Integrated Metabolomic and Transcriptomic Analysis of the Anthocyanin and Proanthocyanin Regulatory Networks in Red Walnut Natural Hybrid Progeny Leaves

BackgroundWalnuts are one of the most important dry fruit crops worldwide, typically exhibiting green leaves and yellow–brown or gray–yellow seed coats. A specific walnut type, red walnut ‘RW-1’, with red leaves and seed coats was selected as the plant material because of its higher anthocyanin and proanthocyanin (PA) contents. Anthocyanins and PAs coprise important secondary defense methods for plants to respond to biotic and abiotic stresses. However, few studies have focused on the molecular mechanism of anthocyanin biosynthesis in walnuts.ResultsFrom the results of widely targeted metabolome and anthocyanidin detection analysis, 395 substances, including 4 PAs and 26 anthocyanins, were identified from the red-leaf walnuts of RW-1 natural hybrid progenies (SR) and the green-leaf walnuts of RW-1 natural hybrid progenies (SG). Among these, all anthocyanin types in SR were significantly upregulated compared with SG. Additionally, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified as the primary components of anthocyanidins because of their higher contents. Nine anthocyanidins, malvidin 3-O-galactoside, malvidin 3-O-arabinoside, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside (Delphin), peonidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside and pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside), were detected only in the SR walnuts. For PAs, proanthocyanin C1 was upregulated in SR compared with SG, while proanthocyanin B1 and proanthocyanin B3 were upregulated in SR-1 and SR-3 but downregulated in SR-2 compared with the controls. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H, UFGTs, LAR and ANR), four MYBs and six WD40s in the anthocyanin and PA biosynthetic pathways were significantly higher in the SR walnut.ConclusionsOur results provide valuable information on anthocyanin and PA metabolites and candidate genes in anthocyanin and PA biosynthesis, which provides new insights into anthocyanin and PA biosynthesis in walnuts.

Regulation of flavonoid biosynthesis in representatives of the tribe Phaseoleae DC.

Flavonoids play a crucial role in plant metabolism. Many of them have antioxidant activity, and they are also pigments that render a variety of colors to plant tissues. Foods rich in flavonoid compounds are considered as functional components of a healthy diet. Currently, there is an increased interest in studying genetic mechanisms underlying the coloration of plants. Flavonoid biosynthesis pathways are controlled by two groups of genes. Structural genes encode enzymes, while regulatory genes are responsible for transcription factors that activate the expression of structural genes. Transcription factors that belong to R2R3-Myb, bHLH-Myc and WDR families form the ternary MBW complex, which is involved in regulating the expression of structural genes of flavonoid biosynthesis. The mechanisms of regulation of the anthocyanins and proanthocyanidin biosynthesis by the MBW complex are described in detail for the model plant Arabidopsis thaliana L. This review summarizes data on the regulation of phenolic pigment biosynthesis and the features of phenolic pigment accumulation in plant tissues in the main representatives of the Phaseoleae tribe: soybean Glycine max (L.) Merr., common bean Phaseolus vulgaris L., adzuki bean Vigna angularis (Willd.) Ohwi & Ohashi, and cowpea V. unguiculata (L.) Walp. The species discussed in this review are the most important food legumes in many countries of the world and they comprise the staple food in diets of millions of people. Identification and characterization of the genes controlling the flavonoid biosynthesis pathways are necessary for successful breeding of modern varieties with an increased dietary value. Identification of the flavonoid accumulation patterns is essential for solving the problem of broadening the diversity of plant products.

The Biases in Flavonoids and Anthocyanin Biosynthesis of a Red Flesh Table Grape Revealed by Metabolome and Transcriptome Co-analysis

Red flesh is a welcomed fruit trait, yet the regulation of red flesh formation in grape is not well understood. ‘Mio Red’ is a seedless table grape variety with light red flesh and blue-purple skin, the flesh color developed in the late stage of berry ripening, remarkably later than the skin coloring at veraison. The flesh and skin flavonoids metabolome and the transcriptome were analyzed. A total of 173 flavonoids including 17 anthocyanins were identified, 68 were found significantly different (Fold change ≥ 2 or ≤ 0. 5, VIP ≥ 1). Quercetin 3-O-glucoside, epicatechin-epiafzelechin, apigenin 6,8-C-diglucoside and hesperetin 5-O-glucoside were of higher content in the flesh, while the rest flavonoids were of higher content in the skin. The main anthocyanin in the flesh was pelargonidin derivatives in contrast to peonidin derivatives in the skin. Transcriptome comparison recruited 3970 differentially expressed genes (DEGs, log2Fold change > = 1, FDR < 0.05, FPKM ≥ 1), among them 57 were structural genes of flavonoid metabolism pathway. Two anthocyanin synthase (ANS) DEGs were annotated, ANS1 (Vitvi11g00565) and ANS2 (Vitvi02g00435) led the expression in the flesh and skin respectively. In the flesh, anthocyanin biosynthesis structural gene UFGT, positive regulators MYBA1/2/3, and anthocyanin transporters GST14 and MATE5 were of significantly lower expression, while negative regulators MYBC2-L1 and MYB3 were of higher transcription. The results of this study provide new information in the coloring mechanism of red flesh grape and assisting breeding of future table grapes having higher content of phytonutrient providing the health benefit as red wines.

