Integrated metabolomic and transcriptomic analysis of the anthocyanin regulatory networks in red walnut

doi:10.21203/rs.3.rs-907324/v1

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Research Article

Integrated metabolomic and transcriptomic analysis of the anthocyanin regulatory networks in red walnut

https://doi.org/10.21203/rs.3.rs-907324/v1

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Background

Walnut is one of the most important dry fruit crops worldwide, typically 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 plant material because of higher anthocyanins contents. Anthocyanins are important colorants with strong antioxidant activity, especially, benefic for human health. However, few studies focused on the molecular mechanism of anthocyanin biosynthesis in walnut.

Results

From the results of Widely Targeted Metabolome and anthocyanidin detection analysis, 395 substances, including 4 procyanidins 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 the anthocyanins in SR were significantly up-accumulated comparing with SG. Also, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified to the primary components of anthocyanidins because of the higher contents. It was noted that 9 anthocyanins including 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 SR walnut. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H and UFGTs), and four MYBs in anthocyanin biosynthetic pathway were significantly higher in SR walnut.

Conclusions

We identified the color formation of SR leaves is due to the accumulation of anthocyanins. And our results obtained the valuable information on the anthocyanin metabolites and candidate genes in anthocyanin biosynthesis, which provided new insights into the anthocyanin biosynthesis in walnuts.

Epigenetics & Genomics

transcriptome

metabolome

anthocyanin

red walnut

Anthocyanin is one of the most important color-presenting materials in flavonoids, mainly existing in leaves, flowers, fruits of higher plants, formed by the combination of anthocyan and glycosyl [1]. Cyanidin (Cy), delphinidin (Dp), pelargonidin (Pg), peonidin (Pn), petunidin (Pt), and malvidin (Mv) are six common anthocyans, and, common glycosyl includes glucose, galactose, sucrose, etc [2]. Different types and amounts of glycosyls bound to different positions of anthocyans resulted in significant increase of anthocyanins compounds [3]. Anthocyanins takes part in protecting plants from ultraviolet rays damage, attracting insect pollination and resisting to low temperature [4]. As a kind of natural food colorants, anthocyanins have been widely used in food and cosmetics due to natural nontoxicity and no side effects [5]. Additionally, numerous studies have demonstrated that anthocyanins have many promising benefits for human health, such as the prevention of cardiovascular disease, neuron disease, cancer, diabetes and inflammation [6–10]. Therefore, anthocyanins have risen to fame for high antioxidant capacity, as well as improving human health and extracting natural food pigments.

The anthocyanin biosynthesis pathways have been well characterized in some plants [11]. Generally, phenylalanine is regarded as the initial of anthocyanin biosynthesis, which is composed of a series of enzymatic reactions, including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), chalcone synthetase (CHS), chalcone isoenzyme (CHI), flavanone 3-hydroxylase (F3H), Flavonoid 3′,5′-Hydroxylase (F3’5’H), dihydroflavonol 4-reductase (DFR), anthocyanin synthetase (ANS), UDP glucose: flavonoid-3-O-glucosyltransferase (UFGT) and so on [12, 13].

Meanwhile, the structural genes in anthocyanin biosynthesis are regulated by multiple transcription factors, among which MYB transcription factors have been widely studied. Grape R2R3-MYB TFs VvMYBA1, VvMYBA6 and VvMYBA7 regulated anthocyanin biosynthesis by activating UFGT and 3AT expressions [14]; The overexpression of MdMYB3 increased the anthocyanin accumulation in tobaccos by activating the gene expressions of CHS, CHI, UFGT and FLS [15]; The overexpression of GhMYB1a in gerbera and tobacco (Nicotiana tabacum) decreased anthocyanin accumulation by upregulating the structural gene expressions of NtCHS, NtF3H, NtDFR, NtANS, and NtUFGT [16].

