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Transcriptome profiling reveals differential gene expression in proanthocyanidin biosynthesis associated with red/green skin color mutant of pear (Pyrus communis L.).

Yang Y, Yao G, Yue W, Zhang S, Wu J - Front Plant Sci (2015)

Bottom Line: Tellingly, higher expression was found for genes encoding ANR and LAR in the green color mutant, promoting the proanthocyanidin (PA) pathway and leading to lower anthocyanin.In addition, qRT-PCR was used to confirm the transcriptome results for 17 DEGs, high correlation of gene expression, further proved that AP2 and WARK regulated the anthocyanin biosynthesis in red skinned "Starkrimson," and ANR and LAR promote PA biosynthesis and contribute to the green skinned variant.This study can serve as a valuable new resource laying a solid foundation for functional gene identification in the anthocyanin pathway of red-skinned pear and provide a good reference for relevant research on molecular mechanisms of color variation in other pear species.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Centre of Pear Engineering Technology Research, Nanjing Agricultural University Nanjing, China.

ABSTRACT
Anthocyanin concentration is the key determinant for red skin color in pear fruit. However, the molecular basis for development of red skin is complicated and has not been well-understood thus far. "Starkrimson" (Pyrus communis L.), an introduced red pear cultivated in the north of China and its green mutant provides a desirable red/green pair for identification of candidate genes involved in color variation. Here, we sequenced and annotated the transcriptome for the red/green color mutant at three stages of development using Illumina RNA-seq technology. The total number of mapped reads ranged from 26 to 46 million in six libraries. About 70.11-71.95% of clean reads could be mapped to the reference genome. Compared with green colored fruit, a total of 2230 differentially expressed genes (DEGs) were identified in red fruit. Gene Ontology (GO) terms were defined for 4886 differential transcripts involved in 15 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Three DEGs were identified as candidate genes in the flavonoid pathway, LAR, ANR, and C3H. Tellingly, higher expression was found for genes encoding ANR and LAR in the green color mutant, promoting the proanthocyanidin (PA) pathway and leading to lower anthocyanin. MYB-binding cis-motifs were identified in the promoter region of LAR and ANR. Based on these findings, we speculate that the regulation of PA biosynthesis might be a key factor for this red/green color mutant. Besides the known MYB and MADS transcription families, two new families, AP2 and WRKY, were identified as having high correlation with anthocyanin biosynthesis in red skinned pear. In addition, qRT-PCR was used to confirm the transcriptome results for 17 DEGs, high correlation of gene expression, further proved that AP2 and WARK regulated the anthocyanin biosynthesis in red skinned "Starkrimson," and ANR and LAR promote PA biosynthesis and contribute to the green skinned variant. This study can serve as a valuable new resource laying a solid foundation for functional gene identification in the anthocyanin pathway of red-skinned pear and provide a good reference for relevant research on molecular mechanisms of color variation in other pear species.

No MeSH data available.


Related in: MedlinePlus

The flavonoid biosynthesis pathway leading to PA production in pear fruit. PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate coenzyme A ligase; CHS, chalcone synthase, CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase; LAR, leucoanthocyanidin reductase; ANS/LDOX, anthocyanidin synthase/leucoanthocyanidin dioxygenase; ANR, anthocyanidin reductase; OMT, O-methyltransferases; UFGT, UDP-glucose: flavonoid-3-O-glucosyltransferase; RT, rhamnosyltransferase; GST, glutathione S- transferase. The blue arrow indicates a process detected between red and green skinned pear, the black arrow was clarified in other plant. In addition, the rhombus shape indicates anthocyanin accumulation, the rectangle shape proanthocyanidin biosynthesis, and the ellipse shape flavonoid biosynthesis.
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Figure 4: The flavonoid biosynthesis pathway leading to PA production in pear fruit. PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate coenzyme A ligase; CHS, chalcone synthase, CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase; LAR, leucoanthocyanidin reductase; ANS/LDOX, anthocyanidin synthase/leucoanthocyanidin dioxygenase; ANR, anthocyanidin reductase; OMT, O-methyltransferases; UFGT, UDP-glucose: flavonoid-3-O-glucosyltransferase; RT, rhamnosyltransferase; GST, glutathione S- transferase. The blue arrow indicates a process detected between red and green skinned pear, the black arrow was clarified in other plant. In addition, the rhombus shape indicates anthocyanin accumulation, the rectangle shape proanthocyanidin biosynthesis, and the ellipse shape flavonoid biosynthesis.

