, Q30, GC content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality. In the transcriptome assembly step, the left files (read1 files) from all libraries/ samples were pooled into one big left.fq file, and right files (read2 files) into one big right.fq file. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity [34, 35] with min_kmer_cov set to 2 by default and all other parameters set default. Gene function was performed as previously described : NCBI blast 2.2.28C  was used for the alignments of unigenes to Nt database with an E-value threshold of 1E-5. The program diamond (v0.8.22)  was selected to perform the comparison against Nr (E-value 1E-5), KOG/COG (E-value 1E-3), and SwissProt (E-value 1E-5) databases. The hmmscan in HMMER 3.0 was operated to search Pfam  with an E-value threshold of 0.01. The GO (Gene Ontology) annotations were carried out in Blast2GO (v2.5)  with an E-value threshold of 1E-6 based on the Nr and Pfam annotations. Pathway analysis was conducted to elucidate significant pathways of DEGs according to the Kyoto Encyclopedia of Gene and Genomes (KEGG) (http://www.genome.jp/kegg) databases. KOBAS software was used to test the statistical enrichment of differential expression genes in KEGG pathways. The pathways with an FDR value of â‰¤0.05 were defined as those with genes that display significant levels of differential expression .
Differential expression analysis
The assembled transcriptome spliced by Trinity was used as reference sequence (ref) , and the clean reads of each sample were mapped back to this ref in RSEM (v1.2.15)  with the Bowtie2 mismatch set 0 as default. The mapping results including read counts of each sample were normalized by calculating the FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions of base pairs sequenced) to obtain relative expression levels of unigenes . Differential expression analysis of different groups was performed using the DESeq2 R package (v1.6.3). Input data of DESeq2 were clean reads obtained by RSEM. DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochbergâ€™s approach for controlling the false discovery rate. Genes with an adjusted P value (padj) <0.05 and |log2FoldChange| >1 were assigned as differentially expressed.
Real time quantitative PCR
To analyse DGEs, total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) from 100 mg of smaples. First strand cDNA was synthesized from 2 Î¼g total RNA using M-MLV Reverse Transcriptase (Promega, WI, USA) according to the manufacturerâ€™s instructions. The reactions (20 Î¼L) were terminated after 40 cycles using the CFX96TM Real Time PCR Detection System (Bio-Rad, CA, USA), and the Actin was used as an internal control. All assays were repeated at least three times. The relevant PCR primer sequences were shown in S1 Table, which were designed based on the CDS sequences of the G. jasmonoides by the Primer Express Software (Applied Biosystems, CA, USA).
Correlation analysis between metabolome and transcriptome data
Pearsonâ€™s correlation coefficients were calculated between the metabolome and transcriptome data. The coefficients were calculated from log2 (fold change) of each metabolite and log2 (fold change) of each transcript with the Excel program. Correlations with a coefficient of R2 > 0.8 were selected.
Metabolome characteristics of different colors and parts of G. jasminoides fruits
Generally, G. jasminoides blossoms from May to June every year. As the flowers wither, the fruit begins to develop. During the development of G. jasminoides fruit, the peel changes from green to red, the sarcocarp changes from white to red (Fig 1), and the metabolite composition and contents of the fruit change greatly. The secondary metabolites present in G. jasminoides during different tissue development stages were investigated based on the widely targeted metabolomics approach by using UPLC-ESI-MS/MS and public and a self-built database (including a MetWare database). A total of 254 secondary metabolites were detected, including 39 terpenoids, 73 flavonoids, 95 phenolic acids, 16 lignans and coumarins, 12 alkaloids, 6 tannins and 13 other metabolites (Fig 2). In the clustering heat map, the accumulation of metabolites displayed clear variation in terms of the pattern of metabolite abundance in different fruit colors and parts, and the green fruit was clearly distinct from the red fruit (Fig 2). We observed that all the biological replicates were grouped together (top side of the figure), indicating high reliability of the metabolome data. In particular, we observed clear separation among GFP, GFS, FP and FS. The above results indicate that there are obvious differences in the metabolic characteristics of the four groups of samples. As shown in Fig 2A, the metabolites detected in the organs of G. jasminoides were mainly terpenoids, phenolic acids and flavonoids. These metabolites may be associated with many factors, such as nutritional components and antioxidants. The biosynthesis of the active components of G. jasminoides is mainly centered on the synthesis of terpenoids in the pathway of secondary metabolites.