It was reported that MYB regulates anthocyanin biosynthesis by either alone or forming MBW complex (MYB-bHLH-WD40) [17, 18]. In MBW complex, bHLH and MYB transcription factors can specifically bind to the promoters of structural genes in anthocyanin biosynthesis pathway, while WD40 protein plays a stable role in MBW complex [17]. The regulatory model of MBW complex included a ternary complex and a binary complex composed of MYB and bHLH. The ternary complex usually directly regulates the transcription of structural genes, but not synthesize intermediate regulators [17]. For example, AcMYB123 and AcbHLH42 in Actinidia chinensis cv. Hongyang promote the anthocyanin accumulation by activating the promoters of AcANS and Ac3FGT1 [19]. PyMYB10 and PyMYB114 co-transformed with PybHLH3 induced the promoters activity of PyDFR, PyANS, PyUFGT, PyGST and PyABC transporter [20]. Therefore, the present study aimed to elucidate the regulation mechanism of main structural genes and transcription factors involving in the anthocyanin accumulation based on red and green walnuts.

Walnut (Juglans regia L.) is an ancient fruit tree from the Juglandaceae family, ranks the top four nuts in the world due to its higher economic value and its health benefits to humans because of rech in oleic acid, linoleic acid, linolenic acid, protein and many trace elements [21]. In addition, walnuts are also considered to be an important ecological tree. The walnut industry has been included as the core point of industrial poverty alleviation in many areas, and plays an important role in helping targeted poverty alleviation and increasing farmers’ income. According to FAO statistics (http://faostat3.fao.org), China's walnut planting area and yield accounted for 48% and 56% of the world, respectively, both ranking the first in the world in 2019. However, phenotype traits of walnut varieties are close, with green leaves and yellow-brown or gray-yellow seed coat. Luckily, a red walnut accession ‘RW-1’ was found by our research group, with the red color leaf, pericarp, seed coat and xylem owing to high anthocyanin content [22], and several bHLH TFs and CHS structural genes were identified in anthocyanin biosynthesis [23, 24]. However, the molecular mechanism of anthocyanin biosynthesis has not yet been clearly elucidated in red walnut.

In recent years, the combination analysis of transcriptome and metabolome has widely used to clarify anthocyanin biosynthesis and accumulation in plants [25–31]. In the current study, the regulatory networks of anthocyanin biosynthesis in walnut were conducted using two ‘RW-1’ natural hybrid accessions, with red and green leaves respectively. The compared analysis of metabolomic and transcriptomics aimed to elucidate the pathway of anthocyanin metabolites and identify the different expressed genes (DEGs) in walnut anthocyanin biosynthesis.

Widely Targeted Metabolome and anthocyanidin detection in red walnut natural hybrid progeny leaves over developmental stages

In order to keep the consistence of genetic background, the RW-1 natural hybrid progenies SG and SR walnuts with different leaves colors were investigated. The SR walnut leaves changed from red to green while the SG walnut leaves displayed green all the time (Fig. 1a). The metabolome of six samples were profiled by Widely Targeted Metabolome and anthocyanidin detection approach. 395 compounds in the walnut leaves were classified into 19 classes (Table S2), including 26 anthocyanins and 4 procyanidins.

Identification of the differentially accumulated metabolites between SR and SG walnut leaves

The PCA chart illustrates the evident separation of the metabolites in the SR and SG, showed that all the biological replicates clustered together, indicating the high reliability of the sequencing data, and the metabolites in SR walnut leaves and SG walnut leaves at different development stages were clearly distinguished (Fig. S2a, b). According to the correlation analysis of population samples, the correlation coefficient of red leaves at different developmental stages was lower, as well as the correlation coefficient between red leaves and green leaves, which suggested the large differences in metabolites from green and red leaves. Meanwhile, the higher correlation of three repetitions indicated that the results in the present study were stable and reliable (Fig. S3).

