- The results showed that the chlorophyll content of normal green leaves was significantly higher than that of mutant yellow leaves of ginkgo. - A total of 283 substances in the metabolic profiles were finally detected, including 50 significantly differentially expressed metabolites (DEMs). - We identified these DEMs and 1361 differentially expressed genes (DEGs), with 37, 4, 3 and 13 DEGs involved in the photosynthesis, chlorophyll, carotenoid, and flavonoid biosynthesis pathways, respectively. - Moreover, integrative analysis of the metabolomes and transcriptomes revealed that the flavonoid pathway contained the upregulated DEM. - Ginkgo (Ginkgo biloba L.) is a one of the oldest dioe- cious gymnosperms [1], with a history dating back to ap- proximately 200 million years ago [2, 3]. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article Wu et al. - Chlorophyll, carotenoids and anthocyanins are the main constituent molecules in the leaves of plants. - Pre- vious studies have provided an understanding of the proteomic, cytological, physiological and transcriptomic changes in the leaves of the xantha mutant of ginkgo [8, 18]. - leaves, the chlorophyll contents of mutant yellow leaves and normal green ginkgo leaves were measured in this study. - The chlorophyll content differed significantly be- tween mutant yellow leaves and normal green leaves of ginkgo. - The levels of chlorophyll a and chlorophyll b in the green leaves of ginkgo were 1.34 mg g − 1 and 0.75 mg g − 1 , respectively. - In contrast, the levels of chlorophyll a and chlorophyll b in the variant yellow leaves were sig- nificantly lower (by 13.43 and 13.33%, respectively) (Table 1).. - The overall distribution among samples and the stability of the entire analytical process were then examined by un- supervised principal component analysis (PCA), followed by supervised orthogonal partial least squares discrimin- ant analysis (OPLS-DA). - Fifty DEMs in the metabolic profiles were identified between the two groups (yellow and green leaves) (Fig. - Via PCA, partial least squares discriminant analysis (PLS-DA), OPLS-DA and permuta- tion of the different leaf color groups, score plots a, b, and c were obtained and are shown in Fig. - The 200- response sorting chart of the OPLS-DA model is shown in Fig. - Using the KEGG pathway mapper function, the metabolic pathway enrichment of the DEMs was based on their up- or downregulation. - Component Green leaves Yellow leaves. - To obtain an overview of the transcriptomes of the dif- ferently colored ginkgo leaves (green and yellow leaves), a large amount of paired-end sample sequencing data were obtained using the Illumina platform. - Given the impact of the data error rate on the results, Trimmo- matic [22] software was used to perform quality pre- treatment of the original data, and the number of reads in the whole quality control process was statistically summarized to obtain 931.52 M clean reads from 6 ginkgo samples. - Then, HISAT v2.0.4 software was used to compare the sequences of clean reads with the cur- rently published ginkgo genome to obtain position infor- mation compared to the reference genome or gene, as well as information regarding the specific sequence char- acteristics of the sequencing sample. - More than 93% of the sequences could be located on the reference genome (Table S1). - The mRNA expression level box-whisker plot and regional distribution map of the 6 samples are shown in Fig. - To verify the unigenes obtained by sequencing and fur- ther analyze the reliability of the RNA-seq data in the present study, 10 unigenes were random selected for qRT-PCR detection. - Overall, the results showed that all the selected genes exhibited similar ex- pression patterns as those observed in the RNA-seq data, despite some differences in expression levels. - In the volcano map, gray and blue indicate no sig- nificant differences, whereas red and green indicate sig- nificant differences in DEGs. - In addition, the hierarchical clustering of DEGs showed that samples of the same type cluster together (Fig. - 2b), indicating that the genes in the same cluster may have similar biological functions.. - 1 Metabolomic profiling of green and yellow leaves of ginkgo. - Principal component analysis (PCA) plots a, partial least squares discriminant analysis (PLS-DA) b, and orthogonal partial least squares discriminant analysis (OPLS-DA) scores c for the analysis of metabolites in green