Integration of Metabolome and Transcriptome Reveals the Relationship of Benzenoid–Phenylpropanoid Pigment and Aroma in Purple Tea Flowers

Tea (Camellia sinensis) flowers are normally white, even though the leaves could be purple. We previously discovered a specific variety with purple leaves and flowers. In the face of such a phenomenon, researchers usually focus on the mechanism of color formation but ignore the change of aroma. The purple tea flowers contain more anthocyanins, which belong to flavonoids. Meanwhile, phenylalanine (Phe), derived from the shikimate pathway, is a precursor for both flavonoids and volatile benzenoid–phenylpropanoids (BPs). Thus, it is not clear whether the BP aroma was attenuated for the appearance of purple color. In this study, we integrated metabolome and transcriptome of petals of two tea varieties, namely, Zijuan (ZJ) with white flowers and Baitang (BT) with purple flowers, to reveal the relationship between color (anthocyanins) and aroma (volatile BPs). The results indicated that in purple petals, the upstream shikimate pathway promoted for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) was elevated. Among the increased anthocyanins, delphinidin-3-O-glucoside (DpG) was extremely higher; volatile BPs, including benzyl aldehyde, benzyl alcohol, acetophenone (AP), 1-phenylethanol, and 2-phenylethanol, were also enhanced, and AP was largely elevated. The structural genes related to the biosynthesis of volatile BPs were induced, while the whole flavonoid biosynthesis pathway was downregulated, except for the genes flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H), which were highly expressed to shift the carbon flux to delphinidin, which was then conjugated to glucoside by increased bronze-1 (BZ1) (UDP-glucose: flavonoid 3-O-glucosyltransferase) to form DpG. Transcription factors (TFs) highly related to AP and DpG were selected to investigate their correlation with the differentially expressed structural genes. TFs, such as MYB, AP2/ERF, bZIP, TCP, and GATA, were dramatically expressed and focused on the regulation of genes in the upstream synthesis of Phe (DAHPS; arogenate dehydratase/prephenatedehydratase) and the synthesis of AP (phenylacetaldehyde reductase; short-chain dehydrogenase/reductase), Dp (F3′H; F3′5′H), and DpG (BZ1), but inhibited the formation of flavones (flavonol synthase) and catechins (leucoanthocyanidin reductase). These results discovered an unexpected promotion of volatile BPs in purple tea flowers and extended our understanding of the relationship between the BP-type color and aroma in the tea plant.

Accumulation Pattern of Flavonoids during Fruit Development of Lonicera maackii Determined by Metabolomics

Lonicera maackii (Caprifoliaceae) is a large, upright shrub with fruits that contain many bioactive compounds. Flavonoids are common active substances in L. maackii. However, there is a dearth of information about the accumulation of these flavonoids and their possible medicinal value. We used targeted metabolomics analysis based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to analyze five developmental stages of L. maackii fruit. A total of 438 metabolites were identified in the five developmental stages, including 81 flavonoids and derivatives. The 81 flavonoids included 25 flavones and derivatives, 35 flavonols and derivatives, two isoflavones, three cyanidins and derivatives, eight procyanidins, and eight flavanones. In addition, we outlined the putative flavonoid biosynthesis pathway and screened their upstream metabolites. More importantly, we analyzed the accumulation patterns of several typical flavones and flavonols. The results reported here improved our understanding of the dynamic changes in flavonoids during fruit development and contributed to making full use of the medicinal value of L. maackii fruit.

Composition of Flavonoids in the Petals of Freesia and Prediction of Four Novel Transcription Factors Involving in Freesia Flavonoid Pathway