As Fig. 1b shown, 87, 20 and 36 DAMs among SG-1 vs SR-1、SG-2 vs SR-2、SG-3 vs SR-3 were identified between pair samples, respectively. And 11, 13 and 12 were only were detected only in SR-1, SR-2 and SR-3 comparing with the controls, respectively; and all of them were anthocyanins (Fig. 2). Furthermore, the volcano maps showed that 30 up-accumulated metabolites while 58 down-accumulated in the first stage (Fig. 1c), 13 up-accumulated while 7 down-accumulated in the second stage (Fig. 1d), and 22 up-accumulated while 14 down-accumulated in the third stages (Fig. 1e), respectively. From Fig. 1f-h, the enrichment analysis of KEGG pathway displayed that the most significant enrichment pathways were anthocyanin biosynthesis, histidine metabolism and pure metabolism in the first period; anthocyanin biosynthesis, isoflavonoid biosynthesis, flavone and flavonol biosynthesis and flavonid biosynthesis in the second period; anthocyanin biosynthesis, pure metabolism and flavonoid biosynthesis in the third period. The above results indicated that the DAMs from the flavonoid biosynthesis pathway were likely to be the key metabolites resulting in the leaves color difference between green and red walnut types.

Components and contents of flavonoids and anthocyanins during walnut leaf development stages

In the work, a total of 88 flavonoid metabolites were identified, including 55 flavonoids, 3 isoflavonoids, 26 anthocyanins and 4 procyanidins.

Among the flavonoid, anthocyanins are the most important colorants in plants [32]. To determine whether the red pigmentation of red walnut is caused by anthocyanins, we analyzed the soluble anthocyanins in green and red leaves using a UPLC-ESI- MS/MS system. A total of 26 anthocyanins were detected in walnut leaves, among them, all the anthocyanins contents were significantly up-accumulated in SR-1 than in SG-1 (Fig. 2). Furthermore, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were the primary components of anthocyanidins. We also found that 9 anthocyanins compounds existed in only red leaves, including 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).

Moreover, 8 down-accumulated flavonoids contents were found to be significantly different in SG-1 vs SR-1, 2 down-accumulated and 1 up-accumulated in second period, while 2 down-accumulated and 3 up-accumulated in third period (Fig. S4). And 3 procyanidins were also showed up-accumulated in SR-1 than in SG-1, while 2 down-accumulated and 1 up-accumulated in second period, and 3 up-accumulated in third period (Fig. S5). In a word, the difference in anthocyanins compounds accumulation was corresponding to that in the leaf colors.

Transcriptome profiles of green- and red- leaves walnuts

RNA-sequencing (RNA-Seq) was used to profile genome-wide gene expression and transcriptome changes during the leaf development. With three biological replicates, the transcriptome sequencing of the 18 walnut leaf samples yielded a total of 134.01 Gb clean data with 95.22% of bases scoring Q30. Furthermore, 92.23–95.27% of the total clean reads were unique matches with the walnut reference genome [23, 24, 33], 4,131 novel genes were identified, including 3,154 annotated genes.

Differentially expressed genes between green- and red- leaves walnuts

From Fig. 3a, 5,708, 1,587 and 703 DEGs were obtained from SG-1 vs SR-1, SG-2 vs SR-2, and SG-3 vs SR-3, respectively, containing 24 identical genes.

To clear the differences in DEGs between SG and SR walnuts, the volcano maps were performed during the leaf developments of walnut. According to the results of transcriptomic analysis, there were 5,708 DEGs between SG-1 vs SR-1, of which 3,513 were up-regulated and 2,195 were down-regulated. There were 1,587 DEGs between SG-2 vs SR-2, including 810 up-regulated genes and 777 down-regulated genes. There were 703 DEGs between SG-3 vs SR-3, including 364 up-regulated genes and 339 down-regulated genes (Fig. 3b-d). The profiles of the DEGs indicated that the differences in genes expression affected leaves color in walnuts.

Weighted gene correlation network analysis (WGCNA)provides insights into the coloration mechanism in red walnut

To identify potential molecular mechanisms of anthocyanin accumulation in SR, WGCNA was used to identify the characteristic genes of the module and the key genes in the module. By WGCNA analysis, the obtained DEGs were divided into 10 modules according to highly correlated genes (FPKM values) from SR and SG (Fig. 4a). The results highlighted genes in the turquoise and lightcyan modules, with significant difference of FPKM values in SR-1 than SG-1 (Fig. 4b). However, turquoise module exhibited a stronger positive relationship with SR-1 than SG-1, and lightcyan module exhibited negative trend. KEGG enrichment analysis based on the modules revealed that 12 genes might be related to the biosynthesis of anthocyanin of SR walnut leaves in the turquoise module, and only one in lightcyan module (Fig. 4c-d, Table S3).

