Widely targeted metabolome combined with transcriptome analysis unravels metabolic and molecular pathways related to red flush and browning of peels in cold-stored ‘Yali’ pear fruit

Purple shoots of tea germplasm Zijuan (Camellia sinensis var. asssamica) have high anthocyanin content and gradually turn green with continued growth and development. To uncover the mechanism of this colour change, we performed transcriptome analysis to identify differentially expressed genes (DEGs). A total of 18 328 DEGs were identified in purple tender shoots of Zijuan (ZJP) compared with mature green leaves of Zijuan (ZJG). These included 85 DEGs associated with phenylpropanoid biosynthesis, 30 DEGs associated with flavonoid biosynthesis, and 3 DEGs involved in flavone and flavonol biosynthesis. We detected higher expression in ZJP than in ZJG of genes encoding the following key enzymes regulating anthocyanin biosynthesis and their corresponding metabolites: chalcone synthase (CHS), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), adenosine triphosphate (ATP)-binding cassette (ABC) transporters, bifunctional 3-dehydroquinate dehydratase (DHQ), chorismate mutase (CM), cinnamoyl-CoA reductase 1 (CCR), and cinnamyl alcohol dehydrogenase (CAD). In addition, upregulated expression of carbohydrate metabolism, glycolysis pathway, sucrose metabolism, and pyruvate metabolism suggested that glycolysis and upregulation of the sucrose synthase (SUS) gene may provide more intermediates as substrates for promoting anthocyanin accumulation in ZJP. Moreover, DEGs involved in anthocyanin biosynthesis, including phenylalanine ammonialyase (PAL), cinnamate acid 4-hydroxylase (C4H), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), ANR, ANS, CHS, and flavonoid 3',5'-hydroxylase (F3'5'H) were significantly (P < 0.01) correlated with the contents of anthocyanin components such as cyanidin (Cy), delphinidin (Dp), peonidin (Pn), petunidin (Pt), and malvidin (Mv). The above results suggested that these upregulated DEGs may contribute to anthocyanin accumulation in ZJP and may play important roles in the colour changes in Zijuan tea. This research provided a foundation for clarifying the mechanisms underlying colour changes in Zijuan tea.

Transcriptome and weighted gene co-expression network analysis reveal key genes involved in the proanthocyanidin biosynthesis in Cerasus humilis

Bahiagrass (Paspalum notatum Flügge) is a warm‐season grass where high lignin content limits its forage quality, negatively affecting animal performance. To create a new breeding method by genome editing, isolation and characterization of lignin biosynthesis genes and identification of their molecular relationships are essential. The caffeic acid O‐methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD) cDNA clones were isolated from bahiagrass, and their protein sequences showed high similarity to other C4 monocot species through phylogenetic analysis. Gene expression analysis of phenylalanine ammonia‐lyase (PAL), COMT and CAD, involved in crucial stages of lignin biosynthesis and lignin content, was conducted at different plant developmental stages. The highest gene expression levels of these genes were observed in the vegetative (V) and early reproductive stages, while the lignin content increased until the middle reproductive stage and remained constant thereafter. Additionally, to further understand the molecular relationships in lignin biosynthesis, the effect of CAD downregulation was analyzed in transgenic bahiagrass lines introduced with sorghum CAD antisense and RNAi vector obtained from a previous report. This led to a reduced lignin content and affected the expression of PAL and COMT working upstream of CAD. In the V stage, PAL expression was lower in transgenic lines compared to wild type (WT), while COMT expression showed no significant differences. However, PAL and COMT expression of transgenic lines in the middle reproductive stage (R2) was significantly higher than in the WT. These findings suggest that the downregulation of CAD gene expression affects PAL and COMT expression and induces a feedback system in the R2 stage. Lignin content influenced the phenotype of the transgenic plants with significantly reduced lignin, exhibiting a dwarf phenotype with shorter plant heights. The findings of this study can be applied to genome editing for the development of practical new breeding materials with improved digestibility in bahiagrass.