Fig 2. Number and heatmap of metabolite profiles of four groups of samples.
(A) Number of different types of metabolites in all samples. (B) Clustering heatmap of the metabolites detected in the total samples. Each example is visualized in a single column and each metabolite is represented by a single row. Red indicates high abundance, whereas low relative metabolites are shown in green. GFP = the peel of green fruit; GFS = the sarcocarp of green fruit; FP = the peel of red fruit; FS = the sarcocarp of red fruit.
Comparison of metabolites produced by the four groups of samples
The differentially accumulated metabolites (DAMs) between pairs of samples (GFP vs GFS, GFP vs FP, GFS vs FS and FP vs FS) were screened based on the criterion of a variable importance in projection (VIP) â‰¥ 1 and a fold change â‰¥2 or â‰¤0.5. The DAM screening results are shown in Fig 3. There were 85 kinds of significant DAMs between FS and FP (including 26 downregulated and 59 upregulated compounds in the FS samples) (Fig 3A), 76 kinds of significant DAMs between GFS and GFP (including 30 downregulated and 46 upregulated compounds in the GFS samples) (Fig 3B), 68 kinds of significant DAM between GFS and FS (including 54 downregulated and 14 upregulated compounds in the GFS samples) (Fig 3C), 73 kinds of significant DAM between GFP and FP (including 50 downregulated and 23 upregulated compounds in the GFP samples) (Fig 3D). As seen from the Venn diagram (Fig 3E), there were 11 common DAMs in the four groups of samples, and 76, 73, 68 and 85 specific DAMs were found in GFP vs GFS, GFP vs FP, GFS vs FS and FP vs FS, respectively.
Fig 3. Differential metabolites analysis.
Volcano plot of metabolites between FS vs FP (A), GFS vs GFP (B), GFS vs FS (C), GFP vs FP (D); (E): Venn diagram representing numbers of DAMs. GFP = the peel of green fruit; GFS = the sarcocarp of green fruit; FP = the peel of red fruit; FS = the sarcocarp of red fruit. Each point in the volcanic plot represents a metabolite, the abscissa represents the logarithm of the quantitative difference multiples of a metabolite in two samples, and the ordinate represents the variable importance in project (VIP) value. The green dots in the figure represent downregulated differentially expressed metabolites, the red dots represent upregulated differentially expressed metabolites, and the black dots represent metabolites detected but that are not significantly different.
Transcriptome characteristics of different colors and different parts of G. jasminoides fruits
In this study, the peel of green fruit (GFP), the sarcocarp of green fruit (GFS), the peel of red fruit (FP) and the sarcocarp of red fruit (FS) of G. jasminoides were used for transcriptome sequencing. A total of 675 million clean reads were obtained, including 101.2 GB of nucleotide sequence, as shown in Table 1. A total of 52,658 unigene sequences with N50 and N90 lengths of 3,215 bp and 642 bp were obtained by assembling the high quality sequences, as shown in S2 Table. The results showed that the quality of the output data was good and that bioinformatics analysis could be carried out. The unigenes were annotated for gene functions in seven databases, including Nr, Nt, Pfam, KOG/COG, Swissprot, KEGG and GO. A total of 4,729 unigenes were annotated in all databases, accounting for 8.98% of the unigenes, and 35,216 unigenes were annotated in at least one database, accounting for 66.87% of the unigenes (Table 2).
