Integrated Metabolomics and Transcriptomics Analyses Reveal Anthocyanin and Carotenoid Biosynthesis Involved in Color Development in Willow Bark

doi:10.21203/rs.3.rs-1740308/v1

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Integrated Metabolomics and Transcriptomics Analyses Reveal Anthocyanin and Carotenoid Biosynthesis Involved in Color Development in Willow Bark

https://doi.org/10.21203/rs.3.rs-1740308/v1

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Background

The rich yellow-orange to vividly deep red bark of willow (Salix spp.) branches have great ornamental and economic value. The mechanism of willow branch color has not been clarified yet.

Results

To study the mechanisms regulating color development, we performed metabolomics and transcriptomics analyses using purple, green, and red willow barks. Seven anthocyanins were isolated; pelargonidin, petunidin 3-O-rutinoside, and cyanin chloride were the most abundant in the red bark and pelargonin chloride in the purple bark. Among the barks, green bark had the highest level of malvidin, but its level was not significantly higher than in the red bark; the purple bark had the highest amount of canthaxanthin, a carotenoid pigment. The integrated biosynthetic pathway of flavonoid, carotenoid, and chlorophyll was constructed for willow barks. Among the three barks, the expression of the structural genes ANS, ANR, and BZ1, involved in anthocyanin synthesis, was highest in the red bark, likely causing anthocyanin accumulation. The highest expression of CrtZ, involved in the carotenoid pathway, was observed in the purple bark, likely causing canthaxanthin accumulation. The high expression of DVR, POR, and CRD1 may be associated with the green pigment synthesis in chlorophyll biosynthetic pathway. Our research findings will facilitate genetic breeding to improve the color and luster of willows.

Conclusions

In conclusion, the purple bark is co-regulated by anthocyanins and carotenoids. The red bark is characterized by anthocyanin accumulation and chlorophyll degradation. The green pigment is regulated by maintaining of chlorophyll synthesis. The key regulation genes BZ1, CrtZ could be candidate genes regulating anthocyanin and canthaxanthin accumulation in red and purple barks. Our research findings facilitate the genetic breeding and cultivation of colorful willows with improved color and luster.

Salix

bark color

metabolomics

transcriptome

anthocyanin

carotenoid

Willows, belonging to the genus Salix in the Salicaceae family, are important landscape trees in China. The characteristics of willow, including the varying forms, branching morphology, and seasonally changing colors, make it increasingly popular in gardening, horticulture, and landscape architecture [1]. Willow has been cultivated for thousands of years, especially the sweeping willow (Salix babylonica). In the past twenty years, the golden sweeping willow with its bright golden branches in winter has become much desirable. The colors of willow bark are diverse, including green, purple, yellow, white, brown, and red [2]. In recent years, the sale of colorful branches has thrived owing to their novelty, while also generating significant economic profits. Therefore, the producing colorful branches of willow have been a key focus of willow breeding. While variation is rich in hybrid generations, an understanding of the genetic mechanism is lacking.

Secondary metabolites determine color development, including three classes of compounds: flavonoids, betalains, and carotenoids [3, 4]. Flavonoids, which include anthocyanins, produce pale yellow, red, violet, and purple to blue colors in flowers, seeds, and fruit skin [5]. The six most common anthocyanins that accumulate in flowers and fruits are cyanidin, petunidin, delphinidin, peonidin, pelargonin, and malvidin [6]. Accordingly, these compounds can change the color of plants from pink to blue-violet [7, 8]. Among these anthocyanins, peonidin and cyanidin color the organ skin purple to red, whereas pelargonin colors it brick red. The presence of delphinidin, malvidin, and petunidin results in blue and purple coloring. Betalains, which originate from tyrosine, are found in plants of the order Caryophyllales, as well as in beetroot where they produce yellow to red coloring [9]. Xanthophylls are the main carotenoids endowing flower petals and fruits with a pale to deep yellow color [10–12]. Combinations of flavonoids and carotenoids in the same organ increases the color variety.