Verticillium wilt (VW) is a soil-borne fungal plant disease. Gossypium hirsutum varieties with the widest planting area are highly susceptible to VW pathogens, because their narrow genetic background of germplasm resources causes difficulties in cultivating VW-resistant varieties through intraspecific breeding. Therefore, G. barbadense cultivars, harboring a natural VW resistance, become ideal donor materials to cultivate high-yield and multi-resistance chromosome segment substitution lines (CSSLs) through hybridization and backcrossing with G. hirsutum receptor and recurrent parent. In order to investigate the molecular mechanism of cotton response to VW infection, a BC5F3:5 CSSL MBI9626 and its parents, CCRI36 (G. hirsutum) and Hai1 (G. barbadense), were chosen to perform transcriptome and metabolome sequencing on their root samples at 0, 7, and 15 days after inoculation (DAI) of V. dahliae V991. In total, 36,564 differentially expressed genes (DEGs) and 102 differentially accumulated metabolites (DAMs) were separately identified from 12 pairwise comparison groups among the 27 samples. Of those, 125 common DEGs were found to participate in the biological processes of oxylipin metabolism, jasmonic acid (JA) biosynthesis/metabolism, and response to wounding in Gene Ontology (GO) enrichment analyses, while most of the DAMs were significantly enriched in tyrosine, purine, and phenylalanine metabolism pathways in enrichment analyses of Kyoto Encyclopedia of Genes and Genomes (KEGG). Having performed a conjoint KEGG analysis of all the DEGs and DAMs, we found two commonly enriched pathways, namely plant hormone signal transduction and flavonoid biosynthesis, which were consistent with the enrichment annotations of the significant model in weighted gene co-expression network analysis on the 2091 DEGs identified by an intersection of the genes in 40 previous QTLs and the total DEGs of this RNA-seq data. Among the ABA signaling pathway, the gene GH_D12G0236 (GHABF3) was selected to be used to perform virus-induced gene silencing (VIGS) verification in CCRI36 and MBI9626, and GHABF3-silenced plants showed a more serious wilting phenotype, an increased disease index (DI), and higher accumulation of fungal biomass compared to their empty-vector plants. These results provide a high-efficiency strategy for screening vital genes affecting cotton VW resistance, and lay a solid foundation for further cotton molecular breeding.

BackgroundCotton boll shedding is one of the main factors adversely affecting the cotton yield. During the cotton plant growth period, low light conditions can cause cotton bolls to fall off prematurely. In this study, we clarified the regulatory effects of low light intensity on cotton boll abscission by comprehensively analyzing the transcriptome and metabolome.ResultsWhen the fruiting branch leaves were shaded after pollination, all of the cotton bolls fell off within 5 days. Additionally, H2O2 accumulated during the formation of the abscission zone. Moreover, 10,172 differentially expressed genes (DEGs) and 81 differentially accumulated metabolites (DAMs) were identified. A KEGG pathway enrichment analysis revealed that the identified DEGs and DAMs were associated with plant hormone signal transduction and flavonoid biosynthesis pathways. The results of the transcriptome analysis suggested that the expression of ethylene (ETH) and abscisic acid (ABA) signaling-related genes was induced, which was in contrast to the decrease in the expression of most of the IAA signaling-related genes. A combined transcriptomics and metabolomics analysis revealed that flavonoids may help regulate plant organ abscission. A weighted gene co-expression network analysis detected two gene modules significantly related to abscission. The genes in these modules were mainly related to exosome, flavonoid biosynthesis, ubiquitin-mediated proteolysis, plant hormone signal transduction, photosynthesis, and cytoskeleton proteins. Furthermore, TIP1;1, UGT71C4, KMD3, TRFL6, REV, and FRA1 were identified as the hub genes in these two modules.ConclusionsIn this study, we elucidated the mechanisms underlying cotton boll abscission induced by shading on the basis of comprehensive transcriptomics and metabolomics analyses of the boll abscission process. The study findings have clarified the molecular basis of cotton boll abscission under low light intensity, and suggested that H2O2, phytohormone, and flavonoid have the potential to affect the shedding process of cotton bolls under low light stress.

