Multi-omics analysis reveals the molecular basis of flavonoid accumulation in fructus of Gardenia (Gardenia jasminoides Ellis).

BACKGROUND: The fruits of Gardenia are rich in flavonoids and geniposides, which have various pharmacological effects such as antioxidant, anti-inflammatory and anticancer. In this study, we analyzed the transcriptome and metabolome of gardenia peel and kernel at different growth stages, revealed the regulatory network related to flavonoid synthesis, and identified the key regulatory genes. RESULTS: The results showed that in terms of flavonoid metabolic pathways, gardenia fruits mainly synthesized cinnamic acid through the phenylpropanoid pathway, and then synthesized flavonoids through the action of catalytic enzymes such as 4-coumaroyl-CoA ligase, chalcone synthase, chalcone isomerase and flavanol synthase, respectively. In addition, we found that the metabolomics data showed a certain spatial and temporal pattern in the expression of genes related to the flavonoid metabolism pathway and the relative content of metabolites, which was related to the development and ripening process of the fruit. CONCLUSIONS: In summary, this study successfully screened out the key genes related to the biosynthesis metabolism of flavonoids in gardenia through the joint analysis of transcriptome and metabolome. This is of certain significance to the in-depth study of the formation mechanism of gardenia efficacy components and the improvement of quality.

Whole-genome resequencing analysis of the medicinal plant Gardenia jasminoides.

Background: Gardenia jasminoides is a species of Chinese medicinal plant, which has high medicinal and economic value and rich genetic diversity, but the study on its genetic diversity is far not enough. Methods: In this study, one wild and one cultivated gardenia materials were resequenced using IlluminaHiSeq sequencing platform and the data were evaluated to understand the genomic characteristics of G. jasminoides. Results: After data analysis, the results showed that clean data of 11.77G, Q30 reached 90.96%. The average comparison rate between the sample and reference genome was 96.08%, the average coverage depth was 15X, and the genome coverage was 85.93%. The SNPs of FD and YP1 were identified, and 3,087,176 and 3,241,416 SNPs were developed, respectively. In addition, SNP non-synonymous mutation, InDel mutation, SV mutation and CNV mutation were also detected between the sample and the reference genome, and KEGG, GO and COG database annotations were made for genes with DNA level variation. The structural gene variation in the biosynthetic pathway of crocin and gardenia, the main medicinal substance of G. jasminoides was further explored, which provided basic data for molecular breeding and genetic diversity of G. jasminoides in the future.

Fructus gardeniae ameliorates anxiety-like behaviors induced by sleep deprivation via regulating hippocampal metabolomics and gut microbiota.

Fructus gardeniae (FG) is a traditional Chinese medicine and health food for thousands of years of application throughout Chinese history and is still widely used in clinical Chinese medicine. FG has a beneficial impact on anxiety, depression, insomnia, and psychiatric disorders; however, its mechanism of action requires further investigation. This study aimed to investigate the effects and mechanisms of FG on sleep deprivation (SD)-induced anxiety-like behavior in rats. A model of SD-induced anxiety-like behavior in rats was established by intraperitoneal injection of p-chlorophenylalanine (PCPA). This was accompanied by neuroinflammation and metabolic abnormalities in the hippocampus and disturbance of intestinal microbiota. However reduced SD-induced anxiety-like behavior and decreased levels of pro-inflammatory cytokines including TNF-α and IL-1ß were observed in the hippocampus of rats after 7 days of FG intervention. In addition, metabolomic analysis demonstrated that FG was able to modulate levels of phosphatidylserine 18, Phosphatidylinositol 18, sn-glycero-3-phosphocholine, deoxyguanylic acid, xylose, betaine and other metabolites in the hippocampus. The main metabolic pathways of hippocampal metabolites after FG intervention involve carbon metabolism, glycolysis/gluconeogenesis, pentose phosphate, and glycerophospholipid metabolism. 16S rRNA sequencing illustrated that FG ameliorated the dysbiosis of gut microbiota in anxious rats, mainly increased the abundance of Muribaculaceae and Lactobacillus, and decreased the abundance of Lachnospiraceae_NK4A136_group. In addition, the correlation analysis demonstrated that there was a close relationship between hippocampal metabolites and intestinal microbiota. In conclusion, FG improved the anxiety behavior and inhibited of neuroinflammation in sleep-deprived rats, and the mechanism may be related to the FG regulation of hippocampal metabolites and intestinal microflora composition.