Analysis of structural genes involved in anthocyanin biosynthesis.

According to the KEGG annotation analysis of all DEGs, 18 DEGs were involved in anthocyanin biosynthesis pathway, of which 17 DEGs were existed in SG-1 vs SR-1. In view of leaves colors difference at Stage 1 on appearance, the combination analysis of transcriptomic and metabolomic data were to explain the anthocyanin biosynthesis pathway in SG and SR walnuts (Fig. 5). It was found that structural genes including C4H genes (gene40343 and gene42522), CHS genes (gene4994, gene39336, gene35863 and gene32601) [24], F3H gene (gene40994), F3’5’H gene (gene4387), ANS gene (gene1297) and UFGT genes (gene28510, gene35146, gene1870, gene24302, gene35144, gene1697, gene36923 and ) were all upregulated and only one UFGT gene (gene35048) was downregulated in red walnut leaves (Fig. 5).

Identification of TFs Related to Anthocyanin Biosynthesis

As we known, anthocyanin biosynthesis is regulated by MBW (MYB-bHLH-WD40) complex. From Fig. 6a, the number of MYB and bHLH ranked the top two of TFs related to anthocyanin biosynthesis in walnut leaves. Based on the Arabidopsis MYB protein domains, 135 putative walnut MYB protein sequences were obtained with default parameters using HMMER and BLASTP (Table S4). A phylogenetic tree was constructed using the 135 JrMYBs and other species MYBs related to anthocyanin biosynthesis (Fig. 6b). Nine subfamilies and 25 JrMYBs were obtained from the current study and 4 differentially expressed MYB genes in red frames, JrMYB1b (gene38312), JrMYB6a (gene32351), JrMYB123 (gene9445), JrTT2 (gene39085) were screened (Fig. 6c). The bHLHs in anthocyanin biosynthesis had been reported by our research group [23].

qPCR analysis of DEGs related to anthocyanin

Based on our above results, 12 differentially expressed structural genes involved in the anthocyanin biosynthetic pathway and 4 MYB genes were analyzed using qPCR methods. The results in Fig. 7 showed that eight structural genes JrC4Hs (gene40343, gene42522), JrF3’5’H (gene4387), JrUFGTs (gene35144, gene35146, gene1697, gene24302 and gene1870) and four MYB genes (gene38312, gene32351, gene9445, gene39085) were highly expressed in red leaves walnut at the first stage, which indicated that the high expression of genes involved in anthocyanin biosynthesis are related the color in red walnut leaves. It was also further proved that the transcriptome data were accurate and consistent with the expression of genes related to anthocyanin biosynthesis in red walnut.

The primary pigments in red skin walnut have been identified as flavonoids particularly anthocyanins [22, 24], and the previous studies on walnut coloration were limited. The current work aimed to explore more comprehensive metabolites involved in the color changes in walnut leaves, and 395 metabolites were obtained from walnut leaves using the widely-targeted metabolomics approach (Table S2). This was the first to present the genome-wide examination of anthocyanins and the gene expression profiles of in walnuts, aiming at providing a more comprehensive landscape of the metabolites involved in the color changes in walnut leaves during development. The transcriptomic and metabolic analysis demonstrated that 17 core genes were identified in the anthocyanin biosynthesis (Fig. 5). These findings provided a theoretical basis for the further study the mechanism of red walnut.