Salinity is one of the most serious threats to agriculture worldwide. Sugar beet is an important sugar-yielding crop and has a certain tolerance to salt; however, the genome-wide dynamic response to salt stress remains largely unknown in sugar beet. In the present study, physiological and transcriptome analyses of sugar beet leaves and roots were compared under salt stress at five time points. The results showed that different salt stresses influenced phenotypic characteristics, leaf relative water content and root activity in sugar beet. The contents of chlorophyll, malondialdehyde (MDA), the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) were also affected by different salt stresses. Compared with control plants, there were 7391 and 8729 differentially expressed genes (DEGs) in leaves and roots under salt stress, respectively. A total of 41 hub genes related to salt stress were identified by weighted gene co-expression network analysis (WGCNA) from DEGs, and a transcriptional regulatory network based on these genes was constructed. The expression pattern of hub genes under salt stress was confirmed by qRT-PCR. In addition, the metabolite of sugar beet was compared under salt stress for 24 h. A total of 157 and 157 differentially accumulated metabolites (DAMs) were identified in leaves and roots, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further indicated that DEGs and DAMs act on the starch and sucrose metabolism, alpha-linolenic acid metabolism, phenylpropanoid biosynthesis and plant hormone signal transduction pathway. In this study, RNA-seq, WGCNA analysis and untargeted metabolomics were combined to investigate the transcriptional and metabolic changes of sugar beet during salt stress. The results provided new insights into the molecular mechanism of sugar beet response to salt stress, and also provided candidate genes for sugar beet improvement.

Integration of transcriptomic and metabolomic data reveals the low temperature response gene regulatory network of germination in soybean (Glycine max L.).

Integrated transcriptomic and metabolomic analysis reveal the molecular and metabolic basis of flavonoids in Chinese sour jujube fruits in different harvest times

Botrytis elliptica, the causal agent of gray mold disease, poses a major threat to commercial Lilium production, limiting its ornamental value and yield. The molecular and metabolic regulation mechanisms of Lilium's defense response to B. elliptica infection have not been completely elucidated. Here, we performed transcriptomic and metabolomic analyses of B. elliptica resistant Lilium oriental hybrid “Sorbonne” to understand the molecular basis of gray mold disease resistance in gray mold disease. A total of 115 differentially accumulated metabolites (DAMs) were detected by comparing the different temporal stages of pathogen infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed the differentially expressed genes (DEGs) and DAMs were enriched in the phenylpropanoid and flavonoid pathways at all stages of infection, demonstrating the prominence of these pathways in the defense response of “Sorbonne” to B. elliptica. Network analysis revealed high interconnectivity of the induced defense response. Furthermore, time-course analysis of the transcriptome and a weighted gene coexpression network analysis (WGCNA) led to the identification of a number of hub genes at different stages, revealing that jasmonic acid (JA), salicylic acid (SA), brassinolide (BR), and calcium ions (Ca2+) play a crucial role in the response of “Sorbonne” to fungal infection. Our work provides a comprehensive perspective on the defense response of Lilium to B. elliptica infection, along with a potential transcriptional regulatory network underlying the defense response, thereby offering gene candidates for resistance breeding and metabolic engineering of Lilium.