and yellow leaves. - The 200-response sorting tests of the OPLS-DA model. - Fifty differentially expressed metabolites (DEMs) between green and yellow leaves. - In the biological process (BP) category, “protein-chromophore linkage” exhibited the highest enrichment (Fig. - The Fisher algorithm was used to perform enrichment ana- lyses of BPs, CCs, and MFs for the DEGs of ginkgo, and the GO terms were enriched in the directed acyclic graph of CC. - On the basis of KEGG pathway enrichment of the DEGs, the most significantly enriched pathway was photosynthesis (ko00195), followed by oxidative phosphorylation (ko00190) (Fig. - To identify the structural genes involved in photosyn- thesis, all the unigenes were searched against the photosynthesis pathway (ko00195) in the KEGG data- base. - A metabolic map was eventually constructed in which multiple transcripts encoding enzymes of the photosynthesis pathway were displayed. - Thirty-seven unigene enrichments were noted in the photosyn- thesis pathway, including upregulated expression of 36 genes and downregulated expression of only one gene, psbA (encoding photosystem II P680 reaction center D1 protein. - Three unigenes were significantly upregulated in yellow leaves compared with green leaves. - Relative to normal green leaves, yellow leaves exhibited seven DEGs that were significantly upregulated in the flavonoid biosynthesis pathway, namely, CHS (chalcone synthase. - 2 Differentially expressed genes (DEGs) between green and yellow leaves of ginkgo. - KEGG path- way analysis of the DEGs and DEMs showed that the fla- vonoid biosynthesis pathway contained the significantly upregulated DEM (−)-epicatechin downstream of ANR (anthocyanidin reductase [EC: 1.3.1.77]. - Moreover, according to the correlation matrix of the top 20 (Fig. - 6) results of the correlation analysis of the DEGs and DEMs, a strong correlation was identified between gene expression level and the response strength of the metabolites. - 3 Analyses of the differentially expressed genes (DEGs). - Gene ontology (GO) analyses of the DEGs. - The x- axis shows the names of the GO terms, and the y-axis presents the -log10 p-values. - 4 Differentially expressed genes in the photosynthesis, chlorophyll metabolism, and carotenoid biosynthesis pathways. - In this study, yellow-green leaf color mutants ap- peared in the same environment, indicating that genetic changes play a decisive role. - Chlorophyll, including chlorophyll a and chlorophyll b, mainly exists in the leaves of plants and is distributed in. - Chlorophyll isprimarily responsible for the capture and photosynthetic transformation of light en- ergy in plants and is the most important pigment in the processes of light morphogenesis and organic accumula- tion in plants [26]. - 5 Differentially expressed genes and metabolites in the flavonoid biosynthesis pathway. - unigenes enriched in the photosynthesis pathway. - Leaf color mutations can damage chloroplast development, and this type of damage is often considered one of the reasons for changes in photosynthesis [8]. - The chloro- plast is an important organelle for photosynthesis in plants and is also the site for the synthesis and accumu- lation of the photosynthetic pigment chlorophyll and ca- rotenoids [29]. - Gb_38719 and Gb_14516), were upregulated in the yellow leaves. - CPOX is a key enzyme in the syn- thesis of porphyrins, which play an important role in pigment formation [30]. - Hence, the significantly upregulated expression of CPOX may be one of the important reasons for the formation of yellow-leaved mutants. - In this study, DWARF27 (Gb_02232), a β-carotene isom- erase with iron, was significantly upregulated in the yel- low leaves. - In addition, flavonoids, a class of polyphenol secondary me- tabolites, are widely present in various plant organs and are involved in the regulation of color formation in flowers, leaves and fruits [36]. - DFR is the first key enzyme in the down- stream pathway of anthocyanin synthesis and is an important regulatory node in the anthocyanin biosyn- thesis pathway [40, 41]. - Integrative analysis of the metabolome and transcriptome. - Through integrative ana- lysis of the metabolomes and transcriptomes, we found that the flavonoid biosynthesis pathway contained the significantly upregulated DEM (−)-epicatechin down- stream of ANR (Fig. - We also found that three DFR genes were significantly upregulated in the yellow leaves.. - The