Anthocyanins are secondary metabolites in plants, such as ornamental plants, fruits, vegetables and medical plants, and play various roles in many biological processes including determining fruit quality and flower colors as well as improving resistance, avoiding UV and strong light damage [34, 35]. In addition to the rich nutrition in kernel, walnut is also an important ecological tree. Red walnut RW-1 possess the great ornamental value, nutritional value and economic benefits. A total of 26 anthocyanins were detected in green- and red- leaves walnuts (Fig. 2), and cyanidin 3-O-galactoside, delphinidin 3-O-galactoside, and delphinidin 3-O-glucoside were the main anthocyanin in SR. Importantly, the anthocyanins content was consistent with leaf color.

Interestingly, 6 anthocyanin compounds including cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin were existed not only in SR but also in SG (Fig. 2). The similar results were also found in white Salvia miltiorrhiza flowers [36]. The color depended on the types and contents of anthocyanins, Co-pigment, chlorophyll, vacuole pH, metal ions [37]. The 6 anthocyanins accumulated lower in green leaves than in red leaves walnuts, suggesting that complete anthocyanin metabolic pathways were also presented in SG. The other factors affecting the color of walnut leaves need to be further studied.

Anthocyanin biosynthesis involves multiple enzymes encoded by early biosynthesis genes (C4H, CHS, CHI and F3H) and anthocyanin-specific biosynthesis genes (F3’ H, F3’5’H, DFR, ANS and UFGT) [37]. The results from this study, the expression levels of C4Hs, F3’5’H and UFGTs genes in SR were significantly higher than those in SG (Fig. 7), suggesting that the genes mainly regulated the red coloration in red leaves walnuts. As the leaf development of walnut, these structural gene are down-regulated, and the color was trending to green. As we known, UFGT gene was involved in the final steps on the flavonoid biosynthetic pathway (biosynthesis and accumulation of anthocyanins). The expression of UFGT genes (gene35144, gene35146, gene1697, gene24302 and gene1870) were higher in SR than in SG at Stage 1, and UFGT gene (gene35038) was the opposite. It was worth noting that the 3 UFGT genes trend during 3 stages were corresponding to the colour change in walnut leaves. The anthocyanidins are extremely unstable and easy to degrade, therefore glycosylation is important to stabilize. As the last step of anthocyanin biosynthesis pathway, UFGTs were considered as the key enzyme to control anthocyanin biosynthesis in many plants and plays an important role in anthocyanin metabolism [12, 38–40]. The present study first explored the expression profile of UFGTs using red walnuts, providing the new sights for study UFGT genes function.

Anthocyanin biosynthesis is regulated by MBW (MYB-bHLH-WD40) complex. In MBW complex, bHLH and MYB transcription factors have DNA binding function, which can specifically bind to the promoters of structural genes in anthocyanin biosynthesis pathway, while WD40 protein plays a stable role in MBW complex [17, 41]. Actinidia chinensis Planch AcMYB123 and AcbHLH42 promote anthocyanin accumulation in the inner pericarp of Actinidia chinensis Planch by activating the promoters of AcANS and AcF3GT1[19]; PyWRKY26 and PybHLH3 acted together on PyMYB114 promoter, and co transfection can activate the expression of PyUFGT to regulate the accumulation and transportation of anthocyanin in pear [20]. In this study, we have identified five MYBs and four bHLHs based on their expression levels and phylogenetic analysis, how the TFs played roles in regulating the structural genes need further explored. These findings can help to elucidate the molecular mechanism and regulatory networks of anthocyanin biosynthesis in walnut and provide a biological basis for breeding new walnut cultivars.

In this study, the metabolomics and transcriptomics were used reveal the anthocyanin biosynthesis metabolic pathway. A total of 26 anthocyanins in SR were significantly up-accumulated comparing with SG, and the transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H and UFGTs), and four MYBs in anthocyanin biosynthetic pathway were significantly higher in SR walnut. Delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified to the primary components of anthocyanidins because of the higher contents. Our results obtained the valuable information on the anthocyanin metabolites and candidate genes in anthocyanin biosynthesis, which provided new insights into the anthocyanin biosynthesis in walnuts.