Cross-talk interactions of sucrose and Fusarium oxysporum in the phenylpropanoid pathway and the accumulation and localization of flavonoids in embryo axes of yellow lupine

Background: Diabetic kidney disease (DKD) is a leading cause of chronic kidney disease in China. Tubular injury contributes to the progression of DKD. Our study was conducted to explore the differential gene expression profiles between kidneys from patients with DKD and kidney living donors (LDs).Methods: In total, seven DKD and eighteen LD gene expression profiles from the GSE104954 dataset were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were analyzed in R with the limma package. DEGs were uploaded to the g:Profiler online database to explore the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Ingenuity pathway analysis (IPA) was carried out using online IPA software. Weighted gene co-expression network analysis (WGCNA) was performed using the WGCNA R package. By integrating DEGs and genes from the top 1 phenotype-gene associated module, we determined the hub gene. We next tested the hub gene, VCAN, in the GSE30122 dataset. We also validated the versican levels in human kidney tissues, explored immune cell type enrichment using an online database xCell, and investigated the correlation between cell types and VCAN expression.Results: A total of 563 DEGs was identified. A large number of pathways were involved in the immune response process according to the results of GO, KEGG, and IPA. Using WGCNA, we selected the lightcyan module in which genes showed the strongest correlation with the phenotype and smallest P-value. We also identified VCAN as a hub gene by integrating DEG analysis and WGCNA. Versican expression was upregulated in human diabetic kidney tissue. Moreover, versican was speculated to play a role in immune injury according to the enrichment of functions and signaling pathways. VCAN transcript levels correlate with the assembly of immune cells in the kidney.Conclusion: Immune processes played an essential role in DKD tubulointerstitium injury. The hub gene VCAN contributed to this process.

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a chronic and crippling bone disease. This study aims to reveal novel diagnostic biomarkers of SONFH.MethodsThe GSE123568 dataset based on peripheral blood samples from 10 healthy individuals and 30 SONFH patients was used for weighted gene co-expression network analysis (WGCNA) and differentially expressed genes (DEGs) screening. The genes in the module related to SONFH and the DEGs were extracted for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Genes with |gene significance| > 0.7 and |module membership| > 0.8 were selected as hub genes in modules. The DEGs with the degree of connectivity ≥5 were chosen as hub genes in DEGs. Subsequently, the overlapping genes of hub genes in modules and hub genes in DEGs were selected as key genes for SONFH. And then, the key genes were verified in another dataset, and the diagnostic value of key genes was evaluated by receiver operating characteristic (ROC) curve.ResultsNine gene co-expression modules were constructed via WGCNA. The brown module with 1258 genes was most significantly correlated with SONFH and was identified as the key module for SONFH. The results of functional enrichment analysis showed that the genes in the key module were mainly enriched in the inflammatory response, apoptotic process and osteoclast differentiation. A total of 91 genes were identified as hub genes in the key module. Besides, 145 DEGs were identified by DEGs screening and 26 genes were identified as hub genes of DEGs. Overlapping genes of hub genes in the key module and hub genes in DEGs, including RHAG, RNF14, HEMGN, and SLC2A1, were further selected as key genes for SONFH. The diagnostic value of these key genes for SONFH was confirmed by ROC curve. The validation results of these key genes in GSE26316 dataset showed that only HEMGN and SLC2A1 were downregulated in the SONFH group, suggesting that they were more likely to be diagnostic biomarkers of SOFNH than RHAG and RNF14.ConclusionsOur study identified that two key genes, HEMGN and SLC2A1, might be potential diagnostic biomarkers of SONFH.