The flavonoid biosynthesis pathway has been well characterized in many plants, including Arabidopsis [13], yam [14], and grapevine [15]. Flavonoids are synthesized as defense metabolites in response to abiotic stress [16], but they also affect fruit quality and nutrition, such as in tomato [17] and kiwi fruit [18]. Anthocyanins, as a subgroup of flavonoids, render color to the plants by modifying the anthocyanidins with sugars and acyl acids. The synthesis of precursors is catalyzed by the enzymes chalcone isomerase (CHI), flavonoid 3’, 5’-hydroxylase (F3’5’H), chalcone synthase (CHS), and flavonoid 3’-hydroxylase (F3’H). Then dihydrokaempferol, dihydroquercetin, and dihydroquercetin were converted to colorless anthocyanins. The catalyzing function of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) produces anthocyanidin [19–20]. Anthocyanins are the pigments generated by this complex pathway through glycosylation reactions catalyzed by UDP-glucosyltransferase and glutathione S-transferase [21].

Chlorophyll is a fat-soluble green pigment that takes part in photosynthesis in plants. Chlorophyll and heme have the same primary biosynthesis steps, beginning at ALA then branching from protoporphyrin Ⅸ catalyzed by the enzyme porphobilinogen deaminase (HEMC, HEMD, HEME, HEMF, and HEMG). Then protoporphyrin Ⅸ combines with Mg2 + forming Mg-protoporphyrin IX by Mg-chelatase CHLH, CHLI1, CHLI2, or CHLD. The catalytic action of divinyl reductase gene (DVR) generates protochlorophyllide (Pchlide), which is converted to chlorophyllide a. Finally, chlorophyllide a was catalyzed by CHLG-producing chlorophyll a or converted to chlorophyll b by chlorophyllide a oxygenase (CAO) [22–23]. Carotenoids are one of the most widely distributed colorants conferring the flowers and fruits red, orange, and yellow [22]. Geranyl–geranyl pyrophosphate (GGPP) is synthesized by the enzyme GGPP synthase. Subsequently, phytoene is produced by phytoene synthase (CtrB). The key enzymes are carotenoid cleavage dioxygenases4 (CCD4), ζ-carotene desaturase (ZDS), β-carotene hydrolase (CHYb), nine cis-epoxycarotenoid dioxygenase (NCED), phytoene desaturase (PDS), and zeaxanthin epoxidase (ZEP). The products of each of these enzymes contribute to coloration [23].

The color variation in willow bark is rich, ranging from green and yellow to red. Red bark is rare in southern China; thus, studying the components and regulation of genes that influence the color of willow bark is of great significance for breeding. In this study, green, purple, and red willow bark were used for metabolome and transcriptome profiling. Metabolites and genes associated with pigment formation in the flavonoid and carotenoid biosynthesis pathways were verified. Our research provides new resources for studying the color development in willow bark and a basis for precise breeding to obtain the desired willow bark color.

Metabolic Analysis Among Different Colored Willow Barks

Green (G), purple (P), and red (R) willow branch barks as shown in Fig. 1 were analyzed in this study. The metabolic components in G, P, and R barks were detected and analysed. A total of 1639 and 1026 metabolites were identified in the positive and negative modes, respectively (Table S1). In positive mode, 23, 11, and 14 differential metabolites were found while 7, 11, and 3 were identified in the negative mode in the three comparisons (Fig. 2A). In the G vs. P, G vs. R, and P vs. R comparisons, 135, 109, and 129 metabolites were detected, respectively. In the G vs. P comparison, 55 (40.7%) metabolites were upregulated, in G vs. R, 68 (62.4%) metabolites were upregulated, and in P vs. R, 70 (54.3%) metabolites were upregulated (Table S2). To verify the function of the differentially accumulated metabolites (DAMs), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was conducted to annotate the DAMs. Metabolites of flavonoid biosynthesis (ko00941) and flavone and flavonol biosynthesis (ko00944) were all enriched in the three groups G vs. P (Fig. 2B), G vs. R (Fig. 2C), and P vs. R (Fig. 2D). In flavone and flavonol biosynthesis, there were 13, 13, and 7 enriched DAMs; in flavonoid biosynthesis, there were 8, 7, and 6 metabolites in the G vs. P, G vs. R, and P vs. R group, respectively.