Functional R2R3-MYB Transcription Factor NsMYB1, Regulating Anthocyanin Biosynthesis, Was Relative To The Fruit Color Differentiation in Nitraria Sibirica Pall

Background Nitraria sibirica Pall. is an economic plant with two kinds of fruit color, widely spreads in the Qinghai Tibet Plateau. The chemical analysis and pharmacological evaluation had been carried out for several tens of years, the mechanism behind the fruit color differentiation is still unclear. Results In this manuscript, the chemical analysis of the extractions showed that the chemical composition of fruit color was anthocyanin, and two kind of Nitraria sibirica Pall. were caused by the content differentiation with the same anthocyanin kinds. Cya-nidin-3-[2ʹ’-(6ʹ’’-coumaroyl)-glucosyl]-glucoside (C3G) was the major anthocyanin. Transcriptome analysis and the qRT-PCR revealed that the structural genes relative to anthocyanin biosynthesis except CHS, F3’5’H and ANS were up-regulated in BF compared with RF, which indicated that transcript factor should be the reason for the expression difference of the structure genes. In the unigenes of the transcript factor MYB and bHLH, relative to anthocyanin, only NsMYB1 (Clus-ter-8422.10600), was high-expression and up-expression in the BF. NsMYB1 encoded the same length protein with four amino acid differences in the RF and BF, and both contained the intact DNA, HTH-MYB and SANT domains. NsMYB1 was close to the AtMYB114, AtMYB113 and AtPAP1, regulating anthocyanin biosynthesis, in phylogenetic relationship. Both NsMYB1r and NsMYB1b could promote the transcript of the structural genes, and induced the anthocyanin accumulation in all tissues of transgenic tobacco. The insertion of ‘TATA’ in the promoter of NsMYB1r gave one more promoter region, and was the reason for higher transcripts in black fruit possibly. Conclusions NsMYB1 was a functional R2R3-MYB transcription factor, regulated the anthocyanin biosynthesis, and leaded to the fruit color differentiation in Nitraria sibirica Pall.

Identification of a Strong Anthocyanin Activator, VbMYBA, From Berries of Vaccinium bracteatum Thunb.

Wufanshu (Vaccinium bracteatum Thunb.), which is a wild member of the genus Vaccinium, accumulates high concentration of anthocyanin in its berries. In this study, the accumulated anthocyanins and their derivatives in Wufanshu berries were identified through UHPLC–MS/MS analysis. Candidate anthocyanin biosynthetic genes were identified from the transcriptome of Wufanshu berries. qRT-PCR analyses showed that the expression of anthocyanin structural genes correlated with anthocyanin accumulation in berries. The R2R3-MYB, VbMYBA, which is a homolog of anthocyanin promoting R2R3-MYBs from other Vaccinium species, was also identified. Transient expression of VbMYBA in Nicotiana tabacum leaves confirmed its role as an anthocyanin regulator, and produced a higher anthocyanin concentration when compared with blueberry VcMYBA expression. Dual-luciferase assays further showed that VbMYBA can activate the DFR and UFGT promoters from other Vaccinium species. VbMYBA has an additional 23 aa at the N terminus compared with blueberry VcMYBA, but this was shown not to affect the ability to regulate anthocyanins. Taken together, our results provide important information on the molecular mechanisms responsible for the high anthocyanin content in Wufanshu berries.

