Dataset Information

Overexpression of herbaceous peony miR156e-3p improves anthocyanin accumulation in transgenic Arabidopsis thaliana lateral branches.

ABSTRACT: microRNAs (miRNAs) play critical regulatory roles in plant growth and development. In the present study, the function of herbaceous peony (Paeonia lactiflora Pall.) miR156e-3p in the regulation of color formation has been investigated. Firstly, P. lactiflora miR156e-3p precursor sequence (pre-miR156e-3p) was isolated. Subsequently, the overexpression vector of pre-miR156e-3p was constructed and transformed into Arabidopsis thaliana. Moreover, the medium screening, GUS staining, polymerase chain reaction (PCR) of the GUS region and real-time quantitative PCR (qRT-PCR) of miR156e-3p all confirmed that the purpose gene had been successfully transferred into Arabidopsis plants and expressed, which resulted in apparent purple lateral branches. And this change in color was caused by the improved anthocyanin accumulation. In addition, expression analysis had shown that the level of miR156e-3p transcript was increased, while transcription level of target gene squamosa promoter binding protein-like gene (SPL1), encoding SPL transcription factor that negatively regulated anthocyanin accumulation, was repressed in miR156e-3p-overexpressing transgenic plants, and its downstream gene dihydroflavonol 4-reductase gene (DFR) that was directly involved in anthocyanin biosynthesis was strongly expressed, which resulted in anthocyanin accumulation of Arabidopsis lateral branches. These findings would improve the understanding of miRNAs regulation of color formation in P. lactiflora.

SUBMITTER: Zhao D

PROVIDER: S-EPMC5653671 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Overexpression of herbaceous peony miR156e-3p improves anthocyanin accumulation in transgenic Arabidopsis thaliana lateral branches.

Zhao Daqiu D Xia Xing X Wei Mengran M Sun Jing J Meng Jiasong J Tao Jun J

3 Biotech 20171023 6

microRNAs (miRNAs) play critical regulatory roles in plant growth and development. In the present study, the function of herbaceous peony (Paeonia lactiflora Pall.) miR156e-3p in the regulation of color formation has been investigated. Firstly, P. lactiflora miR156e-3p precursor sequence (pre-miR156e-3p) was isolated. Subsequently, the overexpression vector of pre-miR156e-3p was constructed and transformed into Arabidopsis thaliana. Moreover, the medium screening, GUS staini ...[more]

PMID: 29071176

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Project description:BACKGROUND: Herbaceous peony (Paeonia lactiflora Pall.) is a traditional flower in China and a wedding attractive flower in worldwide. In its flower colour, yellow is the rarest which is ten times the price of the other colours. However, the breeding of new yellow P. lactiflora varieties using genetic engineering is severely limited due to the little-known biochemical and molecular mechanisms underlying its characteristic formation. RESULTS: In this study, two cDNA libraries generated from P. lactiflora chimaera with red outer-petal and yellow inner-petal were sequenced using an Illumina HiSeq™ 2000 platform. 66,179,398 and 65,481,444 total raw reads from red outer-petal and yellow inner-petal cDNA libraries were generated, which were assembled into 61,431 and 70,359 Unigenes with an average length of 628 and 617 nt, respectively. Moreover, 61,408 non-redundant All-unigenes were obtained, with 37,511 All-unigenes (61.08%) annotated in public databases. In addition, 6,345 All-unigenes were differentially expressed between the red outer-petal and yellow inner-petal, with 3,899 up-regulated and 2,446 down-regulated All-unigenes, and the flavonoid metabolic pathway related to colour development was identified using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of 10 candidate differentially expressed genes (DEGs) involved in the flavonoid metabolic pathway were examined, and flavonoids were qualitatively and quantitatively analysed. Numerous anthoxanthins (flavone and flavonol) and a few anthocyanins were detected in the yellow inner-petal, which were all lower than those in the red outer-petal due to the low expression levels of the phenylalanine ammonialyase gene (PlPAL), flavonol synthase gene (PlFLS), dihydroflavonol 4-reductase gene (PlDFR), anthocyanidin synthase gene (PlANS), anthocyanidin 3-O-glucosyltransferase gene (Pl3GT) and anthocyanidin 5-O-glucosyltransferase gene (Pl5GT). CONCLUSION: Transcriptome sequencing (RNA-Seq) analysis based on the high throughput sequencing technology was an efficient approach to identify critical genes in P. lactiflora and other non-model plants. The flavonoid metabolic pathway and glucide metabolic pathway were identified as relatived yellow formation in P. lactiflora, PlPAL, PlFLS, PlDFR, PlANS, Pl3GT and Pl5GT were selected as potential candidates involved in flavonoid metabolic pathway, which inducing inhibition of anthocyanin biosynthesis mediated yellow formation in P. lactiflora. This study could lay a theoretical foundation for breeding new yellow P. lactiflora varieties.

Project description:Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a well-known pathogen and model organism used to study plant-pathogen interactions and subsequent plant immune responses. Numerous studies have demonstrated the effect of Pst DC3000 on Arabidopsis plants and how type III effectors are required to promote bacterial virulence and pathogenesis. F-Box Nictaba (encoded by At2g02360) is a stress-inducible lectin that is upregulated in Arabidopsis thaliana leaves after Pst DC3000 infection. In this study, a flood inoculation assay was optimized to check the performance of transgenic Arabidopsis seedlings with different expression levels of F-Box Nictaba after bacterial infection. Using a combination of multispectral and fluorescent imaging combined with molecular techniques, disease symptoms, transcript levels for F-Box Nictaba, and disease-related genes were studied in Arabidopsis leaves infected with two virulent strains: Pst DC3000 and its mutant strain, deficient in flagellin ΔfliC. Analyses of plants infected with fluorescently labeled Pst DC3000 allowed us to study the differences in bacterial colonization between plant lines. Overexpression plants showed a reduced bacterial content during the later stages of the infection. Our results show that overexpression of F-Box Nictaba resulted in reduced leaf damage after bacterial infections, whereas knockdown and knockout lines were not more susceptible to Pseudomonas infection than wild-type plants. In contrast to wild-type and knockout plants, overexpressing lines for F-Box Nictaba revealed a significant increase in anthocyanin content, better efficiency of photosystem II (Fv/Fm), and higher chlorophyll content after Pst DC3000 infection. Overexpression of F-Box Nictaba coincided with increased expression of salicylic acid (SA) related defense genes, confirming earlier data that showed that F-Box Nictaba is part of the SA-dependent defense against Pst DC3000 infection. Knockout lines yielded no discernible effects on plant symptoms after Pseudomonas infection suggesting possible gene redundancy between F-Box Nictaba genes.

Dataset Information

Overexpression of herbaceous peony miR156e-3p improves anthocyanin accumulation in transgenic Arabidopsis thaliana lateral branches.

Publications

Overexpression of herbaceous peony miR156e-3p improves anthocyanin accumulation in transgenic <i>Arabidopsis thaliana</i> lateral branches.

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