BackgroundWalnut (Juglans), cultivated globally for their nuts and timber, holds significant economic and ecological value. The eastern black walnut (EBW, J. nigra). a diploid species, an important woody species. Key enzymes in the plant lignin biosynthesis pathway include Phenylalanine ammonia-lyase (PAL) and Cinnamyl alcohol dehydrogenase (CAD). Although the gene families of PAL and CAD have been extensively characterized in various plants, comprehensive genomic resources and expression profiles specific to the EBW remain limited.ResultsBased on RNA sequencing of shells and kernels from black walnut (Juglans nigra) among three development stages, S1 (80 days after flowering, DAF), S2 (111 DAF), and S3(140 DAF) in black walnut shells and kernels, we found the genes related to lignin biosynthesis. genes such as JnPAL, JnC4H, Jn4CL1, Jn4CL2, JnHCT, JnCOMT, JnCAD2, and JnCAD-like exhibited significant differential expression across all three developmental stages. Both PAL and CAD genes were expressed significantly in walnut shells compared to kernels across three developmental stages. We conducted a genome-wide identification and discovered 7 PAL and 3 CAD proteins in J. nigra and 5 PAL and 3 CAD proteins in J. microcarpa genome, respectively. Transcriptome DEGs (differential expressed genes) analysis and identified three candidates PAL (JnPAL1, JnPAL2, and JnPAL6) and 2 CAD genes (JnCAD2 and JnCAD3) were upregulated during the development of walnut kernels and shells. In addition, it was higher expressed of these PAL and CAD genes in the shell than kernel.ConclusionsWe conducted a comprehensive involving genome-wide identification, transcriptome dynamics, and expression regulation of the key lignin biosynthesis gene families PAL and CAD across three development stages of shells. This study not only constitutes a valuable resource for elucidating the role of PAL and CAD genes in determining shell thickness in black walnut but also holds significant potential for informing gene-assisted breeding strategies in walnut cultivation.

In this study, red-blue light (7R3B) was used as the control (CK), while far-red (FR) and ultraviolet-A (UVA) light were supplemented to evaluate their effects on basil growth. The results showed that the FR treatment promoted plant height, stem diameter, and biomass, but reduced chlorophyll and carotenoid content, while the UVA treatment increased stem diameter and chlorophyll b content. Meanwhile, transcriptomic and metabolomic analyses were employed to examine changes in gene expression and metabolite accumulation in basil. The FR treatment reduced the levels of differentially accumulated metabolites (DAMs) in the carotenoid biosynthesis pathway, potentially contributing to the observed decrease in chlorophyll. The FR treatment upregulated the levels of five DAMs (gibberellin, cytokinin, brassinosteroid, jasmonic acid, and salicylic acid) and altered the differentially expressed genes (DEGs) such as gibberellin receptor (GID1) and jasmonate ZIM domain-containing protein (JAZ) in the plant hormone signal transduction pathway, thereby promoting plant growth and shade avoidance responses. The UVA treatment upregulated the 9-cis-epoxycarotenoid dioxygenase (NCED) expression in the carotenoid biosynthesis pathway, possibly indirectly promoting flavonoid synthesis. In the flavonoid biosynthesis pathway, the UVA treatment also promoted flavonoid accumulation by upregulating DEGs including flavonol synthase (FLS), anthocyanidin synthase (ANS), 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase (CYP98A), and flavanone 7-O-glucoside 2″-O-beta-L-rhamnosyltransferase (C12RT1), as well as increasing the levels of DAMs such as kaempferol, luteolin, apigenin, and leucopelargonidin. The accumulation of flavonoids improved antioxidant capacity and nutritional value in basil. Through a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, this study provided valuable insights into the molecular and metabolic mechanisms of the FR and UVA regulation of basil growth, providing guidance for optimizing supplementary lighting strategies in plant factories.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) is an important plantation tree species in China, and seed germination is a key step in forest tree cultivation. To reveal the gene expression network and molecular mechanisms in the germination of Chinese fir seeds, physiological indexes were measured and transcriptome and metabolome analyses were performed on Chinese fir seeds in four stages of germination (imbibition stage, preliminary stage, emergence stage, and germination stage). All six physiological indicators had significant differences at different developmental stages. In transcriptome and metabolome analysis, we identified a large number of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). Gene Ontology (GO) analysis showed a large number of DEGs associated with cell growth, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that DEGs were significantly enriched in the flavonoid biosynthesis, phenylpropanoid biosynthesis, and plant hormone signal transduction pathways. The KEGG enrichment results of DAMs were similar to those of DEGs. The joint analysis of DEGs and DAMs indicated that flavonoid biosynthesis and phenylpropanoid biosynthesis were the key pathways of Chinese fir seed germination. Our study revealed a number of key genes and key metabolites, laying the foundation for further studies on the gene regulatory network of Chinese fir seed germination.