Figure 2 Differentially accumulated metabolite analysis in willow bark. (a) Venn diagram of differentially accumulated metabolites (DAMs) in green vs. purple, green vs. red, and purple vs. red bark comparisons. KEGG pathway orthology of enriched DAMs in (b) green vs. purple bark, (c) green vs. red bark, and (d) purple vs. red bark.

Identification of Flavonoid and Anthocyanins metabolites

Differences in accumulation of metabolites involved in flavonoid, anthocyanins, and flavone and flavonol biosynthesis are illustrated by a heatmap in Fig. 3. Twenty two flavonoids were identified and quantified: 8 were present in the high amounts in the red bark, 5 in the purple bark, and 9 in the green bark. The content of quercetin, apigenin, kaempferide, and myricetin was the highest in purple willow bark, among the three barks. Vitexin, apigenin, laricitrin, and kaempferol-3-O-rutinoside levels were the highest in the green bark. Trifolin levels increased in going from green, to purple, to red bark. Six anthocyanins were detected. Among them, pelargonidin, petunidin 3-O-rutinoside, and cyanin chloride were the most abundant in red bark. Pelargonin chloride was the most abundant in purple bark. The level of malvidin was higher in the green bark than the other two barks. These results imply that the anthocyanins regulate the production of pigment molecules of willow barks.

Transcriptome Profile Characterization

To investigate the putative genes regulating willow bark color, transcriptome sequencing was conducted. A total of 448,839,796 raw reads were obtained with a Q30 > 93.15% (Table S3). After submitting for annotation, 7935, 6896, and 6790 differentially expressed genes (DEGs) were annotated in the G/P, G/R, and P/R comparison groups, respectively, including 4519, 3360, and 2705 upregulated genes and 3416, 3509, and 4085 downregulated genes (Table S4). KEGG pathway functional analysis was conducted, focusing on pigment biosynthesis associated pathways. Flavonoid biosynthesis (ko00941) was significantly enriched in the three group comparisons with 27, 28, and 21 genes enriched in the G/P, G/R, and P/R groups, respectively (Fig. 4). Carotenoid biosynthesis was significantly enriched in G/P and P/R groups, but not in G/R group (Fig. 4B, 4C, 4D). Isoflavonoid biosynthesis was enriched in P/R (Fig. 4D).

Quantitative Real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed genes (DEGs) in Flavonoid and Carotenoid Biosynthesis Pathways.

To conduct quality verification of transcriptome sequencing and genes involved in flavonoid and carotenoid biosynthesis, 12 genes including CHS, CHI, LAR, ctrZ, ZEP, and CCD4 were selected for qRT-PCR. The RNA-sequencing (RNA-seq) gene expression trends were the same as those determined using qRT-PCR for the G, P, and R bark samples (Fig. 5).

Figure 5 Similarity comparison of expression result in fragments per kilobase of exon per million mapped fragments (fpkm) between qRT-PCR and RNA-SEQ data of genes involved in flavonoid and carotenoid biosynthesis. G, green bark; P, purple bark; R, red bark.