Isolation of the Thermostable ß-Glucosidase-Secreting Strain Bacillus altitudinis JYY-02 and Its Application in the Production of Gardenia Blue.

Gardenia blue (GB) is a natural blue pigment widely used in textiles and the pharmaceutical industry. The geniposide in gardenia fruits can be hydrolyzed by ß-glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable ß-glucosidase (EC 3.2.1.21) was isolated from soil and identified as Bacillus altitudinis JYY-02. This strain could potentially be used for GB production from geniposide by fermentation. Optimal fermentation results were achieved at pH 6.5 or 8.0 at 45°C for 45 h with additional sucrose. To obtain a large amount of ß-glucosidase, the whole genome of B. altitudinis JYY-02 was sequenced and annotated; it is 3,727,518 bp long and contains 3,832 genes. The gene encoding ß-glucosidase (bgl) in B. altitudinis JYY-02 was screened from the genome and overexpressed in Escherichia coli BL21(DE3). The recombinant ß-glucosidase was purified by affinity chromatography on a Ni Sepharose 6 fast flow (FF) column. The optimal temperature, pH, and Km values for the recombinant ß-glucosidase were 60°C, pH 5.6, and 0.331 mM, respectively, when p-nitrophenyl-ß-d-glucopyranoside (pNPG) was used as the substrate. The recombinant ß-glucosidase catalyzed the deglycosylation reaction of geniposide, which was then used to produce GB. IMPORTANCE ß-Glucosidases are enzymes capable of hydrolyzing ß-glucosidic linkages present in saccharides and glycosides and have many agricultural and industrial applications. Although they are found in all domains of living organisms, commercial ß-glucosidases are still expensive, limiting their application in industry. In the present study, a thermostable ß-glucosidase-producing strain was obtained for GB production by fermentation, engineered bacteria were constructed for preparing recombinant ß-glucosidase, and a one-step method to purify the recombinant enzyme was established. A large amount of purified ß-glucosidase was easily obtained from the engineered bacteria for industrial applications such as GB production.

Integrated SMRT and Illumina Sequencing Provide New Insights into Crocin Biosynthesis of Gardenia jasminoides.

Crocins are valuable bioactive components of gardenia fruit, and their biosynthesis and accumulation have attracted widespread interest. Studies have investigated the biosynthesis and accumulation of crocin based on Illumina sequencing, but there is a lack of reports based on full-length transcriptome sequencing. Utilising SMRT sequencing and high-performance liquid chromatography (HPLC), we explored crocin biosynthesis and accumulation in the fruit of Gardenia jasminoides. HPLC analysis showed that crocins specifically exist in fruit and that the content of crocins increases gradually during fruit development. SMRT sequencing generated 46,715 high-quality full-length isoforms, including 5230 novel isoforms that are not present in the G. jasminoides genome. Furthermore, a total of 46 genes and 91 lncRNAs were involved in the biosynthesis and accumulation of crocin. The qRT-PCR indicated that genes involved in crocin biosynthesis reached a peak in the NOV stage. These findings contributed to our understanding of crocin biosynthesis and accumulation.

Metabolomics integrated with transcriptomics reveals the distribution of iridoid and crocin metabolic flux in Gardenia jasminoides Ellis.