Metabolic Profiling and Gene Expression Analysis Unveil Differences in Flavonoid and Lipid Metabolisms Between ‘Huapi’ Kumquat (Fortunella crassifolia Swingle) and Its Wild Type

To elucidate the mechanism underlying special characteristic differences between a spontaneous seedling mutant ‘Huapi’ kumquat (HP) and its wild-type ‘Rongan’ kumquat (RA), the fruit quality, metabolic profiles, and gene expressions of the peel and flesh were comprehensively analyzed. Compared with RA, HP fruit has distinctive phenotypes such as glossy peel, light color, and few amounts of oil glands. Interestingly, HP also accumulated higher flavonoid (approximately 4.1-fold changes) than RA. Based on metabolomics analysis, we identified 201 differential compounds, including 65 flavonoids and 37 lipids. Most of the differential flavonoids were glycosylated by hexoside and accumulated higher contents in the peel but lower in the flesh of HP than those of RA fruit. For differential lipids, most of them belonged to lysophosphatidycholines (LysoPCs) and lysophosphatidylethanolamines (LysoPEs) and exhibited low abundance in both peel and flesh of HP fruit. In addition, structural genes associated with the flavonoid and lipid pathways were differentially regulated between the two kumquat varieties. Gene expression analysis also revealed the significant roles of UDP-glycosyltransferase (UGT) and phospholipase genes in flavonoid and glycerophospholipid metabolisms, respectively. These findings provide valuable information for interpreting the mutation mechanism of HP kumquat.

Weighted gene coexpression correlation network analysis reveals a potential molecular regulatory mechanism of anthocyanin accumulation under different storage temperatures in ‘Friar’ plum

Background Flesh is prone to accumulate more anthocyanin in postharvest ‘Friar’ plum (Prunus salicina Lindl.) fruit stored at an intermediate temperature. However, little is known about the molecular mechanism of anthocyanin accumulation regulated by storage temperature in postharvest plum fruit. Results To reveal the potential molecular regulation mechanism of anthocyanin accumulation in postharvest ‘Friar’ plum fruit stored at different temperatures (0 °C, 10 °C and 25 °C), the fruit quality, metabolite profile and transcriptome of its flesh were investigated. Compared to the plum fruit stored at 0 °C and 25 °C, the fruit stored at 10 °C showed lower fruit firmness after 14 days and reduced the soluble solids content after 21 days of storage. The metabolite analysis indicated that the fruit stored at 10 °C had higher contents of anthocyanins (pelargonidin-3-O-glucoside, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and quercetin-3-O-rutinose), quercetin and sucrose in the flesh. According to the results of weighted gene coexpression correlation network analysis (WGCNA), the turquoise module was positively correlated with the content of anthocyanin components, and flavanone 3-hydroxylase (F3H) and chalcone synthase (CHS) were considered hub genes. Moreover, MYB family transcription factor APL (APL), MYB10 transcription factor (MYB10), ethylene-responsive transcription factor WIN1 (WIN1), basic leucine zipper 43-like (bZIP43) and transcription factor bHLH111-like isoform X2 (bHLH111) were closely related to these hub genes. Further qRT–PCR analysis verified that these transcription factors were specifically more highly expressed in plum flesh stored at 10 °C, and their expression profiles were significantly positively correlated with the structural genes of anthocyanin synthesis as well as the content of anthocyanin components. In addition, the sucrose biosynthesis-associated gene sucrose synthase (SS) was upregulated at 10 °C, which was also closely related to the anthocyanin content of plum fruit stored at 10 °C. Conclusions The present results suggest that the transcription factors APL, MYB10, WIN1, bZIP43 and bHLH111 may participate in the accumulation of anthocyanin in ‘Friar’ plum flesh during intermediate storage temperatures by regulating the expression of anthocyanin biosynthetic structural genes. In addition, the SS gene may play a role in anthocyanin accumulation in plum flesh by regulating sucrose biosynthesis.

Combined transcriptome and metabolome integrated analysis of Acer mandshuricum to reveal candidate genes involved in anthocyanin accumulation

The red color formation of Acer mandshuricum leaves is caused by the accumulation of anthocyanins primarily, but the molecular mechanism researches which underlie anthocyanin biosynthesis in A. mandshuricum were still lacking. Therefore, we combined the transcriptome and metabolome and analyzed the regulatory mechanism and accumulation pattern of anthocyanins in three different leaf color states. In our results, 26 anthocyanins were identified. Notably, the metabolite cyanidin 3-O-glucoside was found that significantly correlated with the color formation, was the predominant metabolite in anthocyanin biosynthesis of A. mandshuricum. By the way, two key structural genes ANS (Cluster-20561.86285) and BZ1 (Cluster-20561.99238) in anthocyanidin biosynthesis pathway were significantly up-regulated in RL, suggesting that they might enhance accumulation of cyanidin 3-O-glucoside which is their downstream metabolite, and contributed the red formation of A. mandshuricum leaves. Additionally, most TFs (e.g., MYBs, bZIPs and bHLHs) were detected differentially expressed in three leaf color stages that could participate in anthocyanin accumulation. This study sheds light on the anthocyanin molecular regulation of anthocyanidin biosynthesis and accumulation underlying the different leaf color change periods in A. mandshuricum, and it could provide basic theory and new insight for the leaf color related genetic improvement of A. mandshuricum.

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.

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.