Integrated Map of Metabolite and Transcript Analysis

Integration of the results illustrating changes in metabolite and transcript levels can help to better understand the regulatory pathway(s) of color development in willow bark. Anthocyanin biosynthesis and flavone and flavonol biosynthesis were significantly enriched in the three compared groups of bark color. As shown in Fig. 6, gene expression levels of CHI, ANS, and BZ1 were higher in the red bark than in the purple or green bark. The expression of FLS was higher in the purple bark than in the other two barks. The expression levels of the downstream regulatory genes ANR and ANS were consistent with the content of pelargonidin and epicatechin. In addition, the expression level of LAR was high in the green and purple barks, and epigallocatech content was the highest in green bark. These results support different regulatory pathways for different colored pigments. In our research, higher levels of pelargonidin, trifolin, phlorizin, and hesperetin accumulated in the red bark than in the purple or green bark (Fig. 6). Levels of epicatechin, dihydromyricetin, and kaempferol were higher in both the purple and red bark than in the green bark. Higher levels of quercetin, rutin, syringetin, myricetin, neohesperidin, phloretin, and xanthohumol were detected in the purple bark than in the green or red bark.

Figure 6 Integrated map of flavonoid biosynthesis based on metabolite and transcript analyses. (A) Reconstruction of the willow bark flavonoid biosynthesis pathway using the annotated DEGs and DAMs. The heatmap of the genes refers to the fpkm value of the samples, and the color scale from minimum (green) to maximum (red) corresponds with low to high gene expression. The nine samples were divided into three groups (n = 3 each) in order for green bark (G), purple bark (P), and red bark (R); CHS, chalcone synthase; ANS, anthocyanidin synthase; DFR, dihydroflavonol 4-reductase; BZ1, bronze-1; LAR, leucoanthocyanidin reductase; and FLS, flavonol synthetase;F3H, flavanone 3-hydroxylase.

Annotated Genes in the Carotenoid Biosynthesis Pathway and Porphyrin and Chlorophyll Metabolism Pathway

In porphyrin and chlorophyll metabolism pathway, six metabolites were identified including pheophorbide a, L-stercobilin, biliverdin, 5, 6-dimethylbenzimidazole, L-threonine, and L-glutamic acid (Fig. 7A, 7C). Ten DEGs were enriched; among them, one HCAR, one CHLI, one CAO have the highest level of expression in the purple bark compared with the other two (Fig. 7A, 7D). In the carotenoid biosynthetic pathway, 14 DEGs were annotated, including one CrtB, one PDS, four ZDS, one DWARF 27, three ZEP, three NECD, and one AAO3, in the colored willow bark samples (Fig. 7E). Four genes were expressed the most in the purple bark: CrtZ, one ZEP, two NECD, and AAO3. Meanwhile, the levels of the metabolites canthaxanthin and abscisic acid glucose ester were the highest in purple bark, compared with the other barks, supporting the role of various genes in the synthesis of carotenoids and their derivatives in different colors of bark.

Correlation Between DEGs and Metabolites

To investigate the correlation between pigment associated metabolites and DEGs involved in flavonoid, chlorophyll, and carotenoid biosynthesis pathway, the correlation network was constructed (Fig. 8). Malvidin, pelargonin, and cyanidin-3-O-glucoside were associated with many genes indicating the influence of these genes on them. Cyanidin-3-O-glucoside and canthaxanthin were both associated with the genes such as ANR, DFR, CHS, and PDS. Malvidin shares the same regulatory genes with canthaxanthin. Plargonin was the hub metabolite connected with malvidin, canthaxanthin, cyanidin-3-O-glucoside, and petunidin (Fig. 8).

Figure 8 Correlation between differentially expressed gens and metabolites

Integrated metabolome and transcriptome analyses are increasingly applied in research on color formation in plants [24, 25]. This study is the first to perform these analyses to elucidate the regulatory mechanism of willow bark color. The mining of DAMs and DEGs involved in pigmentation in willow bark could provide a fundamental basis for genetic breeding for colorful willows.