Gardenia jasminoides Ellis (G. jasminoides) fruits are used as a resource for obtaining natural colorants and in traditional Chinese herbal medicine. However, G. jasminoides presents a relatively long flowering period and different ripening periods, so there are significant differences in the accumulation of metabolites in fruits of different colors. In addition, the complete metabolic pathways of iridoidsand crocins, which are used as medicinal composition of G. jasminoides, are poorly understood at present. In this research, we comprehensively compared the transcriptome and metabolites profiles of the developmental stages and locations of iridoid and crocin biosynthesis. A large number of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were detected in four groups of samples, and clear variation in the pattern of metabolite abundance and gene expression were observed among different fruit colors and parts. Geniposide and gardenoside mainly accumulated in the sarcocarp of green fruit (GFS) and the sarcocarp of red fruit (FS), respectively. Crocin mainly accumulated in the peel and sarcocarp of red fruits. In the iridoid pathway, we hypothesized that there was a transport mechanism from the sarcocarp to the peel of G. jasminoides because of the inconsistent expression of G8O, 10-HGO and IS associated with differences in fruit ripening. UGTs play an important role in the biosynthesis of the active components of G. jasminoides. Combined transcriptome and metabonomics analysis showed a negative correlation between the biosynthesis of geniposide and crocin. The redirection of the metabolic flux and the regulation of key enzymes may be the main reasons for the changes in the biosynthesis of iridoid and crocin in G. jasminoides fruit. Our study expended valuable information for functional genomic library and provided new insights for metabolic engineering of secondary metabolite in G. Jasminoides.

Tandem gene duplications drive divergent evolution of caffeine and crocin biosynthetic pathways in plants.

BACKGROUND: Plants have evolved a panoply of specialized metabolites that increase their environmental fitness. Two examples are caffeine, a purine psychotropic alkaloid, and crocins, a group of glycosylated apocarotenoid pigments. Both classes of compounds are found in a handful of distantly related plant genera (Coffea, Camellia, Paullinia, and Ilex for caffeine; Crocus, Buddleja, and Gardenia for crocins) wherein they presumably evolved through convergent evolution. The closely related Coffea and Gardenia genera belong to the Rubiaceae family and synthesize, respectively, caffeine and crocins in their fruits. RESULTS: Here, we report a chromosomal-level genome assembly of Gardenia jasminoides, a crocin-producing species, obtained using Oxford Nanopore sequencing and Hi-C technology. Through genomic and functional assays, we completely deciphered for the first time in any plant the dedicated pathway of crocin biosynthesis. Through comparative analyses with Coffea canephora and other eudicot genomes, we show that Coffea caffeine synthases and the first dedicated gene in the Gardenia crocin pathway, GjCCD4a, evolved through recent tandem gene duplications in the two different genera, respectively. In contrast, genes encoding later steps of the Gardenia crocin pathway, ALDH and UGT, evolved through more ancient gene duplications and were presumably recruited into the crocin biosynthetic pathway only after the evolution of the GjCCD4a gene. CONCLUSIONS: This study shows duplication-based divergent evolution within the coffee family (Rubiaceae) of two characteristic secondary metabolic pathways, caffeine and crocin biosynthesis, from a common ancestor that possessed neither complete pathway. These findings provide significant insights on the role of tandem duplications in the evolution of plant specialized metabolism.

Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Crocin Biosynthesis in Gardenia jasminoides Ellis (Rubiaceae).

Crocins, enriched in Gardenia jasminoides fruits, have a pharmacological activity against central nervous system diseases, cardiovascular diseases, and cancer cell growth. The biosynthesis of crocins has been widely explored, but its regulatory mechanism remains unknown. Here, the basic helix-loop-helix (bHLH) transcription factors related to crocin biosynthesis were systematically identified on the basis of the genome of G. jasminoides. A total of 95 GjbHLH transcription factor genes were identified, and their phylogenetic analysis indicated that they could be classified into 23 subfamilies. The combination of gene-specific bHLH expression patterns, the coexpression analysis of biosynthesis genes, and the analysis of promoter sequences in crocin biosynthesis pathways suggested that nine bHLHs in G. jasminoides might negatively regulate crocin biosynthesis. This study laid a foundation for understanding the regulatory mechanism of crocin biosynthesis and the improvement and breeding of G. jasminoides varieties.

In Vivo Production of Five Crocins in the Engineered Escherichia coli.