Freesia hybrida is rich in flower colors with beautiful flower shapes and pleasant aroma. Flavonoids are vital to the color formation of its flowers. In this study, five Freesia cultivars with different flower colors were used to study on the level of accumulation of their flavonoids and expression of flavonoid-related genes and further explore new novel transcription factor (TF). Ultra-high-performance liquid chromatography and VION ion mobility quadrupole time-of-flight mass spectrometer (UPLC-Q-TOF-MS) were used to determine the flavonoids. Combined with transcriptome sequencing technology, the molecular mechanism of the flavonoid metabolism difference in Freesia was revealed. A total of 10 anthoxanthin components and 12 anthocyanin components were detected using UPLC-Q-TOF-MS. All six common anthocyanin aglycones in high plants, including cyanidin, delphinidin, petunidin, peonidin, malvidin, and pelargonidin, were detected in Freesia at first time in this study. In orange, yellow, and white cultivars, anthoxanthins gradually decreased with the opening of the petals, while in red and purple cultivars, anthoxanthins first increased and then decreased. No anthocyanin was detected in yellow and white cultivars, while anthocyanins increased with the opening of the petals and reached their maximum at the flowering stage (S3) in other three cultivars. The correlation analysis revealed that the color of Freesia petals was closely related to the composition and content of anthoxanthins and anthocyanins. Petals of five cultivars at S3 were then selected for transcriptome sequencing by using the Illumina Hiseq 4000 platform, and a total of 100,539 unigenes were obtained. There were totally 5,162 differentially expressed genes (DEGs) when the four colored cultivars were compared with the white cultivar at S3. Comparing all DEGs with gene ontology (GO), KEGG, and Pfam databases, it was found that the genes involved in the flavonoid biosynthesis pathway were significantly different. In addition, AP2, WRKY, and bHLH TF families ranked the top three among all differently expressed TFs in all DEGs. Quantitative real-time PCR (qRT-PCR) technology was used to analyze the expression patterns of the structural genes of flavonoid biosynthesis pathway in Freesia. The results showed that metabolic process was affected significantly by structural genes in this pathway, such as CHS1, CHI2, DFR1, ANS1, 3GT1, and FLS1. Cluster analysis was performed by using all annotated WRKY and AP2 TFs and the above structural genes based on their relatively expression. Four novel candidate TFs of WRKY and AP2 family were screened. Their spatiotemporal expression patterns revealed that these four novel TFs may participate in the regulation of the flavonoid biosynthesis, thus controlling its color formation in Freesia petals.

Transcriptomic and Metabolomic Differences Between Two Saposhnikovia divaricata (Turcz.) Schischk Phenotypes With Single- and Double-Headed Roots

Saposhnikovia divaricata is derived from the dried roots of Saposhnikovia divaricata (Turcz.) Schischk and used as a Chinese herbal medicine for treating respiratory, immune, and nervous system diseases. The continuously increasing market demand for traditional Chinese medicine requires the commercial cultivation of Saposhnikovia divaricata using standardized methods and high yielding genotypes, such as double-headed root plants, for achieving consistent quality and a reliable supply. In this study, we aimed to identify the quantitative differences in chromone, a precursor of flavonoid biosynthesis, between plants with single- and double-headed roots using high-performance liquid chromatography and further explore the two phenotypes at the transcriptomic and metabolomic levels. Our results showed that the chromone content was significantly higher in plants with double-headed roots than in those with single-headed roots. Transcriptomic analysis revealed six significantly differentially expressed genes between the two phenotypes, including five key genes in the flavonoid biosynthesis pathway (4-coumarate-CoA ligase, chalcone synthase 1, vinorine synthase, chalcone-flavonone isomerase 1, and flavanone 3 beta-hydroxylase) and one key gene in the abscisic acid biosynthetic pathway (zeaxanthin epoxidase). Moreover, metabolomic analysis showed that the 126 differentially expressed metabolites were mainly enriched in the biosynthesis of secondary metabolites and phytohormones. Overall, our results suggest that plants with double-headed roots have higher medicinal value than those with single-headed roots, probably due to differences in various biosynthetic pathways. These data might help select the genotypes with superior yield and therapeutic properties.

Toward understanding of the methoxylated flavonoid biosynthesis pathway in Dracocephalum kotschyi Boiss

Nowadays, with the development and advancement of next-generation sequencing technologies, a new path has been provided for transcriptomic studies. In this study, the transcriptome of Dracocephalum kotschyi Boiss., as an endemic and endangered plant which is contained a large amount of valuable secondary metabolites with antioxidant and anticancer properties, was sequenced. Then functional annotation and gene ontology analysis for 165,597 assembled transcripts were performed, most were associated with the metabolic pathways. This might be because there are various active biochemical pathways in this plant. Furthermore, after comprehensive transcript annotation, the putative genes involved in the main metabolic pathways of D. kotschyi were identified. Then, the biosynthetic pathway of its valuable methoxylated flavones was proposed. Finally, the accumulations of important methoxylated-flavone metabolites in three different tissues were quantified by HPLC. The relative expression of the genes involved in the proposed pathway was investigated by qRT-PCR, which indicated high expression levels in the bud tissue. The present results may lead to the design strategies to preserve the genetic diversity of endangered D. kotschyi plants and apply the new methods for engineering its valuable methoxylated-flavones pathway.