Plant colors are the products of flavonoid, chlorophyll, and carotenoid synthesis [26, 27]. Among the various colors, purple, blue, and red depend on the content of flavonoids/anthocyanins, including cyanidin and peonidin in common [28]. Chlorophylls present in the photosynthetic reaction systems are responsible for the green coloration [29] and the yellow to orange colors are attributed to carotenoids [30]. However, the regulation of pigment biosynthesis is complex and influenced by species and environmental conditions, as well as their interactions. In willow bark, four kinds of anthocyanin derivatives were identified including cyanidin, pelargonidin, petunidin, and malvidin. Pelargonidin is a kind of anthocyanin that commonly produces red pigment in plants [31], and in our study, the accumulation of pelargonidin correlated with red bark color in willows. It was found that cyanidin-3-O-glucoside is the red-brown component of ligulate flower [32].The cyanidin-3-O-glucoside is the purple pigment encoded by dominant Pp alleles in black rice [33]. In willow bark, there is no significant difference of cyanidin-3-O-glucoside in purple and red. The stability and types of the pigments are always influenced by temperature, hydrogen ion concentration (pH), illuminated environment, and structure of anthocyanin [34]. Petunidin 3-O-rutinoside can be changed from dark red to purple, and blue to green and to light yellow according to the pH, which provides us a new way to regulate the red color based on the soil pH. Pelargonidin mostly exhibited red, while pelargonidin chloride purple. Cyanin chloride mostly exhibited red, while cyanidin O-rutinoside equally exhibited purple and green and cyanidin-3-O-glucoside red and purple. This indicates that structural differences in the same anthocyanin leads to different colors in willow bark.

Carotenoids are also natural pigments, coloring vegetables, fruits, and flowers yellow, orange, and red and act as a supplement to the flavonoid/anthocyanin content when their levels decline. Canthaxanthin is a kind of ketocarotenoid, which is rarely synthesized in plants, and is not yet detected in plants [35]. The reddish-orange coloration in trout flesh, flamingo feathers, egg yolks, and koi carp skin is attributed to canthaxanthin [36, 37]. Canthaxanthin was the only carotenoid detected in willow bark in this study. Its content in the purple bark was 10 and 6 times higher than that in the green and red barks. The purple color was probably determined by the kind of carotenoid and anthocyanin combinations, and canthaxanthin plays a major role in the purple variety. Color mutations can be caused by damage to the chlorophyll biosynthesis pathway. Stercobilin, an intermediate in the chlorophyll biosynthesis pathway and a brown pigment, was detected in the red bark, while L-glutamic acid, an intermediate product, was higher in the red bark than in the green or purple barks.

Stable purple and red barks in willow are rare and have ornamental and economic value. The structural genes involved in color change in flavonoid biosynthesis are CYP73S, ANS, ANR, CHI, CHS, DFR, and F3H [38]. In total, 13 structural genes were identified in the three colors of willow bark. CHS catalyzes the first step in the synthesis of chalcone, and CHI catalyzes the stereospecific cyclization of chalcone and 6-deoxychalcone to (2S)-liquiritigenin and (2S)-naringenin [39]. The expression of CHS and CHI could affect the accumulation of anthocyanins as a branch point [40]. One CHI gene (IMY05_010G0169800) and two CHS (IMY05_003G0132300, MSTRG.5771) genes were expressed at high levels in the red bark, while CHI (IMY05_019G0069500) and two other CHS (IMY05_014G0118800, IMY05_016G0041700) genes were expressed at equal levels. These genes may play different roles in flavonoid biosynthesis including compounds such as rutin (R bark), luteolin (P bark), apigenin (G bark), and hesperetin (R). The change of DFR enzyme activity could produce red transgenic gentian flower through reduction of DHK. The low DHK metabolite content and the high pelargonidin content indicate a role of DHK in red pigment. ANS genes could directly convert the substrate of leucoanthocyanidins into colorful anthocyanidins. The activity of ANS and DFR could result in the accumulation abundance of pigmentation. ANS and ANR were significantly higher in R than in G and P barks, which may result in the accumulation of anthocyanin in the colored bark. The BZ1 gene encodes the final enzyme that converts UDP-glucose to the water soluble 3-hydroxyl group anthocyanins to achieve a more stable state [41]. The BZ1 and ANS genes are responsible for anthocyanin accumulation in red-skinned pear [42]. The expression level of ANS in the R bark was 11 times higher than that in the G bark. For the two BZ1 genes, IMY05_013G0103500 in the R bark was 147 times and 44 times higher than that in the G and P barks, respectively. The second BZ1 gene, IMY05_013G0118300, was 27–28 times higher in the R and P barks than in the G bark. This indicated that the expression abundance of ANS and BZ1 are decisive of the red pigment accumulation in willow bark.