PCA of the samples based on the fragments per kilobase of exon per million fragments mapped (FPKM) values showed that all the biological replicates clustered together, indicating the high reliability of our results (Fig 4A). According to the results, four groups of samples were clearly distinguished in the PC1 dimension of the PCA score graph (35.3% variation) and were further differentiated in the PC2 dimension, indicating that gene expression varied significantly in different fruit stages and parts. The FPKM method  was used to calculate the expression of all unigenes to remove the effects of length differences and sequencing depth. DEGs were defined as genes that were significantly enriched or depleted in one sample relative to another according to DESeq  (padj < 0.05 and log2 (fold change) > 1). Compared with FP, 2,351 unigenes in FS showed significant differences; compared with GFP, 4,095 unigenes in GFS showed significant differences; compared with FS, 5,137 unigenes in GFS showed significant changes in their expression levels; and compared with FP, 5,357 unigenes in GFP showed significant differences (Fig 4Bâ€“4E).
Fig 4. Differential expressed genes (DEGs) in G. jasmonoides fruits during ripening.
(A) Principal component analysis of the twelve samples based on the gene expression profiles; Volcano plots displaying the upregulated, downregulated and noregulated genes between FS vs FP (B), GFS vs GFP (C), GFS vs FS (D), GFP vs FP (E). GFP = the peel of green fruit; GFS = the sarcocarp of green fruit; FP = the peel of red fruit; FS = the sarcocarp of red fruit. The green dots in the figure represent downregulated DEGs, the red dots represent upregulated DEGs, and the blue dots represent genes detected but that are not significantly different.
KEGG enrichment of DEGs in different groups of samples
KEGG pathway enrichment analysis revealed that these DEGs were mainly enriched in several secondary metabolite metabolism and signal transduction. The results for the top 20 KEGG pathways enriched in each group are represented in scatter plots (Fig 5). According to the enriched pathway of which related to iridoid and crocin biosynthesis (S3 Table), FS vs FP (Fig 5A) mainly included ubiquinone and other terpenoid-quinone biosynthesis, Sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis. GFS vs GFP (Fig 5B) mainly contained phenylpropanoid biosynthesis, monoterpenoid biosynthesis. GFS vs FS (Fig 5C) mainly included phenylpropanoid biosynthesis, carotenoid biosynthesis, terpenoid backbone biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, sesquiterpenoid and triterpenoid biosynthesis. GFP vs FP (Fig 5D) mainly concentrated on phenylpropanoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, diterpenoid biosynthesis, phenylalanine, diterpenoid biosynthesis, carotenoid biosynthesis. This analysis preliminarily identified obvious differences in the above substances in different developmental stages and parts of fruits and provided basic data for our subsequent research.
Fig 5. Top 20 KEGG pathway enrichment of DEGs between (A) FS vs FP, (B) GFS vs GFP, (C) GFS vs FS, (D) GFP vs FP.
GFP = the peel of green fruit; GFS = the sarcocarp of green fruit; FP = the peel of red fruit; FS = the sarcocarp of red fruit. The dot color represents the qvalue, and the dot size represents the number of differential expressed genes. Red star denotes the metabolic pathway related to the synthesis of iridoids and crocins in Gardenia jasminoides Ellis.
