Transcriptomic and Metabolomic Analysis of Anthocyanin Synthesis in Purple Leaf Brassica napus

doi:10.7668/hbnxb.20194754

Abstract

Abstract:

By screening differentially expressed genes and metabolites in the green and purple leaves of Brassica napus,it lays a theoretical foundation for the analysis of mechanism of anthocyanin synthesis.Phenotypic observation of GL and PL seedlings showed that PL exhibited a mottled distribution of purple when two true leaves were grown(3 weeks),as the purple gradually deepened during development,it gradually became lighter and eventually disappeared after the bolting stage(>16 weeks),while GL leaves remained green throughout the entire period;transcriptome analysis identified 2 523 co-differentially expressed genes at weeks 6,13,and 16,and these differentially expressed genes were significantly enriched in the anthocyanin synthesis pathway.Twenty-four genes related to anthocyanin synthesis were significantly differentially expressed,including 3 MYBL2,1 C4H,1 F3H,2 F3'H,2 TT8,3 DFR,4 ANS,5 UGT,and 3 TT19.The expression levels of these genes in PL leaves were higher than those in GL at 6 and 13 weeks.Eight differentially expressed genes were selected for qRT-PCR validation and the results were consistent with the trend of transcriptome analysis data.A total of 50 anthocyanins were detected in the anthocyanin-targeted metabolome,of which 29 showed significant difference in accumulation;compared to GL,PL had 16 types of anthocyanin accumulation upregulated and 13 types of anthocyanin accumulation downregulated.The up-regulation of TT8 and its target genes(DFR,ANS,UGT,TT19)in early development(6—13 weeks)promoted the accumulation of cyanidin-based anthocyanins and the decrease of petunian-based anthocyanins in PL.The expression levels of differentially expressed genes and metabolites in the anthocyanin synthesis pathway reached significant levels.

Key words: Brassica napus, Anthocyanins, Transcriptome, Metabolome, Leaf color

ZHOU Hongfeng, ZHU Siying, HE Dan, LIU Lili, CHEN Daozong, TAN Chen, ZHANG Dawei, YAN Mingli. Transcriptomic and Metabolomic Analysis of Anthocyanin Synthesis in Purple Leaf Brassica napus[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 25-34. doi: 10.7668/hbnxb.20194754.

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Figures/Tables 10

Tab.1 The corresponding primers of qRT-PCR

基因名称 Gene ID	正向引物序列(5'—3') Forward primer(5'—3')	反向引物序列(5'—3') Reverse primer(5'—3')
BnaC09G0215200ZS	TCTCACGATGCGACGATTCT	ATTGTCATCACTGCCCGGAA
BnaA09G0187400ZS	ATCCAGAATATAACGTGCCTT	CAGATTTCAAATGTTCCGGTA
BnaC01G0158400ZS	ATGCACATTGGAGATACGTT	TTAGCCGGAGACTCAACACT
BnaA01G0127300ZS	AGATCTCTCACTTTGGCCTA	ACTTCATTCTCTAGACGGTCA
BnaA03G0469300ZS	CCAAAAGAATACATCCGTCCA	CATAGCAGCCTTCTTGAGC
BnaC07G0445600ZS	AAGCCACAGGAAAGATCCAA	CGTGGCTTCTATGTAATCACT
BnaC09G0362400ZS	ATTCCGCTGATTGGATACCTG	CTTCCCGTCAATGATCTCTCG
BnaA06G0190700ZS	CTTGCGGTTGCAATGAACCAA	CTTCCTCACTAGAGGCGGTTTA
BnaA10G0103400ZS	TCCCCAAGAAAGCTCTAGACCA	ACGCAGAATTGTCTCTACCTC
BnaA02G0068400ZS	ACTATGCGACCAAATACTCCA	CGTCACATTCTTCGCCTAACCTG
BnaC05G0016900ZS	AGCTAGGCTTGGTGAGTCTAAGTAT	AACCCAAGCACTGACATGTGG
Actin	CTTCCTCACGCTATCCTCCG	AGCCGTCTCCAGCTCTTGC

Fig.1 Phenotypes of PL and GL in different periods and the contents of total anthocyanin in young leaves A.Comparison of PL and GL phenotypes(3 weeks);B.Comparison of PL and GL phenotypes(6 weeks);C.Accumulation of total anthocyanin contents in PL and GL young leaves.Different lowercase letters indicate significant differences(P<0.05).The same as Fig.4—5.

Tab.2 Statistics of transcriptome sequencing of Brassica napus

样本ID Sample ID	Read数量/M Number of read	碱基数量/Gb Number of base	Q30碱基数量/Gb (Q30百分比/%) Q30 bases(Q30 percentage)	Q20碱基数量/Gb (Q20百分比/%) Q20 bases(Q20 percentage)	比对率/% Mapping rate
GL_6Wk_r1	52.068	7.731	7.604(98.35)	7.349(95.06)	96.21
GL_6Wk_r2	49.644	7.391	7.269(98.35)	7.024(95.03)	96.89
GL_6Wk_r3	50.693	7.546	7.418(98.30)	7.162(94.91)	97.04
PL_6Wk_r1	49.655	7.393	7.268(98.31)	7.018(94.92)	93.55
PL_6Wk_r2	45.563	6.785	6.673(98.35)	6.449(95.04)	93.90
PL_6Wk_r3	40.911	6.082	5.948(97.81)	5.684(93.47)	92.93
GL_13Wk_r1	47.623	7.116	6.974(98.00)	6.701(94.16)	96.79
GL_13Wk_r2	51.203	7.649	7.503(98.09)	7.221(94.41)	96.87
GL_13Wk_r3	42.116	6.297	6.175(98.06)	5.939(94.32)	96.90
PL_13Wk_r1	50.268	7.514	7.369(98.07)	7.089(94.34)	92.77
PL_13Wk_r2	45.394	6.781	6.649(98.05)	6.394(94.29)	92.82
PL_13Wk_r3	42.250	6.317	6.193(98.04)	5.953(94.23)	92.72
GL_16Wk_r1	49.853	7.452	7.276(97.64)	6.950(93.26)	96.41
GL_16Wk_r2	41.980	6.280	6.129(97.59)	5.849(93.14)	96.60
GL_16Wk_r3	45.819	6.860	6.695(97.59)	6.388(93.12)	96.72
PL_16Wk_r1	48.443	7.251	7.074(97.56)	6.748(93.07)	92.80
PL_16Wk_r2	43.808	6.554	6.393(97.53)	6.094(92.97)	92.77
PL_16Wk_r3	46.205	6.904	6.745(97.68)	6.442(93.30)	92.90