In the carotenoid synthesis pathway, most carotenoid pigments are derived from phytoene, which is synthesized in the first reaction of the pathway catalyzed by CtrB. Red lycopene is subsequently synthesized by the actions of two enzymes, PDS and ZDS. Lycopene cyclization produces β-carotene, α-carotene and their derivatives in two branches [43]. In the primary synthesis steps, the expression of crtB is much higher in the red bark. Subsequently, one PDS and four ZDS genes were highly expressed in the green bark and were partially expressed in high levels in the red bark, indicating that the branching of the carotenoid synthesis pathway produces different products. Only one gene, ctrZ (IMY05_017G0118300), was expressed in high levels in the purple bark compared with the other barks. This was consistent with the content of canthaxanthin. The overexpression of the β-carotene hydroxylase gene (crtZ) from Agrobacterium aurantiacum resulted in the accumulation of more canthaxanthin [44]. The protein encoded by DWARF27 (D27) participates in strigolactone biosynthesis, regulating rice branching [45]. Thus, D27 be a potential candidate gene to regulate the branching and phenotype of colorful willows.

It is likely that the purple color of willow barks is because of the co-pigmentation interaction of carotenoid and anthocyanin, with carotenoid being a major factor. Most key structural genes involved in chlorophyll biosynthesis pathway, including hemH, hemB, por, CRD1, DVR, and CLH2 were the highest in the green bark indicating that the green color was determined by chlorophyll synthesis. POR is a key enzyme catalyzing the conversion of protochlorophyllide to chlorophyllide in chlorophyll synthesis [46]. The CRD1 mainly function in chloroplast movement. The higher expression of POR and CRD1 in the green bark may explain the reason for maintenance of green pigmentation. The stay-green (SGR) protein was overexpressed in melon affecting CHL degradation and leaf yellowing [47]. Among the three barks, the highest expression of SGR (IMY05_005G0141100) was observed in the red bark and other SGR (IMY05_016G0094700) was silenced in the green bark, causing the green coloration. Meanwhile, degradation metabolites were found in high levels in the red bark, indicating that chlorophyll was degraded in the red bark by the SGR. Chlorophyllide a oxygenase (CAO) is vital in regulating Chl a/b ratio and the expression of HMChl a reductase (HCAR) could limit Chl b degradation [48]. Overexpression of 7-hydroxymethyl chlorophyll (Chl) a reductase (HCAR) accelerated chlorophyll degradation in cucumber [48]. Therefore, the highest expression of CAO and HCAR in the purple bark, among the three barks, suggests that chlorophyll degradation occurs by converting Chl a to Chl b.