Crocins are highly valuable medicinal compounds for treating human disorders, and they also serve as spices and coloring agents. However, the supply of crocins from plant extractions is insufficient for current demands, and using synthetic biology to produce crocins remains a big challenge. Here, we report the in vivo production of five types of crocins in E. coli with GjUGT94E13 and GjUGT74F8, which are responsible for the glycosylation of crocetin, from the crocin-producing plant Gardenia jasminoides. Subsequently, native UDP-glucose biosynthesis in E. coli is strengthened by the overexpression of pgm and galU. The optimization of catalytic reactions has demonstrated that 50 mM NaH2PO4-Na2HPO4 buffer (pH 8.0) plus 5% glucose is the best medium to use for the efficient glycosylation of crocetin. In engineered E. coli, the conversion rate of crocin III and crocin V from crocetin (50 mg/L) by the catalysis of GjUGT74F8 was increased to 66.1%, and the conversion rate of five types of crocins from crocetin (50 mg/L) via GjUGT94E13 and GjUGT74F8 was 59.6%, much higher than the catalytic activity of the reported microbial UGTs. This study not only sheds light on the in vivo biosynthesis of crocins in E. coli, but also provides important genetic tools for the de novo synthesis of crocins.

Construction of Escherichia coli cell factories for crocin biosynthesis.

BACKGROUND: Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, ß-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. RESULTS: In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS. CONCLUSIONS: A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Genetic Authentication of Gardenia jasminoides Ellis var. grandiflora Nakai by Improved RAPD-Derived DNA Markers.

The evergreen shrub, Gardenia jasminoides Ellis var. grandiflora Nakai is one of the most popular garden-plants, with significant ornamental importance. Here, we have cloned improved random amplified polymorphic DNA (RAPD) derived fragments into T-vector, and developed sequence-characterized amplified region (SCAR) markers. These markers have been deposited in GenBank database with the accession numbers KP641310, KP641311, KP641312 and KP641313 respectively. The BLAST search of database confirmed the novelty of these markers. The four SCAR markers, namely ZZH11, ZZH31, ZZH41 and ZZH51 can specifically recognize the genetic materials of G. jasminoides from other plant species. Moreover, SCAR marker ZZH31 can be used to distinguish G. jasminoides Ellis var. grandiflora Nakai from other G. jasminoides on the market. Together, this study has developed four stably molecular SCAR markers by improved RAPD-derived DNA markers for the genetic identification and authentication, and for ecological conservation of medicinal and ornamental plant G. jasminoides.

Genetic characterization and authentication of Gardenia jasminoides in different regions of China by using improved RAPD analysis.

Gardenia jasminoides is a common garden medicinal plant known for its anticancer, anti-inflammatory, anti-thrombic, anti-fibrotic, antiviral, hepatoprotective, lung-protective, renal-protective, retina-protective and neuroprotective activities. It is found in several regions of the world, including China, but information about its genetic characteristics is limited. Here, we employed an improved method of random amplified polymorphic DNA (RAPD) analysis (with increased RAMP time) to investigate the genetic link between G. jasminoides samples collected from six different regions of Southern China. Total 26 RAPD primers were selected randomly, among which 23 primers generated reproducible polymorphic amplification bands. A total of 174 bands were obtained, where each primer had amplified 5-13 bands with an average of 7.56 bands per primer. The band size ranged approximately 150-2200 bp. Cluster dendrogram was obtained based on the improved RAPD amplification profiles, which showed that the similarity coefficients among six varieties of G. jasminoides ranged 0.67-0.88. To our knowledge, this is the first report of genetic characterization of G. jasminoides using improved RAPD analysis, which may be useful for the preservation of genetic diversity and identification of Gardenia population.

De novo transcriptome analysis of petal senescence in Gardenia jasminoides Ellis.