Regulation of MVA/MEP/iridoid pathway genes during fruit development
Previous studies have shown that iridoid are derived from terpenoid, which are synthesized by the upstream MVA and MEP pathways; these pathways produce early precursors of iridoid . In this work, the accumulation rule of iridoid as active components in different maturation stages and different parts of gardenia fruit was studied. This study identified a large number and variety of genes in G. jasminoides that participate in the biosynthesis of iridoid. Combined with the results of previous studies, we reconstructed the iridoid biosynthesis pathway and obtained candidate unigenes screened from the DEGs. To obtain a systematic view of the iridoid biosynthesis pathway, we observed the abundance of 24 transcripts encoding 15 key enzyme genes involved in iridoid biosynthesis. The whole pathway could be divided into the MAP pathway, MEP pathway and iridoid pathway. As shown in Fig 6, some key enzymes were encoded by more than one unigene. In the MEP pathway, three unigenes were predicted to encode DXS (1-deoxy-D-xylulose-5-phosphate synthase), one unigene was predicted to encode DXR (2-C-methyl-D-erythritol-4-phosphate reductoisomerase), one unigene was predicted to encode HDR (4-hydroxy-3-methyl-2-(E)-butenlyl diphosphate reductase), one unigene was predicted to encode GPPS (geranyl diphosphate synthase) and one unigene was predicted to encode geranylgeranyl diphosphate synthase (GGPPS). We performed cluster analysis of all the differentially expressed genes involved in the MEP pathway and found that the red and green fruits were divided into two groups. According to the change trend of gene expression, 5 genes were divided into two categories: one group showed high expression levels and another group showed low expression levels (Fig 7D).
Fig 6. Transcriptional regulations during the G. jasmonoides fruit development in the biosynthesis of active components.
GFP = the peel of green fruit; GFS = the sarcocarp of green fruit; FP = the peel of red fruit; FS = the sarcocarp of red fruit. Gene expression is displayed as heat map depicting the FPKM values. Rectangles marked with yellow and blue background represent increased and reduced expression of genes, respectively. Color scale indication log2 (FPKM values). Multiple transcripts matching each gene are shown side by side near the gene names.
Fig 7. Heatmap and hierarchial clustering showing differentially expressed genes (DEGs) in four pathways.
(A) gene regulation during biosynthesis of iridoids; (B) The ion abundance of iridoids in G. jasmonoides; (C) The ion abundance of crocins in G. Jasmonoides; (D) DEGs involved in MEP pathway; (E) DEGs involved in MVA pathway; (F)DEGs involved in iridoid pathway; (G) DEGs involved in crocin pathway; GFP = the peel of green fruit; GFS = the sarcocarp of green fruit; FP = the peel of red fruit; FS = the sarcocarp of red fruit. Rectangles marked with red (yellow) and blue background represent increased and reduced expression of genes, respectively.
The key enzymes in the MVA pathway mainly include HMGS (3-hydroxy-3-methylglutaryl CoA synthase), HMGR (3-hydroxy-3-methyl glutaryl CoA reductase), MK (mevalonate kinase), FPPS (farnesyl diphosphate synthase) and SS (squalene synthase) (Fig 6). The same as MEP pathway, a part of genes showed high expression levels and the others showed low levels (Fig 7E). In addition to SS-1, HMGR-1 andHMGR-3, the expression levels of different predicted genes in the pathway were higher than others. The main enzymes involved in the iridoid pathway were GES (geraniol synthase), G10H (geraniol 10-hydroxylase), G8O (8-hydroxygeraniol dehydrogenase), 10-HGO (10-hydroxygeraniol oxidoreductase) and IS (iridoid synthase). Most genes involved in the iridoid pathway showed an expression pattern similar to those of the genes in the MEP pathway (Figs 6 and 7Dâ€“7F). The predicted DXS-1, HDR-1, DXR-1, G10H-1, 10-HGO-1, GES-1 and IS-1 genes were shown high expression levels in the sarcocarp of green fruit (GFP) compared with their expression in the other three sample types (Fig 6). These expression of predicted genes were consistent with the accumulation of geniposide and are worthy of attention (Fig 7B). Hereafter, we will focus on the genes that were highly expressed in the peel of green fruit (GFP). The opposite expression patterns between the members of certain families indicated the existence of multigene families that may differentially regulate iridoid biosynthesis in G. jasminoides. The FPKM values of the 24 selected genes are illustrated in S4 Table.
