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Comparative Transcriptome Analysis Revealed the Key Genes Regulating Ascorbic Acid Synthesis in Actinidia.

ABSTRACT: Actinidia (kiwifruit) is known as 'the king of vitamin C' due to its rich ascorbic acid (AsA) concentration, which makes it an important model for studying the regulation of AsA metabolism. Herein, transcriptomic analysis was employed to identify candidate genes that regulate AsA synthesis in Actinidia species with 100-fold variations in fruit AsA content (A. latifolia and A. rufa). Approximately 1.16 billion high-quality reads were generated, and an average of 66.68% of the data was uniquely aligned against the reference genome. AsA-associated DEGs that predominately respond to abiotic signals, and secondary metabolic pathways were identified. The key candidate genes, for instance, GDP-L-galactose phosphorylase-3 (GGP3), were explored according to integrated analysis of the weighted gene co-expression network and L-galactose pathway. Transgenic kiwifruit plants were generated, and the leaves of GGP3 (OE-GGP3) overexpressing lines had AsA contents 2.0- to 6.4-fold higher than those of the wild type. Transcriptomic analysis of transgenic kiwifruit lines was further implemented to identify 20 potential downstream target genes and understand GGP3-regulated cellular processes. As a result, two transcription factors (AcESE3 and AcMYBR) were selected to carry out yeast two-hybrid and BiFC assays, which verified that there were obvious AcESE3-AcMYBR and AcESE3-AcGGP3 protein-protein interactions. This study provides insight into the mechanism of AsA synthesis and provides candidate factors and genes involved in AsA accumulation in kiwifruit.

SUBMITTER: Liu X

PROVIDER: S-EPMC8657573 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Actinidia (kiwifruit) is an emerging fruit plant that is severely affected by salt stress in northern China. Plants have evolved several signaling network mechanisms to cope with the detrimental effects of salt stress. To date, no reported work is available on metabolic and molecular mechanisms involved in kiwifruit salt tolerance. Therefore, the present study aims to decipher intricate adaptive responses of two contrasting salt tolerance kiwifruit species Actinidia valvata [ZMH (an important genotype), hereafter referred to as R] and Actinidia deliciosa ['Hayward' (an important green-fleshed cultivar), hereafter referred to as H] under 0.4% (w/w) salt stress for time courses of 0, 12, 24, and 72 hours (hereafter refered to as h) by combined transcriptome and metabolome analysis. Data revealed that kiwifruit displayed specific enrichment of differentially expressed genes (DEGs) under salt stress. Interestingly, roots of R plants showed a differential expression pattern for up-regulated genes. The KEGG pathway analysis revealed the enrichment of DEGs related to plant hormone signal transduction, glycine metabolism, serine and threonine metabolism, glutathione metabolism, and pyruvate metabolism in the roots of R under salt stress. The WGCNA resulted in the identification of five candidate genes related to glycine betaine (GB), pyruvate, total soluble sugars (TSS), and glutathione biosynthesis in kiwifruit. An integrated study of transcriptome and metabolome identified several genes encoding metabolites involved in pyruvate metabolism. Furthermore, several genes encoding transcription factors were mainly induced in R under salt stress. Functional validation results for overexpression of a candidate gene betaine aldehyde dehydrogenase (AvBADH, R_transcript_80484) from R showed significantly improved salt tolerance in Arabidopsis thaliana (hereafter referred to as At) and Actinidia chinensis ['Hongyang' (an important red-fleshed cultivar), hereafter referred to as Ac] transgenic plants than in WT plants. All in all, salt stress tolerance in kiwifruit roots is an intricate regulatory mechanism that consists of several genes encoding specific metabolites.

Project description:Organic acids and sugars are the primary components that determine the quality and flavor of loquat fruits. In the present study, major organic acids, sugar content, enzyme activities, and the expression of related genes were analyzed during fruit development in two loquat cultivars, 'JieFangZhong' (JFZ) and 'BaiLi' (BL). Our results showed that the sugar content increased during fruit development in the two cultivars; however, the organic acid content dramatically decreased in the later stages of fruit development. The differences in organic acid and sugar content between the two cultivars primarily occured in the late stage of fruit development and the related enzymes showed dynamic changes in activies during development. Phosphoenolpyruvate carboxylase (PEPC) and mNAD malic dehydrogenase (mNAD-MDH) showed higher activities in JFZ at 95 days after flowering (DAF) than in BL. However, NADP-dependent malic enzyme (NADP-ME) activity was the lowest at 95 DAF in both JFZ and BL with BL showing higher activity compared with JFZ. At 125 DAF, the activity of fructokinase (FRK) was significantly higher in JFZ than in BL. The activity of sucrose synthase (SUSY) in the sucrose cleavage direction (SS-C) was low at early stages of fruit development and increased at 125 DAF. SS-C activity was higher in JFZ than in BL. vAI and sucrose phosphate synthase (SPS) activities were similar in the two both cultivars and increased with fruit development. RNA-sequencing was performed to determine the candidate genes for organic acid and sugar metabolism. Our results showed that the differentially expressed genes (DEGs) with the greated fold changes in the later stages of fruit development between the two cultivars were phosphoenolpyruvate carboxylase 2 (PEPC2), mNAD-malate dehydrogenase (mNAD-MDH), cytosolic NADP-ME (cyNADP-ME2), aluminum-activated malate transporter (ALMT9), subunit A of vacuolar H+-ATPase (VHA-A), vacuolar H+-PPase (VHP1), NAD-sorbitol dehydrogenase (NAD-SDH), fructokinase (FK), sucrose synthase in sucrose cleavage (SS-C), sucrose-phosphate synthase 1 (SPS1), neutral invertase (NI), and vacuolar acid invertase (vAI). The expression of 12 key DEGs was validated by quantitative reverese transcription PCR (RT-qPCR). Our findings will help understand the molecular mechanism of organic acid and sugar formation in loquat, which will aid in breeding high-quality loquat cultivars.