Plant material and growth conditions

The red walnut (Juglans regia L. accession RW-1, Germplasm resource number: JUREG4108210002) was introduced from Taihang Mountain, China (Fig. S1). In order to maintain a relatively consistent genetic background, different color phenotypes of natural hybrid plants (SG for green leaf natural hybrid plants, and SR for red leaf natural hybrid plants) grown in the Fruit Tree Experimental Station of Horticulture College, Henan Agricultural University, Zhengzhou, Henan, China. And the sampling permission has been obtained from the public land management agency of Henan Agricultural University. The leaves were collected according to the color changing of SR walnut leaves, as full red period (SR-1), red-green period (SR-2), and full green period (SR-3), the leaves of SG walnut in the same period were collected as the control[24] (Fig. 1a). All samples were immediately frozen in liquid nitrogen and stored at -80°C until RNA and metabolite extraction.

Extraction and quantification of metabolites

The sample Widely Targeted Metabolome and anthocyanidin detection were performed at Wuhan MetWare Biotechnology Co., Ltd. (Wuhan, China) as described previously [28, 42–44].

In brief, 100 mg leaves were crushed into powder and extracted with 0.6 mL of 70% aqueous methanol overnight at 4°C [45]. Following centrifugation at 10,000 g for 10 min, the supernatant was filtered through a microporous membrane (0.22 µm) for subsequent liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Quality control (QC) samples were prepared using mixing sample extracts. The extracts were filtrated and analyzed by Ultra Performance Liquid Chromatography coupled to Electrospray Ionization Tandem Mass Spectrometry (UPLC-ESI-MS/MS). Principal component analysis (PCA) were performed to verify the differences and reliability of metabolites in all the samples. Differentially accumulated metabolites (DAMs) between groups were filtered with VIP value ≥ 1 and absolute Log₂FC (fold change) ≥ 1. Subsequently, the differentially expressed metabolites with the significant enrichment were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg) and Plant Metabolic Network (PMN, https://plantcyc.org/) were used to analyze phenylalanine metabolism pathway, flavonoid metabolism pathway and anthocyanin biosynthesis pathway [45, 46]. We constructed metabolic pathways based on the KEGG database.

RNA extraction, sequencing and transcriptome data analysis

The total RNA from walnut leave was extracted using the Omega Plant RNA Kit (Omega Bio-tek, Norcross, Georgia) according to the manufacturer’s instructions. The integrity and quality of total RNA was examined on 1% agarose gels, and the RNA concentrations were measured by a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). cDNA library was constructed and sequenced on a HiSeq 2,500 platform (Illumina, San Diego, CA, United States) (NCBI accession PRJNA688391) by Biomarker Biotechnology Corp. (Beijing, China), following paired-end reads were produced after cluster generation.

The expression abundance of unigene was represented as FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) value, and the DEGs [false discovery rate (FDR) < 0.05, and | Log₂FC | ≥ 1.5] between red leaves and green leaves were obtained using the DESeq 1.8.3 package [47]. Function annotation was performed using Gene Ontology (GO) and the pathways with significant enrichment were identified based on KEGG pathway employing the cluster Profiler R package [48]. WGCNA (The Weighted Gene Co-Expression Network Analysis) was performed as previously described [49].

Quantitative Real-time (qRT) PCR Assay

The expression of structural genes and transcription factors genes in the anthocyanin biosynthetic pathway were examined by qRT-PCR method. The first-stand cDNA was synthesized using the FastQuant RT Kit (with gDNase) (Tiangen Biotech, Beijing, China), and qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on anABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, United States), Jr18S (GenBank accession No. XM_019004991.1) as the house-keeping gene[10]. Quantification was evaluated using the 2 − ^ΔΔCt method. All the primers were shown in Table S1.

Statistical analysis

All data were analyzed using SPSS 22.0 software, expressing as the mean ± standard deviation (SD) of three replicates. Significant differences were carried out using a one-sided paired t-test (^**p < 0.01, ^*p < 0.05) between red leaf and green leaf samples.