Key genes involved in crocin biosynthesis were shown significantly high expression level with the development of the fruits
The biosynthesis pathway of crocin in G. jasminoides is generally decomposed into the MEP pathway, carotenoid pathway and crocin pathway, and iridoid and crocin biosynthesis share the MEP pathway (Fig 6). Eighteen DEGs related to these transcriptomic results were screened as participants in the biosynthesis pathway of crocin (Fig 6). In the carotenoid synthesis pathways, one unigene was predicted to encode phytoene synthase (PSY), one unigene was predicted to encode phytoene desaturase (PDS), one unigene was predicted to encode zeta-carotene isomerase (Z-ISO), and one unigene was predicted to encode zeta-carotene desaturase (ZDS) (Fig 6). The expression of carotenoid isomerase (CRTISO) and lycopene cyclase (LCYB) did not differ significantly in any of the four groups of samples. In addition to UGTs-1, the predicted genes showed high expression level in the ripening of fruits. In particular, the expression levels of PSY-1, Z-ISO-1 and ZDS-1 in the peel of red fruits were significantly higher than those in the sarcocarp. The crocin pathway included four key enzymes: ALDH, CCD, NCED and UGTs (Fig 6). A total of 14 key DEGs were selected in this study, including 3 CCD, 2 NCED, 5 ALDH and 4UGT genes. In addition to NCED-1, the remaining genes showed an high expression trend in the transformation from green fruit to red fruit. In green fruit, the expression levels of ALDH-2, ALDH-3, ALDH-5, CCD-2 and CCD-3 in the peel were higher than those in the sarcocarp. Similarly, most genes in red fruit showed higher expression in the peel than in the sarcocarp (Fig 6). In particular, the FPKM value of CCD-2 in the red fruit peel was 146.83 times that in green fruit peel and 313.4 times that in green sarcocarp. The metabonomics results indicated that the total ion abundance of crocin-I, crocin-II, and crocin-III in red fruits was higher than that in green fruits, and the abundance in the peel was slightly higher than that in the sarcocarp. The expression trends of ALDH-1, ALDH-2, ALDH-3, ALDH-4, ALDH-5, UGTs-4, CCD-2 and CCD-3 were similar to the ion abundance trend of crocin, and it is speculated that they are closely related to the biosynthesis of crocin. Among the candidate genes of CCD, the homology of CCD-1 and CCD-2 with the functional gene CCD4a (KY631925.1) which reported by Xu et al. was 84.64% and 99.79%. Among the candidate genes of UGT, UGT-4 had 99.93% homology with UGT60 (KY631935.1) which reported by Xu et al. Among the candidate genes of ALDH, ALDH-1, ALDH-2, ALDH-3 have 100.0%, 90.16% and 82.41% homology with ALDH12 (KY631926.1) which reported by Xu et al., respectively. Especially, the expression trend of the above genes in red or green fruits was similar to Xu et al., which further verified the reliability of transcriptome results (Table 3). The FPKM values of the 18 selected genes are illustrated in S5 Table.
UGT plays an important role in the biosynthesis of the active component of G. jasmonoides
In G. jasminoides, UGTs are involved in a variety of biosynthesis of secondary metabolite pathways and are also the key downstream enzymes in crocin biosynthesis. In the inferred iridoid pathway (Fig 7A), 7-deoxyloganetic acid generates different iridoid derivatives under the action of various enzymes, and geniposide is finally generated via the action of P450, in which different UGTs participate in a multistep reaction. Fig 7A shows that UGT participates in geniposidic acid, shanzhiside, geniposide and genipin-1-gentiobioside synthesis. As observed at the transcription level, the expression level of the unigene encoding UGT in GFS was higher than other groups, and the expression level in FP was the lowest. The metabonomic results showed that besides geniposide, Shanzhiside methylester and Shanzhiside, other iridoids presented higher ion abundance in red fruits than in green fruits. Geniposide and Shanzhiside methylester showed higher ion abundance in GFS than other groups, and showed that Shanzhiside in the peel was higher than that in the sarcocarp (Fig 7C, S6 Table). Comprehensive analysis showed that the expression patterns of UGTS-I-1, UGTS-I-2 and UGTS-I-3 were positively correlated with the ion abundance of geniposide in sarcocarp (Fig 7B). These results suggested that the three genes were responsible for the biosynthesis of geniposide, and these genes were selected for further analysis. In the crocin pathway, with the exception of UGT-3, the remaining UGTs were positively correlated with the total ion abundance of the three types of crocin, which was higher in the red fruit than in the green fruit, and that in the sarcocarp was higher than that in the peel (Fig 7C and 7G, S6 Table). We hypothesize that these genes are involved in and regulate the biosynthesis of crocin. In summary, the enzyme activity of UGTs directly affects the biosynthesis of iridoids and crocin, and we will conduct further studies on these genes.