Project description:BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers worldwide. However, the molecular mechanisms involved in HCC remain unclear and are in urgent need of elucidation. Therefore, we sought to identify biomarkers in the prognosis of HCC through an integrated bioinformatics analysis.MethodsMessenger RNA (mRNA) expression profiles were obtained from the Gene Expression Omnibus database and The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) for the screening of common differentially expressed genes (DEGs). Function and pathway enrichment analysis, protein-protein interaction network construction and key gene identification were performed. The significance of key genes in HCC was validated by overall survival analysis and immunohistochemistry. Meanwhile, based on TCGA data, prognostic microRNAs (miRNAs) were decoded using univariable and multivariable Cox regression analysis, and their target genes were predicted by miRWalk.ResultsEleven hub genes (upregulated ASPM, AURKA, CCNB2, CDC20, PRC1 and TOP2A and downregulated AOX1, CAT, CYP2E1, CYP3A4 and HP) with the most interactions were considered as potential biomarkers in HCC and confirmed by overall survival analysis. Moreover, AURKA, PRC1, TOP2A, AOX1, CYP2E1, and CYP3A4 were considered candidate liver-biopsy markers for high risk of developing HCC and poor prognosis in HCC. Upregulation of hsa-mir-1269b, hsa-mir-518d, hsa-mir-548aq, hsa-mir-548f-1, and hsa-mir-6728, and downregulation of hsa-mir-139 and hsa-mir-4800 were determined to be risk factors of poor prognosis, and most of these miRNAs have strong potential to help regulate the expression of key genes.ConclusionsThis study undertook the first large-scale integrated bioinformatics analysis of the data from Illumina BeadArray platforms and the TCGA database. With a comprehensive analysis of transcriptional alterations, including mRNAs and miRNAs, in HCC, our study presented candidate biomarkers for the surveillance and prognosis of the disease, and also identified novel therapeutic targets at the molecular and pathway levels.

Project description:Stigma color is an important morphological trait in many flowering plants. Visual observations in different field experiments have shown that a green stigma in melons is more attractive to natural pollinators than a yellow one. In the current study, we evaluated the characterization of two contrasted melon lines (MR-1 with a green stigma and M4-7 with a yellow stigma). Endogenous quantification showed that the chlorophyll and carotenoid content in the MR-1 stigmas was higher compared to the M4-7 stigmas. The primary differences in the chloroplast ultrastructure at different developmental stages depicted that the stigmas of both melon lines were mainly enriched with granum, plastoglobulus, and starch grains. Further, comparative transcriptomic analysis was performed to identify the candidate pathways and genes regulating melon stigma color during key developmental stages (S1-S3). The obtained results indicated similar biological processes involved in the three stages, but major differences were observed in light reactions and chloroplast pathways. The weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) uncovered a "black" network module (655 out of 5302 genes), mainly corresponding to light reactions, light harvesting, the chlorophyll metabolic process, and the chlorophyll biosynthetic process, and exhibited a significant contribution to stigma color. Overall, the expression of five key genes of the chlorophyll synthesis pathway-CAO (MELO03C010624), CHLH (MELO03C007233), CRD (MELO03C026802), HEMA (MELO03C011113), POR (MELO03C016714)-were checked at different stages of stigma development in both melon lines using quantitative real time polymerase chain reaction (qRT-PCR). The results exhibited that the expression of these genes gradually increased during the stigma development of the MR-1 line but decreased in the M4-7 line at S2. In addition, the expression trends in different stages were the same as RNA-seq, indicating data accuracy. To sum up, our research reveals an in-depth molecular mechanism of stigma coloration and suggests that chlorophyll and related biological activity play an important role in differentiating melon stigma color.

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Comparative Transcriptome Analysis Revealed the Key Genes Regulating Ascorbic Acid Synthesis in Actinidia.

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