The pigmentation regulation of bark color in Salix were characterized by integrated metabolome and transcriptome technology. The red bark contains more pelargonidin, petunidin 3-O-rutinoside, and cyanin chloride than the purple and green barks. The purple bark contains more pelargonin chloride than the red and green barks. Malvidin was present in the highest level in the green bark but not significantly higher than the red bark. Canthaxanthin, a carotenoid metabolite, was detected in the purple bark. The structural genes CHS, ANS, DFR, ANR, and BZ1 were annotated in the flavonoid biosynthesis pathway. The high expression of ANS and BZ1 in the red bark probably lead to the accumulation of anthocyanin. CrtZ has higher expression level in the purple bark than the other two barks, which may be associated with the canthaxanthin accumulation in the purple bark. The high expression of hemH, hemB, por, CRD1, DVR, and CLH2 may explain the green pigmentation in the green bark. The high expression of SGR in the red bark is probably associated with the accumulation of stercobilin in the chlorophyll degradation pathway. The highest expression of CAO and HCAR in the purple bark, compared with the other two barks, suggesting that chlorophyll degradation occurs through converting Chl a to Chl b and may lead to the production of pheophorbide A. The connection network of anthocyanin and DEGs also show that the purple bark is co-regulated by anthocyanins and carotenoids. The red bark is characterized by anthocyanin accumulation and chlorophyll degradation. The green pigment is regulated by maintaining of chlorophyll synthesis. Our research findings facilitate the genetic breeding and cultivation of colorful willows with improved color and luster.

Plant Material

The three Salix species were collected and deposited in the willow nursery in Da Feng District, Yan Cheng City, Jiangsu Province (N 33.20-33.35 E 120.15-120.47). The species were preserved by the authors who have permissions to collect the willow barks. The P bark was from ‘Salix×Jiangsunesis 1053’, G bark from S. integra, and R bark from S. miyabeana. The voucher specimens of ‘Salix×Jiangsunesis 1053’, S. integra, and S. miyabeana were deposited at the herbarium of Jiangsu Academy of Forestry under the voucher numbers 1053, P61, and P78, respectively. The email of the person who is in charge of the sample collection is [email protected].

Sample Preparation

The willow bark was scraped from branches using scalpels in the nursery. The bark of each color was collected from six individuals of the same species. The samples were put into eppendorf tubes and fast frozen using liquid nitrogen, then stored at -80°C until required for metabolite and RNA isolation. Six biological repeats were performed to determine the metabolites and three biological repeats were used to determine the transcripts.

Metabolite Extraction and UHPLC-MS/MS Analysis

Willow bark powder (100 mg), which was grounded with liquid nitrogen, was taken into an Eppendorf tube, and 500 µL water solution with 80% methanol was added. The mixture was resuspended and vortexed. It was then incubated for 5 min, and the powder was centrifuged for 20 min at 1.5 × 103 g, 4°C. The supernatant was transferred to a clean eppendorf tube containing LC-MS grade water with 53% methanol and centrifuged for 20 min. Then the supernatant was absorbed and analyzed using the LC-MS system. The chromatographic column was from Hypesil Gold column (C18) with the following parameters: column temperature gradient 40°C and flow rate 0.2 mL/min. The component of eluent A was 0.1% formic acid in the positive mode and 5 mM ammonium acetate in the negative mode. The eluent B was methanol in both modes. The negative mode was eluent A, pH 9.0, and eluent B, methanol. Vanquish UHPLC system (ThermoFisher, Germany) and Orbitrap Q ExactiveTMHF-X mass spectrometer (Thermo Fishe) were used for UHPLC-MS/MS detection in Gene Denovo Co., Ltd. (Guangzhou, China).

Metabolite Profile Analysis

The raw data were filtered by Compound Discoverer 3.1 (CD3.1, Thermo Fisher). After peak extraction and peak area quantitation, the prediction of molecular formula was performed using molecular ion peaks and fragment ions peaks. By blasting with the Masslist, mzVault and mzCloud databases, the relative and accurate quantitative of final metabolites were identified. Principal component analysis (PCA) [49], orthogonal partial least squares discriminant analysis (OPLS-DA), heatmap, cluster analysis, volcano map, and metabolic pathway analysis involving different compounds were carried out using R (http://www.r-project.org/) [50, 51]. The screening criteria of differential metabolites and function annotation followed the method of Zhang [52].

