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. 2020 Mar 27;20(1):129.
doi: 10.1186/s12870-020-02344-0.

Transcriptomic and metabolomic analysis provides insights into anthocyanin and procyanidin accumulation in pear

Zhen Zhang  1   2 , Changping Tian  3 , Ya Zhang  1   2 , Chenzhiyu Li  1   2 , Xi Li  1   2 , Qiang Yu  3 , Shuo Wang  1   2 , Xinyu Wang  1   2 , Xuesen Chen  1   2 , Shouqian Feng  4   5
Affiliations

Transcriptomic and metabolomic analysis provides insights into anthocyanin and procyanidin accumulation in pear

Zhen Zhang et al. BMC Plant Biol. .

Abstract

Background: Pear is one of the most important fruit crops worldwide. Anthocyanins and procyanidins (PAs) are important secondary metabolites that affect the appearance and nutritive quality of pear. However, few studies have focused on the molecular mechanism underlying anthocyanin and PA accumulation in pear.

Results: We conducted metabolome and transcriptome analyses to identify candidate genes involved in anthocyanin and PA accumulation in young fruits of the pear cultivar 'Clapp Favorite' (CF) and its red mutation cultivar 'Red Clapp Favorite' (RCF). Gene-metabolite correlation analyses revealed a 'core set' of 20 genes that were strongly correlated with 10 anthocyanin and seven PA metabolites. Of these, PcGSTF12 was confirmed to be involved in anthocyanin and PA accumulation by complementation of the tt19-7 Arabidopsis mutant. Interestingly, PcGSTF12 was found to be responsible for the accumulation of procyanidin A3, but not petunidin 3, 5-diglucoside, opposite to the function of AtGSTs in Arabidopsis. Transformation with PcGSTF12 greatly promoted or repressed genes involved in anthocyanin and PA biosynthesis, regulation, and transport. Electrophoretic mobility shift and luciferase reporter assays confirmed positive regulation of PcGSTF12 by PcMYB114.

Conclusion: These findings identify a core set of genes for anthocyanin and PA accumulation in pear. Of these, PcGSTF12, was confirmed to be involved in anthocyanin and PA accumulation. Our results also identified an important anthocyanin and PA regulation node comprising two core genes, PcGSTF12 and PcMYB114. These results provide novel insights into anthocyanin and PA accumulation in pear and represent a valuable data set to guide future functional studies and pear breeding.

Keywords: Anthocyanin; Metabolome and transcriptome analyses; PcGSTF12; PcMYB114; Pear; Procyanidin.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Changes in pigment (anthocyanins and procyanidins; PAs) contents in ‘CF’ and ‘RCF’. a Changes in fruit pigmentation of ‘CF’ and ‘RCF’. b Concentrations of anthocyanins and PAs in peel of ‘CF’ and ‘RCF’. ‘CF1’ and ‘RCF1’ refer to fruits of ‘CF’ and ‘RCF’ collected at 2 days after full bloom (DAFB), respectively, ‘CF2’ and ‘RCF2’ refer to fruits of ‘CF’ and ‘RCF’ collected at 5 DAFB, respectively. Numbers refer to -fold change in metabolite contents
Fig. 2
Fig. 2
Number of differentially expressed genes (DEGs) identified by RNA-seq analysis. a Numbers of DEGs. b Venn diagram representing numbers of DEGs. c Number of DEGs in group 2–1 categorized into DNA binding, plant hormone signal transduction, flavonoid biosynthesis, phenylpropanoid biosynthesis, brassinosteroid biosynthesis, zeatin biosynthesis, flavonoid metabolism, phenylalanine metabolism, glutathione metabolism and ABC transporters. d Number of DEGs in group RCF-CF involved in DNA binding, plant hormone signal transduction, phenylpropanoid biosynthesis, flavonoid biosynthesis, glutathione metabolism and phenylalanine metabolism. e Overlapping DEGs between group 2–1 and group RCF-CF. f Transcript levels of overlapping DEGs between group 2–1 and RCF-CF as determined by RNA-seq
Fig. 3
Fig. 3
Connection network between core genes and anthocyanin and procyanidin (PA) metabolites. a Networks between 20 core genes and 10 anthocyanins and seven PAs. b Networks between PcGSTF12 (PCP025171) and seven anthocyanins and seven PAs. PcGSTF12 is shown in red font
Fig. 4
Fig. 4
Transcript levels of anthocyanin- and procyanidin-related genes. Each experiment had three biological replicates
Fig. 5
Fig. 5
Functional analysis of PcGSTF12 in anthocyanin and procyanidin (PA) accumulation. a Phylogenetic analyses of PcGSTF12 and its paralogs. The accession numbers are MdGST (AEN84869), Riant2 (KT312848), LcGST4 (KT946768), AN9 (Y07721), FvRAP-L1 (gene28763), VvGST4 (AAX81329), CsGST (ABA42223), AtGSTF2 (At4G02520), AtGSTF5 (At1G02940), AtGSTF3 (At2G02930), bronze-2 (AAV64226), FvRAP-L7 (gene10552), AtGSTF12/TT19 (At5G17220), AtGSTF14 (At1G49860), AtGSTF7 (At1G02920), VvGST1 (AAN85826), FvRAP (gene31672), FvRAP-L4 (gene10549), FvRAP-L2 (gene08595), FvRAP-L3 (gene22014), AtGSTF10 (At2G30870), FvRAP-L5 (gene10550), AtGSTF11 (At3G03190), AtGSTF13 (At3G62760), FvRAP-L6 (gene10551), AtGSTF8 (At2G47730), AtGSTF4 (At1G02950), AtGSTF6 (At1G02930), AtGSTF9 (At2G30860). b Phenotypes of tt19–7-OE seedlings and fresh seeds. c Heat map of anthocyanins and PAs of 7-day-old seedlings of tt19–7 mutant, tt19–7-OE, and WT (wild-type). tt19–7-OE refers to 35S::PcGSTF12 transgenic lines. Scale bars: 5 mm (B–C). Differentially accumulated metabolites between WT and tt19–7-OE marked by red star. d Correlation analyses between transcript levels of PcGSTF12 and PcMYB114. e EMSA assays. Probe was biotin-labeled fragment containing MBS motif. Competitor probe was non-labeled probe. Mutant probe contained two nucleotide mutations. Competitors, and mutant probes at a 10×, 25×, and 50× molar excess were present (+) or absent (−) in each reaction. Black arrows indicate increasing multiples. f Effects of PcMYB114 on promoter activities as demonstrated by luciferase reporter assays. Empty-vector was used as a control. Values are means ± SD of three independent biological replicates. Statistical significance: **P < 0.01
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