BACKGROUND: The petal senescence of ethylene insensitive species has not been investigated thoroughly while little is known about the temporal and tissue specific expression patterns of transcription factors (TFs) in this developmental process. Even less is known on flower senescence of the ornamental pot plant Gardenia jasminoides, a non climacteric flower with significant commercial value. RESULTS: We initiated a de novo transcriptome study to investigate the petal senescence in four developmental stages of cut gardenia flowers considering that the visible symptoms of senescence appear within 4 days of flower opening. De novo assembly of transcriptome sequencing resulted in 102,263 contigs with mean length of 360 nucleotides that generated 57,503 unigenes. These were further clustered into 20,970 clusters and 36,533 singletons. The comparison of the consecutive developmental stages resulted in 180 common, differentially expressed unigenes. A large number of Simple Sequence Repeats were also identified comprising a large number of dinucleotides and trinucleotides. The prevailing families of differentially expressed TFs comprise the AP2/EREBP, WRKY and the bHLH. There are 81 differentially expressed TFs when the symptoms of flower senescence become visible with the most prevailing being the WRKY family with 19 unigenes. No other WRKY TFs had been identified up to now in petal senescence of ethylene insensitive species. A large number of differentially expressed genes were identified at the initiation of visible symptoms of senescence compared to the open flower stage indicating a significant shift in the expression profiles which might be coordinated by up-regulated and/or down-regulated TFs. The expression of 16 genes that belong to the TF families of WRKY, bHLH and the ethylene sensing pathway was validated using qRT--PCR. CONCLUSION: This de novo transcriptome analysis resulted in the identification of TFs with specific temporal expression patterns such as two WRKYs and one bHLH, which might play the role of senescence progression regulators. Further research is required to investigate their role in gardenia flowers in order to develop tools to delay petal senescence.

Iridoid-specific glucosyltransferase from Gardenia jasminoides.

Iridoids are one of the most widely distributed secondary metabolites in higher plants. They are pharmacologically active principles in various medicinal plants and key intermediates in the biosynthesis of monoterpenoid indole alkaloids as well as quinoline alkaloids. Although most iridoids are present as 1-O-glucosides, the glucosylation step in the biosynthetic pathway has remained obscure. We isolated a cDNA coding for UDP-glucose:iridoid glucosyltransferase (UGT85A24) from Gardenia jasminoides. UGT85A24 preferentially glucosylated the 1-O-hydroxyl group of 7-deoxyloganetin and genipin but exhibited only weak activity toward loganetin and no activity toward 7-deoxyloganetic acid. This suggests that, in the biosynthetic pathway of geniposide, a major iridoid compound in G. jasminoides, glucosylation occurs after methylation of 7-deoxyloganetic acid. UGT85A24 showed negligible activity toward any acceptor substrates other than iridoid aglycones. Thus, UGT85A24 has a remarkable specificity for iridoid aglycones. The mRNA level of UGT85A24 overlaps with the marked increase in genipin glucosylation activity in the methyl jasmonate-treated cell cultures of G. jasminoides and is related to iridoid accumulation in G. jasminoides fruits.

Crocin and geniposide profiles and radical scavenging activity of gardenia fruits (Gardenia jasminoides Ellis) from different cultivars and at the various stages of maturation.

The major components of gardenia fruits are geniposide and water soluble pigment crocins. In this study, we investigate crocins and geniposide profiles of gardenia fruits from different cultivars and at the various stages of maturation. DPPH scavenging activity of gardenia fruits from different cultivars and at the various stages of fruit maturation was also assayed. Quantitative determination of crocins in the gardenia at the various stages of maturation revealed a significant increase when ripening. However, geniposide content was negatively correlated with ripening stages. A significant difference was observed when comparing crocin content of different gardenia from various cultivars and geniposide content also showed marked variety. Current study indicated no relationship between crocin and geniposide content in gardenia fruits at the various stages of maturation and DPPH radical scavenging activity. Data showed that, although crocins feature markedly less DPPH scavenging activity than gardenia ethanol extract, total crocin content of gardenias collected in various cultivars correlate, to a certain degree, with radical scavenging effects of the Chinese traditional medicine (r=0.75).

[Molecular ecology of Gardenia jasminoides authenticity].

By using amplified fragment length polymorphism (AFLP) markers, this paper studied the genetic relationships among five wild or cultivated Gardenia jasminoides Ellis populations in Jiangxi Province. Chemical fingerprint was also built with HPLC method. The results showed that there was a great genetic difference among these samples. The UPGMA obtained with NTSYS-PC 2. 10e software suggested that there were seven branches of population, and the population from near geographical location clustered firstly. The geniposide content of these branches was not correlated with UPGMA. It could be concluded that the authenticity was resulted from the co-action of genotype and environmental change. The microelements content in G. jasminoides fruit measured by inductively coupled plasma showed that there was a negative correlation between Zn and geniposide contents.