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      The MYB transcription factor PbMYB12b positively regulates flavonol biosynthesis in pear fruit

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          Abstract

          Background

          As a class of natural antioxidants in plants, fruit flavonol metabolites are beneficial to human health. However, the regulatory networks for flavonol biosynthesis in most fruits are largely unknown. Previously, we reported a spontaneous pear bud sport ‘Red Zaosu’ ( Pyrus bretschneideri Rehd.) with a high flavonoid content in its fruit. The identification of the flavonol biosynthetic regulatory network in this mutant pear fruit is crucial for elucidating the flavonol biosynthetic mechanism in fruit.

          Results

          Here, we demonstrated the PbMYB12b positively regulated flavonols biosynthesis in ‘Red Zaosu’ fruit. Initially, we investigated the accumulation patterns of four major quercetin glycosides and two major isorhamnetin glycosides in the fruit of ‘Red Zaosu’ and its wild-type ‘Zaosu’. A PRODUCTION OF FLAVONOL GLYCOSIDES (PFG)-type MYB transcription factor PbMYB12b was also screened for because of its correlation with flavonol accumulation in pear fruit. The biofunction of PbMYB12b was verified by transient overexpression and RNAi assays in pear fruit and young leaves. Overexpression of PbMYB12b enhanced the biosynthesis of quercetin glycosides and isorhamnetin glycosides by positively regulating a general flavonoids biosynthesis gene PbCHSb and a flavonol biosynthesis gene PbFLS. This finding was also supported by dual-luciferase transient expression assay and transient β-glucuronidase (GUS) reporter assay.

          Conclusions

          Our study indicated that PbMYB12b positively regulated flavonol biosynthesis, including four major quercetin glycosides and two major isorhamnetin glycosides, by promoting the expression of PbCHSb and PbFLS in pear fruit.

          Electronic supplementary material

          The online version of this article (10.1186/s12870-019-1687-0) contains supplementary material, which is available to authorized users.

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          Most cited references24

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          Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

          Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.
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            The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis.

            Comprehensive functional data on plant R2R3-MYB transcription factors is still scarce compared to the manifold of their occurrence. Here, we identified the Arabidopsis (Arabidopsis thaliana) R2R3-MYB transcription factor MYB12 as a flavonol-specific activator of flavonoid biosynthesis. Transient expression in Arabidopsis protoplasts revealed a high degree of functional similarity between MYB12 and the structurally closely related factor P from maize (Zea mays). Both displayed similar target gene specificity, and both activated target gene promoters only in the presence of a functional MYB recognition element. The genes encoding the flavonoid biosynthesis enzymes chalcone synthase, chalcone flavanone isomerase, flavanone 3-hydroxylase, and flavonol synthase were identified as target genes. Hence, our observations further add to the general notion of a close relationship between structure and function of R2R3-MYB factors. High-performance liquid chromatography analyses of myb12 mutant plants and MYB12 overexpression plants demonstrate a tight linkage between the expression level of functional MYB12 and the flavonol content of young seedlings. Quantitative real time reverse transcription-PCR using these mutant plants showed MYB12 to be a transcriptional regulator of CHALCONE SYNTHASE and FLAVONOL SYNTHASE in planta, the gene products of which are indispensable for the biosynthesis of flavonols.
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              The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed.

              In Arabidopsis, proanthocyanidins specifically accumulate in the endothelium during early seed development. At least three TRANSPARENT TESTA (TT) genes, TT2, TT8, and TTG1, are necessary for the normal expression of several flavonoid structural genes in immature seed, such as DIHYDROFLAVONOL-4-REDUCTASE and BANYULS (BAN). TT8 and TTG1 were characterized recently and found to code for a basic helix-loop-helix domain transcription factor and a WD-repeat-containing protein, respectively. Here the molecular cloning of the TT2 gene was achieved by T-DNA tagging. TT2 encoded an R2R3 MYB domain protein with high similarity to the rice OsMYB3 protein and the maize COLORLESS1 factor. A TT2-green fluorescent protein fusion protein was located mostly in the nucleus, in agreement with the regulatory function of the native TT2 protein. TT2 expression was restricted to the seed during early embryogenesis, consistent with BAN expression and the proanthocyanidin deposition profile. Finally, in gain-of-function experiments, TT2 was able to induce ectopic expression of BAN in young seedlings and roots in the presence of a functional TT8 protein. Therefore, our results strongly suggest that stringent spatial and temporal BAN expression, and thus proanthocyanidin accumulation, are determined at least partially by TT2.
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                Author and article information

                Contributors
                zhongdishaonian@sina.com
                2215039628@qq.com
                1007707251@qq.com
                y-j@nwafu.edu.cn
                lixieyu@qq.com
                594071069@qq.com
                1612386694@qq.com
                2974870893@qq.com
                cqyang@nwsuaf.edu.cn
                wzhg001@163.com
                fwm64@sina.com
                86-029-87081023 , lingfxu2013@sina.com
                Journal
                BMC Plant Biol
                BMC Plant Biol
                BMC Plant Biology
                BioMed Central (London )
                1471-2229
                21 February 2019
                21 February 2019
                2019
                : 19
                : 85
                Affiliations
                [1 ]ISNI 0000 0004 1760 4150, GRID grid.144022.1, College of Horticulture, , Northwest A&F University, ; Taicheng Road NO.3, Yangling, Shaanxi Province China
                [2 ]ISNI 0000 0004 1760 4150, GRID grid.144022.1, State Key Laboratory of Crop Stress Biology for Arid Areas, , Northwest A&F University, ; Taicheng Road NO.3, Yangling, Shaanxi Province China
                Article
                1687
                10.1186/s12870-019-1687-0
                6385385
                30791875
                6b155d23-3be4-4a0d-897f-e58d4df2ee97
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 19 November 2018
                : 18 February 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31572086
                Award ID: 31171925
                Award ID: 31401845
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2019

                Plant science & Botany
                flavonol,myb12,fruit,pear
                Plant science & Botany
                flavonol, myb12, fruit, pear

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