Integrated Transcriptomic and Metabolomic Analyses of the Molecular Mechanisms of Two Highland Barley Genotypes with Differential Pyroxsulam Responses

Background: Highland barley is one of the few crops that can be grown at high elevations, making it a key resource within the Tibet Plateau. Weeds are a significant threat to highland barley production and new herbicides and tolerant barley varieties are needed to control this ever-growing problem. Results: A better understanding of existing herbicide resistance mechanisms is therefore needed. In this study, transcriptomic and metabolomic analyses were used to identify molecular and physiological changes in two highland barley genotypes with differing sensitivities to pyroxsulam. We identified several stress-responsive metabolites, including flavonoids and antioxidants, which accumulated to significantly higher levels in the pyroxsulam-resistant genotype. Additionally, we found key genes in both the flavonoid biosynthesis pathway and the antioxidant system were up-regulated in pyroxsulam-resistant barley. Conclusions: This work significantly expands on the current understanding of the molecular mechanisms underlying differing pyroxsulam tolerance among barley genotypes and provides several new avenues to explore for breeding or engineering tolerant barley.

Transcriptome and metabolome analyses reveal pathways associated with fruit color in plum (Prunus salicina Lindl.)

Background In order to reveal the mechanism of fruit color changes in plum, two common plum cultivars Changli84 (Ch84, red fruit) and Dahuangganhe (D, yellow fruit) in Northeast China were selected as plant materials. Transcriptome sequencing and metabonomic analyzing were performed at three different developmental stages: young fruit stage, colour-change stage, and maturation stage. Results “Flavonoid biosynthesis” was significantly enriched in the KEGG analysis. Some DEGs in “Flavonoid biosynthesis” pathway had an opposite trend between the two cultivars, such as CHS , DFR and FLS . Also, transcriptional control of MBW (MYB–bHLH–WD) protein complexes showed a close relationship with plum fruit color, especially the expression of MYBs and bHLHs . In the current study, procyanidin B1 and B2 had the highest level at young fruit stage in Ch84 and the content of procyanidin B2 decreased sharply at the color change stage. Conversely, the content of cyanidin increased with the growth of fruit and reached the peak at the maturation stage. Conclusion The content of procyanidin B1 and B2 in plums at young fruit stage might be the leading factors of the matured fruit color. At the maturation stage, the cyanidin produced by procyanidins keeps the color of the fruit red. Correspondingly, genes in “flavonoid biosynthesis” pathway play critical roles in regulating the accumulation of anthocyanin in plum.

Comparative Analysis of Flavonoid Metabolites in Foxtail Millet (Setaria italica) with Different Eating Quality

Foxtail millet (Setaria italica) is an important minor cereal crop in China. The yellow color of the de-husked grain is the most direct aspect for evaluating the foxtail millet quality. The yellow pigment mainly includes carotenoids (lutein and zeaxanthin) and flavonoids. To reveal the diversity and specificity of flavonoids in foxtail millet, we chose three high eating quality and two poor eating quality varieties as research materials. A total of 116 flavonoid metabolites were identified based on Ultra Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC-ESI-MS/MS) system. The tested varieties contained similar levels of flavonoid metabolites, but with each variety accumulating its unique flavonoid metabolites. A total of 33 flavonoid metabolites were identified as significantly discrepant between high eating quality and poor eating quality varieties, which were mainly in the flavonoid biosynthesis pathway and one of its branches, the flavone and flavonol biosynthesis pathway. These results showed the diversified components of flavonoids accumulated in foxtail millets and laid the foundation for further research on flavonoids and the breeding for high-quality foxtail millet varieties.

Origin of black glume color in the Triticum aestivum lines with introgressions from wheat wild relatives

ABSTRACT Dark color of a glume in Triticinae is a known morphological marker with studied genetic control. However, the pigments that provide the color development, molecular basis of their biosynthesis, and mechanisms of its regulation are still unknown. Dark color of glumes is a common trait in the Triticum aestivum lines with alien introgressions. To find out the basis of this trait, we studied nature of the compounds that color the glumes by analyzing the total phenolic content (TPC) in the dark glumed introgression lines (ILs) and comparing it to their parents. TPC in the dark ILs was found to be higher than in their parents and in the light lines. All studied genotypes were screened for the expression of eight genes encoding the flavonoid biosynthesis pathway enzymes. Expression of three flavonoid genes in the pathway were changed in the ILs compared to their parents. Firstly, the expression of F3’H and FNS was absent in the lines with introgressions from Aegilops spp. The expression of three genes, F3’H, FLS, and FNS, was found to be altered in the light lines with introgressions from A. muticum. No expression of the F3’H was detected in the T. aestivum / A. muticum lines with light and dark plants, and the other genes of the pathway were found to be expressed at the parental levels. No correlation between the expression profiles and the phenotype was found in the T. aestivum / A. muticum lines. So we hypothesized that other undetected changes are present in these lines.