upregulated expression of these DFR genes might be the main cause of the increase in the downstream (−)-epicatechin content. - DFR is also an important regu- latory node in the anthocyanin biosynthesis pathway [42]. - Therefore, DFR can directly or indirectly affect the synthetic and metabolic pathways of anthocyanins, lead- ing to changes in the (−)-epicatechin content in plants, which ultimately leads to leaf color variation. - Integrative analysis of the transcriptome and metabolome can be used not only to study the genetic characteristics of. - In addition, chlorophyll, a green pigment that is cru- cial for photosynthesis, absorbs energy from sunlight in the antenna system and transfers the energy to the reac- tion center [26, 44]. - This study provides an ideal reference for investigations of the chlorophyll biosynthesis pathway and photosynthetic mechanism of ginkgo. - To determine which of the DEGs in yellow leaves can be used as marker genes, we must confirm the main or key genes regulating ginkgo in the future.. - Notably, 37 of the identified DEGs were enriched in the photo- synthesis pathway, 4 in the chlorophyll pathway, 3 in the carotenoid pathway, and 13 in the flavonoid biosynthesis pathway, suggesting that the ginkgo mutant exhibits spe- cific differences in the regulation or alteration of pig- ment biosynthesis. - Integrative analysis of the metabolome and transcriptome showed that the flavon- oid pathway contained the significantly upregulated DEM (−)-epicatechin downstream of ANR. - The iden- tification of these differentially accumulated genes and metabolites suggests the presence of a complex regula- tory mechanism in the mutant yellow leaves of ginkgo.. - This branch is thought to result from a bud mutation that composes a quarter of the crown, with the remainder of the crown branches pro- ducing green leaves (Fig. - Several green leaf and yellow leaf scions were grafted onto rootstocks in the ginkgo germplasm nursery at Nanjing Forestry Uni- versity Base. - The phenotypes of mutant yellow leaves and normal green leaves are shown in Fig. - In this study, three leaves were sampled per replicate, with three biological replicates for each group in the same period. - The DEMs were selected on the basis of both a statistically signifi- cant threshold of variable influence on projection (VIP) values obtained from an OPLS-DA model and p-values from a two-tailed Student’s t-test of the normalized peak. - 7 Phenotypes of normal green leaves and mutant yellow leaves of ginkgo. - The phenotype of mutant yellow leaves. - The results of the transcriptional analysis were vali- dated by qRT-PCR. - Results of the comparison of sequences to the reference genome.. - Different colors in the figure represent FPKM values in dif- ferent ranges. - qRT-PCR validation of 10 putative genes in the ginkgo transcriptome. - Histogram showing the qPCR results of 10 unigenes between green leaves and yellow leaves of ginkgo. - YW participated in the data analysis, performed the experiments and wrote the manuscript. - This study was supported by the Special Fund for Forest Scientific Research in the Public Welfare the Agricultural Science and Technology Independent Innovation Funds of Jiangsu Province (CX the National Key Research and Development Program of China. - The funding bodies had no role in the design of the study, the collection, analysis, and interpretation of data, or in writing the manuscript.. - Sampling was permitted by the Ginkgo Engineering Technology Research Center of the State Forestry Administration. - Yellow and green leaf scions were grafted onto rootstocks in the ginkgo germplasm nursery at Nanjing Forestry University Base.. - Variation in the concentrations of major secondary metabolites in Ginkgo leaves from different geographical populations. - Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase. - Compensatory alterations in the photochemical apparatus of a photoregulatory, chlorophyll b-deficient mutant of maize. - Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hybrida. - Characterization of the 820-nm transmission signal paralleling the chlorophyll a fluorescence rise (OJIP) in pea leaves. - Metabolomics integrated with transcriptomics reveals redirection of the phenylpropanoids metabolic flux in Ginkgo biloba. - Analysis of mulberry leaf components in the treatment of diabetes using network pharmacology
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