RNA isolation and transcriptome sequencing

Total RNA extraction kit was Omega plant RNA kit (Omega Bio-Tek, CA, USA). The cDNA libraries were constructed using the NEB#7530 kit (#E7530, New England Biolabs). The library quality test was performed by DNA 1000 assay Kit. The sequencing platform used was Illumina HiSeq2500 of Gene Denovo Biotechnology Co. (Guangzhou, China). DEGs analysis was performed using DESeq2 software (and by edgeR within a group between two samples). Annotation of DEGs and KEGG functional enrichment followed the method of Zhou [53].

Quantitative Real-time PCR

The RNA used for qRT-PCR was reverse transcribed using Hifair R III 1st Strand cDNA Synthesis SuperMix (gDNA digester plus; Yeasen Biotechnology [Shanghai] Co., Ltd.). qRT-PCR was performed using Genious 2×SYBR Green Fast qPCR Mix (ABclonal, Wuhan, China) by LightCycler® 480 II (Roche, German). The two-step cycling procedure was performed at 95 °C, 5 min; 40 cycles at 95 °C, 10 s; 60 °C, 30 s, and 40 cycles at 72 °C for one-point sensing. The melting curve cycle conditions followed 95 °C (15 s), 60 °C (60 s), and 95 °C (15 s) for the continuous test signal. The expression values were analyzed using the 2-ΔΔCt method.

Ethics approval and consent to participate

Not applicable. The study was fully compliant with local and national regulations on plant usage.

Consent for publication

Not applicable.

Availability of data and materials

The transcriptome sequence data were submitted to GenBank of NCBI (https://www.ncbi.nlm.nih.gov/) under the accession No. SRP375909. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA839146, SAMN28511960, SAMN28511961, SAMN28511962, SAMN28511963, SAMN28511964, SAMN28511965, SAMN28511966, SAMN28511967, SAMN28511968; The metabolome data were submitted in Metabolights (https://www.ebi.ac.uk/metabolights/); the uploaded data number is MTBLS4820. The metabolome data will be available immediately after the Metabolights database audit.

Competing interests

The authors declare that they have no competing interest.

Funding

This research was funded by Jiangsu Agricultural Science and Technology Independent Innovation Fund (CX(20)3042). Independent innovation projects of Jiangsu academy of forestry (ZZKY202101), The key research and development program of national forestry and grassland administration (GLM[2021]83).

Authors’ Contributions

Conceptualization, J.Z.; methodology, J.H.G., X.D.H; software, J.Z.; validation, J.Z., J.H.G., Pu Wang writing—original draft preparation, J.Z.; writing—review and editing, J.Z.; visualization, J.Z. B.S.W.; Z.G.Q.; funding acquisition, J.Z. B.S.W., Q.S.C All authors have read and agreed to the published version of the manuscript.

Acknowledgements

Not applicable.

Authors’ information

1 National willow engineering technology research center, Jiangsu Academy of Forestry, Nanjing 211153, Jiangsu, China; 2 Nanjing Forestry university, Nanjing 210037, Jiangsu, China.

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Supplementalmaterial.pdf
Additional file 1: Supplemental materialTable S1: Information of all the metabolitesTable S2: The up and down-regulated metabolites Table S3: The RNA-seq sequencing profilesTable S4: The number of up- and down-regulated DEGs

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Integrated Metabolomics and Transcriptomics Analyses Reveal Anthocyanin and Carotenoid Biosynthesis Involved in Color Development in Willow Bark

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Abstract

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Introduction

Results

Identification of Flavonoid and Anthocyanins metabolites

Integrated Map of Metabolite and Transcript Analysis

Annotated Genes in the Carotenoid Biosynthesis Pathway and Porphyrin and Chlorophyll Metabolism Pathway

Correlation Between DEGs and Metabolites

Discussion

Conclusion

Methods

Declarations

References

Additional Declarations

Supplementary Files

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Version 1