Expression analysis of DEGs by qRT-PCR
To validate the subsequent splicing and assembly and the FPKM expression results, we selected the 51 DEGs for verification of their expression levels by qRT-PCR. Primers were designed in the homologous regions of the unigenes that were transcribed from the same gene, and those with high similarity annotations were retained. 9/51(18%) of candidate gene were found to be inconsistent with respect to expression between the qRT-PCR and RNA-Seq data. Besides deleted genes, t the relative expression levels of the 42 genes in four groups of samples were shown correlations with the Illumina analysis (S1 Fig). The results were within the normal range and the transcriptome results were accurate and efficient.
Conjoint analysis of DEGs and DAMs associated with other metabolic pathways in four samples
According to the combined analysis of DAMs and DEGs in this study, the different genes and different metabolites in the same group were simultaneously mapped to the KEGG pathway map to better understand the relationships between the genes and metabolites. Table 4 shows that the six groups were also associated with seven metabolic pathways, which included phenylpropanoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, ubiquinone and other terpenoidquinone biosynthesis, tyrosine metabolism, folate biosynthesis and isoquinoline alkaloid biosynthesis. In each group, the numbers of DEGs and DAMs that were enriched in the phenylalanine biosynthesis pathway were greatest. The major DAMs were concentrated in the p-coumaryl alcohol, coniferyl alcohol, scopoletin, 4-hydroxybenzoic acid, syringin and syringetin pathways. These results showed that in addition to the iridoids and crocins present in G. jasminoides fruit, other DAMs also showed significant differences in different fruit parts and samples from different maturation stages, which also provided basic data for our study on the biosynthesis of other metabolites in G. jasminoides. Additionally, upstream of the MEP and MVA pathways, the significant DAMs and DEGs identified from the phenylalanine biosynthesis pathway also indicate new research directions.
Localization of active components in different parts of Gardenia jasminoides fruit
G. jasminoides fruits are widely used in Asian countries to obtain natural colorants and in traditional Chinese herbal medicine since they exhibit homeostatic, hepatoprotective, analgesic, antiphlogistic, antipyretic, and hypolipidemic effects . Studies have shown that iridoids and crocins are the main active substances in G. jasminoides fruit and show high medicinal value . As iridoid glycosides such as geniposide and gardenoside are the main components of G. jasminoides fruit, their pharmacological activity and biosynthesis have been reported . Studies on the biosynthesis pathways of iridoids from G. jasminoides have been conducted by Ye et al. . The genes involved in iridoid biosynthesis were expressed mainly in flowers and fruits, whereas iridoids, such as gardenoside and geniposide, accumulated mainly in fruits. In this research, we selected the main iridoid glycoside organ of accumulation, the fruit, and divided it into pericarp and pulp sample to conduct transcriptome and metabolomics studies. In the present work, using the widely targeted metabolomics approach, the obtained ion abundance results showed that geniposide and shanzhiside methyl ester accumulated mainly in the sarcocarp of green fruit, gardenoside accumulated mainly in the sarcocarp of red fruit, and the other detected iridoid glycosides accumulated mainly in the peel of red fruit, indicating that the distribution of different iridoids differed among the organs. Additionally, crocins has been found in the stigmas of Crocus sativus L. and the fruit of G. jasminoides [21, 46, 47]. Because of the complex harvesting process and low yield of crocins, researchers have focused on G. jasminoides crocins. A transcriptome analysis of the leaves, green fruits, and red fruits of G. jasminoides was recently conducted to identify and predict the genes that encode key enzymes responsible for crocins production via comparison with Crocus sativus L. . Our results showed that crocins mainly accumulated in the peel and sarcocarp of red fruits and that the ion abundance in green fruits was significantly lower than that in red fruits. Understanding the distribution pattern of active ingredients in different parts of Gardenia fruits will provide a theoretical basis for our follow up screening of fine germplasms and for understanding the molecular mechanisms of active ingredient synthesis.