Mentions: Flavonoid biosynthesis is a dynamic and complex processes catalyzed by a series of enzymes. Flavonoids are a diverse group of plant secondary metabolites with various biological functions that play important roles during plant development. Proanthocyanidins (PAs, also known as condensed tannins) are components of metabolites synthesized through the general flavonoid biosynthesis pathway (Figure 4). In a previous study, it was reported that leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS; also called leucoanthocyanidin dioxygenase, LDOX), and anthocyanidin reductase (ANR; in Arabidopsis, the product of the BANYULS gene) were the three principal enzymes for flavan-3-ols biosynthesis. The synthesis of PAs and anthocyanins share common steps leading to flavan- 3,4-diols (such as leucoanthocyanidin), which can be converted to catechin (2,3-trans-flavan-3-ol) by LAR (Tanner et al., 2003) or to anthocyanidin by ANS (Abrahams et al., 2003). Anthocyanidin then either serves as the substrate for the synthesis of epicatechin (2,3-cis-flavan-3-ol) by ANR (Xie et al., 2003) or can otherwise be converted to anthocyanin by glycosylation (Schijlen et al., 2004). Recently, it was proven that transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in an Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin (Liu et al., 2013a,b). In strawberry, it was demonstrated that redirection of the anthocyanin pathway to flavan-3-ols was observed by the down-regulation of an anthocyanin glucosyltransferase in ripening strawberry fruit (Griesser et al., 2008), while the down-regulation of anthocyanin reductase (ANR) induced a redirection of the proanthocyanidin pathway, leading to premature, and ectopic anthocyanin biosynthesis via enzymatic glycosylation as the alternative pathway (Fischer et al., 2014). In our study, ANR showed relatively high expression levels in green color mutant of “Starkrimson” pear, leading to unstable anthocyanidin through ANS catalysis. This could not be converted to stable colored anthocyanin by 3,5-glycoside flavonoid transferase (UFGT), and thus anthocyanin did not accumulate in the pear fruit. Therefore, high ANR expression might be a main reason leading to the skin color change from red to green in this mutant. However, further study is needed to verify gene function and reveal the regulatory mechanism underlying the phenomenon. In addition, we detected significantly expressed p-coumarate 3-hydroxylase (C3H), which is in the flavonoid biosynthesis pathway. In a previous study, C3H was found to be a key gene playing an important role in lignin biosynthesis metabolism. Dardick et al. (2010) reported that p-coumarate 3-hydroxylase is involved in stone formation in peach fruit by microarray studies using a developmental series from young fruits. Recently, Xue et al. (2014) reported synthesis and codon-optimiztion of an Arabidopsis thaliana ref8 gene encoding a C3H for enhanced expression in Synechocystis. This heterologous pathway enabled Synechocystis to produce caffeic acid. As this is the first report of a C3H being related to flavonoid metabolism in pear, the gene function still needs further study.


Transcriptome profiling reveals differential gene expression in proanthocyanidin biosynthesis associated with red/green skin color mutant of pear (Pyrus communis L.).

Yang Y, Yao G, Yue W, Zhang S, Wu J - Front Plant Sci (2015)

The flavonoid biosynthesis pathway leading to PA production in pear fruit. PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate coenzyme A ligase; CHS, chalcone synthase, CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase; LAR, leucoanthocyanidin reductase; ANS/LDOX, anthocyanidin synthase/leucoanthocyanidin dioxygenase; ANR, anthocyanidin reductase; OMT, O-methyltransferases; UFGT, UDP-glucose: flavonoid-3-O-glucosyltransferase; RT, rhamnosyltransferase; GST, glutathione S- transferase. The blue arrow indicates a process detected between red and green skinned pear, the black arrow was clarified in other plant. In addition, the rhombus shape indicates anthocyanin accumulation, the rectangle shape proanthocyanidin biosynthesis, and the ellipse shape flavonoid biosynthesis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588701&req=5