Fig.2 Statistical analysis of DEGs between GL and PL at different developmental stages and enrichment analysis of co-differentially expressed genes A.The number of DEGs among GL and PL in different periods:Up-regulated expression indicates genes were up-regulated in PL compared with GL,Down-regulated expression indicates genes were Down-regulated in PL compared with GL;B.Venn diagrams of differentially expressed genes between PL and GL shared at different developmental stages:G0.DEGs at week 6,G1.DEGs at week 13,G2.DEGs at week16;C.Enrichment analysis of genes expression in pathway:1.Translation factors,2.SNARE interactions in vesicular transport,3.Sesquiterpenoid and triterpenoid biosynthesis,4.Secretion system,5.Ribosome biogenesis in eukaryotes,6.Ribosome,7.Purine metabolism,8.Photosynthesis proteins,9.Photosynthesis,10.Phenylalanine,tyrosine and tryptophan biosynthesis,11.Nucleotide excision repair,12.Mismatch repair,13.Homologous recombination,14.Exosome,15.DNA replication proteins,16.DNA replication,17.Chromosome and associated proteins,18.Chaperones and folding catalysts,19.Arginine and proline metabolism,20.Anthocyanin biosynthesis.

Fig.3 Heat map of gene expression levels of anthocyanin biosynthesis pathway related gene Fold-fold analysis of genes related to anthocyanin synthesis,Log₂ FPKM;-.log₂FC≤1,+.1<log₂FC≤log₂100,++.log₂FC>log₂100,P.Only expressed in PL,G.Only expressed in GL.

Fig.4 Verification results of qRT-PCR of differentially expressed gene UGT.BnaC09G0362400ZS;GST.BnaA02G0068400ZS;ANS1.BnaC01G0158400ZS;ANS2.BnaA01G0127300ZS;ANS3.BnaA03G0469300ZS;ANS4.BnaC07G0445600ZS;DFR1.BnaC09G0215200ZS;DFR2.BnaA09G0187400ZS.

Fig.5 Anthocyanin metabolome analysis A.Accumulation of major metabolites related to anthocyanin synthesis,different lowercase letters indicate significant differences(P<0.05);B.Heat map of anthocyanin differential metabolites(Log₂ (anthocyanin metabolites content)):1.Cyanidin-3-(6-O-p-caffeoyl)-glucoside,2.Cyanidin-3-O-sophoroside,3.Cyanidin-3-O-5-O-(6-O-coumaroyl)-diglucoside,4.Cyanidin-3,5,3'-O-triglucoside,5.Cyanidin-3-(6″-caffeylsophoroside)-5-glucoside,6.Peonidin-3,5-O-diglucoside,7.Kaempferol-3-O-rutinoside,8.Cyanidin-3-O-sambubioside-5-O-glucoside,9.Cyanidin-3-O-sambubioside,10.Delphinidin-3-O-rhamnoside,11.Delphinidin-3-O-5-O-(6-O-coumaroyl)-diglucoside,12.Malvidin-3-O-(6-O-malonyl-beta-D-glucoside),13.Pelargonidin-3-O-5-O-(6-O-coumaroyl)-diglucoside,14.Peonidin-3-(caffeoyl-glucosyl-glucoside)-5-glucoside,15.Peonidin-3-O-(6-O-malonyl-beta-D-glucoside),16.Procyanidin B1,17.Cyanidin-3-O-xyloside,18.Cyanidin-3-O-glucoside,19.Petunidin-3-O-galactoside,20.Petunidin-3-O-glucoside,21.Petunidin-3-O-sophoroside,22.Naringenin-7-O-glucoside,23.Quercetin-3-O-glucoside,24.Cyanidin-3-O-rutinoside,25.Delphinidin-3-O-(6-O-malonyl-beta-D-glucoside),26.Delphinidin-3-O-sophoroside,27.Delphinidin-3-O-glucoside,28.Petunidin-3-O-5-O-(6-O-coumaroyl)-diglucoside,29.Petunidin-3-O-(6-O-p-coumaroyl)-glucoside.

Fig.6 Analysis of differential metabolite multiples of PL relative GL

Fig.7 Differential metabolite KEGG classification

Fig.8 Joint analysis of anthocyanin biosynthesis in transcriptome and metabolome The red gene.The up-regulated expression of the gene in PL compared with GL;The black gene.There is no significant difference in the expression of PL and GL;The red ascending arrow.The up-regulated accumulation of the metabolite in PL;The blue down arrow.Down-regulated accumulation of this metabolite in PL.

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