Characterization of the iridoids biosynthetic pathway
The developing technologies for largescale metabolite identification and transcriptome sequencing have made metabolomics a powerful tool for investigating the biological processes and active ingredients involved in plant development [48, 49]. Because of the pharmacological and economic importance of iridoids, there has been intense interest in understanding their biosynthetic mechanisms to develop biotechnological approaches for their targeted production . In a previous transcriptomic and metabonomic study, researchers identified several unigenes bearing high sequence similarity to candidate genes for crocin and iridoid biosynthesis [10, 12, 20, 44]. The upstream MVA and MEP pathways produce early precursors of iridoid. In our study, the expression of DXS and DXR was found to vary, which indicated that there were multiple copies of the genes that regulate these enzymes, and their upstream regulation did not shown consistent changes. Therefore, future work is needed to isolate and clone the genes regulating these enzymes and to explore the function of the genes. In the iridoid pathway, GPPS, GGPPS and GES catalyze the synthesis of geraniol from geranyl pyrophosphate; geraniol is then transformed into 10-oxogeranial catalyzed by GES, G10H and 10-HGO, and the transformation of iridotrial into downstream substances carried out. In our study, the genes encoding the enzymes of the G8O pathway downstream showed high expression level in green fruits, and their expression was higher in the sarcocarp than in the peel. According to our metabolomics results, we hypothesized that a transport mechanism existed in the peel and sarcocarp of G. jasminoides through which precursor substances generated in the sarcocarp were transported to the peel to initiate subsequent synthesis.
A negative correlation between the biosynthesis of geniposide and crocin
Combined with the results of recent studies, we successfully constructed a metabolic map of the main active components of G. jasminoides and divided it into 5 pathways. Iridoids are derived from terpenoids, which are synthesized via the upstream MVA and MEP pathways and by the related enzymes in the iridoid pathway. The other portions of this map corresponded to crocin biosynthesis, including the upstream MEP pathway, the midstream carotenoid pathway, and the downstream crocin pathway. In the MVA and MEP pathways, HMGS-1, FPPS-1, FPPS-2, DXS-3, and DXR-1 were shown lower expression level with the development of fruit, while other candidate unigenes were increased. In the iridoid pathway, in addition to G10H-1 and G10H-2, other unigenes were shown high expression level when the fruit became red. Additionally, G80H-1, G80H-2, were shown high expression level in the sarcocarp of the green fruit, which showed the same trend as the ion abundance of geniposide. As the fruit develops, the genes related to the synthesis of crocins were shown higher expression level. Additionally, the change trend of key enzyme genes involved in the crocin pathway was positively correlated with the change in the ion abundance of crocins. In particular, the expression levels and ion abundance in the peel were both high. In summary, candidate genes showed different expression trends during fruit development. When the fruits were young (green), iridoid biosynthesis dominated the metabolic pathways. High levels of iridoids, especially geniposide, are synthesized in the fruit. When the fruit turns red, key enzyme encoding genes related to the biosynthesis of crocin were shown higher expression level, leading to a significant increase in the ion abundance of crocins in the sarcocarp. It was demonstrated that different active ingredients and genes exhibit fruit tissue specific accumulation and expression in fruit. The redirection of metabolic flux and regulation of key enzymes may be the main reasons for the changes in the biosynthesis of iridoids and crocins in G. jasminoides fruit.