Figure 4: The flavonoid biosynthesis pathway leading to PA production in pear fruit. PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate coenzyme A ligase; CHS, chalcone synthase, CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase; LAR, leucoanthocyanidin reductase; ANS/LDOX, anthocyanidin synthase/leucoanthocyanidin dioxygenase; ANR, anthocyanidin reductase; OMT, O-methyltransferases; UFGT, UDP-glucose: flavonoid-3-O-glucosyltransferase; RT, rhamnosyltransferase; GST, glutathione S- transferase. The blue arrow indicates a process detected between red and green skinned pear, the black arrow was clarified in other plant. In addition, the rhombus shape indicates anthocyanin accumulation, the rectangle shape proanthocyanidin biosynthesis, and the ellipse shape flavonoid biosynthesis.
Mentions: Flavonoid biosynthesis is a dynamic and complex processes catalyzed by a series of enzymes. Flavonoids are a diverse group of plant secondary metabolites with various biological functions that play important roles during plant development. Proanthocyanidins (PAs, also known as condensed tannins) are components of metabolites synthesized through the general flavonoid biosynthesis pathway (Figure 4). In a previous study, it was reported that leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS; also called leucoanthocyanidin dioxygenase, LDOX), and anthocyanidin reductase (ANR; in Arabidopsis, the product of the BANYULS gene) were the three principal enzymes for flavan-3-ols biosynthesis. The synthesis of PAs and anthocyanins share common steps leading to flavan- 3,4-diols (such as leucoanthocyanidin), which can be converted to catechin (2,3-trans-flavan-3-ol) by LAR (Tanner et al., 2003) or to anthocyanidin by ANS (Abrahams et al., 2003). Anthocyanidin then either serves as the substrate for the synthesis of epicatechin (2,3-cis-flavan-3-ol) by ANR (Xie et al., 2003) or can otherwise be converted to anthocyanin by glycosylation (Schijlen et al., 2004). Recently, it was proven that transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in an Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin (Liu et al., 2013a,b). In strawberry, it was demonstrated that redirection of the anthocyanin pathway to flavan-3-ols was observed by the down-regulation of an anthocyanin glucosyltransferase in ripening strawberry fruit (Griesser et al., 2008), while the down-regulation of anthocyanin reductase (ANR) induced a redirection of the proanthocyanidin pathway, leading to premature, and ectopic anthocyanin biosynthesis via enzymatic glycosylation as the alternative pathway (Fischer et al., 2014). In our study, ANR showed relatively high expression levels in green color mutant of “Starkrimson” pear, leading to unstable anthocyanidin through ANS catalysis. This could not be converted to stable colored anthocyanin by 3,5-glycoside flavonoid transferase (UFGT), and thus anthocyanin did not accumulate in the pear fruit. Therefore, high ANR expression might be a main reason leading to the skin color change from red to green in this mutant. However, further study is needed to verify gene function and reveal the regulatory mechanism underlying the phenomenon. In addition, we detected significantly expressed p-coumarate 3-hydroxylase (C3H), which is in the flavonoid biosynthesis pathway. In a previous study, C3H was found to be a key gene playing an important role in lignin biosynthesis metabolism. Dardick et al. (2010) reported that p-coumarate 3-hydroxylase is involved in stone formation in peach fruit by microarray studies using a developmental series from young fruits. Recently, Xue et al. (2014) reported synthesis and codon-optimiztion of an Arabidopsis thaliana ref8 gene encoding a C3H for enhanced expression in Synechocystis. This heterologous pathway enabled Synechocystis to produce caffeic acid. As this is the first report of a C3H being related to flavonoid metabolism in pear, the gene function still needs further study.

Bottom Line: Tellingly, higher expression was found for genes encoding ANR and LAR in the green color mutant, promoting the proanthocyanidin (PA) pathway and leading to lower anthocyanin.In addition, qRT-PCR was used to confirm the transcriptome results for 17 DEGs, high correlation of gene expression, further proved that AP2 and WARK regulated the anthocyanin biosynthesis in red skinned "Starkrimson," and ANR and LAR promote PA biosynthesis and contribute to the green skinned variant.This study can serve as a valuable new resource laying a solid foundation for functional gene identification in the anthocyanin pathway of red-skinned pear and provide a good reference for relevant research on molecular mechanisms of color variation in other pear species.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Centre of Pear Engineering Technology Research, Nanjing Agricultural University Nanjing, China.

ABSTRACT
Anthocyanin concentration is the key determinant for red skin color in pear fruit. However, the molecular basis for development of red skin is complicated and has not been well-understood thus far. "Starkrimson" (Pyrus communis L.), an introduced red pear cultivated in the north of China and its green mutant provides a desirable red/green pair for identification of candidate genes involved in color variation. Here, we sequenced and annotated the transcriptome for the red/green color mutant at three stages of development using Illumina RNA-seq technology. The total number of mapped reads ranged from 26 to 46 million in six libraries. About 70.11-71.95% of clean reads could be mapped to the reference genome. Compared with green colored fruit, a total of 2230 differentially expressed genes (DEGs) were identified in red fruit. Gene Ontology (GO) terms were defined for 4886 differential transcripts involved in 15 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Three DEGs were identified as candidate genes in the flavonoid pathway, LAR, ANR, and C3H. Tellingly, higher expression was found for genes encoding ANR and LAR in the green color mutant, promoting the proanthocyanidin (PA) pathway and leading to lower anthocyanin. MYB-binding cis-motifs were identified in the promoter region of LAR and ANR. Based on these findings, we speculate that the regulation of PA biosynthesis might be a key factor for this red/green color mutant. Besides the known MYB and MADS transcription families, two new families, AP2 and WRKY, were identified as having high correlation with anthocyanin biosynthesis in red skinned pear. In addition, qRT-PCR was used to confirm the transcriptome results for 17 DEGs, high correlation of gene expression, further proved that AP2 and WARK regulated the anthocyanin biosynthesis in red skinned "Starkrimson," and ANR and LAR promote PA biosynthesis and contribute to the green skinned variant. This study can serve as a valuable new resource laying a solid foundation for functional gene identification in the anthocyanin pathway of red-skinned pear and provide a good reference for relevant research on molecular mechanisms of color variation in other pear species.

No MeSH data available.


Related in: MedlinePlus