Used

ANS

anthocyanin synthetase

C4H

cinnamate 4-hydroxylase

CHI

chalcone isoenzyme

CHS

chalcone synthetase

Cyanidin

DAMs

differentially accumulated metabolites

DEGs

the different expressed genes

DFR

dihydroflavonol 4-reductase

delphinidin

F3H

flavanone 3-hydroxylase

F3’5’H

Flavonoid 3′,5′-Hydroxylase

FDR

false discovery rate

FPKM

Fragments Per Kilobase of transcript per Million fragments mapped

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

LC-MS/MS

liquid chromatography/tandem mass spectrometry

malvidin

PAL

phenylalanine ammonia lyase

PCA

principal component analysis

Ethics approval and consent to participate

The collection of wild red walnut were completed under the support of the project ‘Investigation and cataloguing of walnut genetic resources in Henan Province (2016) (Grant ID:GR-2016-06)’, and all the experimental research and field studies on plants comply with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its additional files.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the National Key R&D Program of China (2018YFD1000604) and the Support Plan of Scientific and Technological Innovation Team in Colleges and Universities of Henan Province, China (19IRTSTHN009). The funding organizations provided the financial support to the research projects, but had no role in the design of the study, data collection and analysis, and writing of the manuscript.

Authors’ Contributions

GW and JS designed the research. LL and WZ performed experimental works and data analysis, prepared the original draft. LL and LW participated in data analysis; HM, GZ and WW helped review and editing the draft; LW provided support for projects and funds, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

Not applicable.

Accession Codes

We have deposited our raw data in the NCBI Sequence Read Archive (SRA) under accession numbers PRJNA688391 (https://www.ncbi.nlm.nih.gov/).

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No competing interests reported.

SupplementalFiguresS1S5.docx
Figure S1. Phenotypic comparison between common green walnut 'Zhonglin 1' and red walnut ‘RW-1’. (a-d) leaves, fruits, pistillate flower and longitudinal section of fruit in 'Zhonglin 1'. (e-h) leaves, fruits, pistillate flower and longitudinal section of fruit of ‘RW-1’. Figure S2. Two dimensional and three-dimensional maps of principal component analysis of metabolites in total samples. Figure S3. Correlation analysis of population samples. The heatmap represents the correlation coefficient between different blade samples, and from green to red in the heatmap indicates the correlation coefficient raning from low to high. SR: Seedling progenies-Red leaves; The leaves were collected according to the color changing of SR walnut leaves, as full red period (SR-1), red-green period (SR-2), and full green period (SR-3), the leaves of SG walnut in the same period were collected as the control. Figure S4. Concentrations of flavonoids and isoflavonoids in leaves of SR and SG. SG: Seedling progenies-Green leaves; SR: Seedling progenies-Red leaves; The leaves were collected according to the color changing of SR walnut leaves, as full red period (SR-1), red-green period (SR-2), and full green period (SR-3), the leaves of SG walnut in the same period were collected as the control. The red frames indicated up-accumulated metabolites, and the green frames for down-accumulated metabolites. Figure S5. Concentrations of procyanidins in leaves of SR and SG. SG: Seedling progenies-Green leaves; SR: Seedling progenies-Red leaves; The leaves were collected according to the color changing of SR walnut leaves, as full red period (SR-1), red-green period (SR-2), and full green period (SR-3), the leaves of SG walnut in the same period were collected as the control. The red frames indicated up-accumulated metabolites, and the green frames for down-accumulated metabolites.
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Integrated metabolomic and transcriptomic analysis of the anthocyanin regulatory networks in red walnut

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Identification of the differentially accumulated metabolites between SR and SG walnut leaves

Components and contents of flavonoids and anthocyanins during walnut leaf development stages

Transcriptome profiles of green- and red- leaves walnuts

Differentially expressed genes between green- and red- leaves walnuts

Weighted gene correlation network analysis (WGCNA)provides insights into the coloration mechanism in red walnut

Identification of TFs Related to Anthocyanin Biosynthesis

qPCR analysis of DEGs related to anthocyanin

Discussion

Conclusions

Methods

Plant material and growth conditions

Extraction and quantification of metabolites

RNA extraction, sequencing and transcriptome data analysis

Quantitative Real-time (qRT) PCR Assay

Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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