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      PpGST1, an anthocyanin‐related glutathione S‐transferase gene, is essential for fruit coloration in peach

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          Summary

          Anthocyanins have crucial biological functions and affect quality of horticultural produce. Anthocyanins accumulate in ripe peach fruit; differential accumulation is observed in deep coloured cultivar ‘Hujingmilu’ and lightly pigmented cultivar ‘Yulu’. The difference was not fully explained by accumulation of total flavonoids and expression of anthocyanin biosynthetic genes. Expression analysis was conducted on a glutathione S‐transferase gene ( PpGST1), and it was found that the expression correlated well with anthocyanin accumulation in peach fruit tissues. Functional complementation of the Arabidopsis tt19 mutant indicated that PpGST1 was responsible for transport of anthocyanins but not proanthocyanidins. PpGST1 was localized in nuclei and the tonoplast, including the sites at which anthocyanin vacuolar sequestration occurred. Transient overexpression of PpGST1 together with PpMYB10.1 in tobacco leaves and peach fruit significantly increased anthocyanin accumulation as compared with PpMYB10.1 alone. Furthermore, virus‐induced gene silencing of PpGST1 in a blood‐fleshed peach not only resulted in a reduction in anthocyanin accumulation but also a decline in expression of anthocyanin biosynthetic and regulatory genes. Cis‐element analysis of the PpGST1 promoter revealed the presence of four MYB binding sites (MBSs). Dual‐luciferase assays indicated that PpMYB10.1 bound to the promoter and activated the transcription of PpGST1 by recognizing MBS1, the one closest to the ATG start codon, with this trans‐activation being stronger against the promoter of deep coloured ‘Hujingmilu’ compared with lightly coloured cultivar ‘Yulu’. Altogether, our data provided molecular evidence supporting coordinative regulatory roles of PpGST1 and PpMYB10.1 in anthocyanin accumulation in peach.

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          Selection of reliable reference genes for gene expression studies in peach using real-time PCR

          Background RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach. Results In this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT), cyclophilin (CYP2), RNA polymerase II (RP II), phospholipase A2 (PLA2), ribosomal protein L13 (RPL13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), tubblin beta (TUB), tubblin alpha (TUA), translation elongation factor 2 (TEF2) and ubiquitin 10 (UBQ10). All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results. Conclusion Our study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls. GAPDH and ACT also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of TEF2/UBQ10/RP II and TEF2/RP II were suggested for use in all samples and subsets, respectively.
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            Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.

            Anthocyanin pigmentation is an important consumer trait in peach (Prunus persica). In this study, the genetic basis of the blood-flesh trait was investigated using the cultivar Dahongpao, which shows high levels of cyanidin-3-glucoside in the mesocarp. Elevation of anthocyanin levels in the flesh was correlated with the expression of an R2R3 MYB transcription factor, PpMYB10.1. However, PpMYB10.1 did not co-segregate with the blood-flesh trait. The blood-flesh trait was mapped to a 200-kb interval on peach linkage group (LG) 5. Within this interval, a gene encoding a NAC domain transcription factor (TF) was found to be highly up-regulated in blood-fleshed peaches when compared with non-red-fleshed peaches. This NAC TF, designated blood (BL), acts as a heterodimer with PpNAC1 which shows high levels of expression in fruit at late developmental stages. We show that the heterodimer of BL and PpNAC1 can activate the transcription of PpMYB10.1, resulting in anthocyanin pigmentation in tobacco. Furthermore, silencing the BL gene reduces anthocyanin pigmentation in blood-fleshed peaches. The transactivation activity of the BL-PpNAC1 heterodimer is repressed by a SQUAMOSA promoter-binding protein-like TF, PpSPL1. Low levels of PpMYB10.1 expression in fruit at early developmental stages is probably attributable to lower levels of expression of PpNAC1 plus the presence of high levels of repressors such as PpSPL1. We present a mechanism whereby BL is the key gene for the blood-flesh trait in peach via its activation of PpMYB10.1 in maturing fruit. Partner TFs such as basic helix-loop-helix proteins and NAC1 are required, as is the removal of transcriptional repressors.
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              Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves

              Background Botrytis cinerea Pers. Fr. is an important pathogen causing stem rot in tomatoes grown indoors for extended periods. MicroRNAs (miRNAs) have been reported as gene expression regulators related to several stress responses and B. cinerea infection in tomato. However, the function of miRNAs in the resistance to B. cinerea remains unclear. Results The miRNA expression patterns in tomato in response to B. cinerea stress were investigated by high-throughput sequencing. In total, 143 known miRNAs and seven novel miRNAs were identified and their corresponding expression was detected in mock- and B. cinerea-inoculated leaves. Among those, one novel and 57 known miRNAs were differentially expressed in B. cinerea-infected leaves, and 8 of these were further confirmed by quantitative reverse-transcription PCR (qRT-PCR). Moreover, five of these eight differentially expressed miRNAs could hit 10 coding sequences (CDSs) via CleaveLand pipeline and psRNAtarget program. In addition, qRT-PCR revealed that four targets were negatively correlated with their corresponding miRNAs (miR319, miR394, and miRn1). Conclusion Results of sRNA high-throughput sequencing revealed that the upregulation of miRNAs may be implicated in the mechanism by which tomato respond to B. cinerea stress. Analysis of the expression profiles of B. cinerea-responsive miRNAs and their targets strongly suggested that miR319, miR394, and miRn1 may be involved in the tomato leaves’ response to B. cinerea infection. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0410-4) contains supplementary material, which is available to authorized users.
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                Author and article information

                Contributors
                chjxu@zju.edu.cn
                Journal
                Plant Biotechnol J
                Plant Biotechnol. J
                10.1111/(ISSN)1467-7652
                PBI
                Plant Biotechnology Journal
                John Wiley and Sons Inc. (Hoboken )
                1467-7644
                1467-7652
                19 November 2019
                May 2020
                : 18
                : 5 ( doiID: 10.1111/pbi.v18.5 )
                : 1284-1295
                Affiliations
                [ 1 ] Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology Zhejiang University Hangzhou China
                [ 2 ] New Zealand Institute for Plant & Food Research Limited Auckland New Zealand
                [ 3 ] School of Biological Sciences University of Auckland Auckland New Zealand
                Author notes
                [*] [* ] Correspondence (Tel +86‐571‐8898‐2289; fax +86-571-8898-2224; email chjxu@ 123456zju.edu.cn )

                Author information
                https://orcid.org/0000-0001-6284-6878
                Article
                PBI13291
                10.1111/pbi.13291
                7152611
                31693790
                f8846088-24c3-4688-bf53-0ef7496e4ce2
                © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                History
                : 10 August 2019
                : 14 October 2019
                : 27 October 2019
                Page count
                Figures: 7, Tables: 0, Pages: 12, Words: 9071
                Funding
                Funded by: National Natural Science Foundation in China
                Award ID: 31572102
                Funded by: Fundamental Research Funds for the Central Universities
                Award ID: 2019FZA6010
                Funded by: 111 Project
                Award ID: B17039
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                May 2020
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.0 mode:remove_FC converted:12.04.2020

                Biotechnology
                anthocyanins,peach,transport,glutathione s‐transferase,myb10.1,promoter
                Biotechnology
                anthocyanins, peach, transport, glutathione s‐transferase, myb10.1, promoter

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