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Identification of CaAN3 as a fruit-specific regulator of anthocyanin biosynthesis in pepper (Capsicum annuum)

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Abstract

Key Message

The novel gene CaAN3 encodes an R2R3 MYB transcription factor that regulates fruit-specific anthocyanin accumulation.

Abstract

The key regulatory gene CaAN2 encodes an R2R3 MYB transcription factor that regulates anthocyanin biosynthesis in various tissues in pepper (Capsicum annuum). However, CaAN2 is not expressed in certain pepper accessions showing fruit-specific anthocyanin accumulation. In this study, we identified the novel locus CaAN3 as a regulator of fruit-specific anthocyanin biosynthesis, using an F2 population derived from a hybrid cultivar with purple immature fruits and segregating for CaAN3. We extracted total RNA, assembled two RNA pools according to fruit color, and carried out bulked segregant RNA sequencing. We aligned the raw reads to the pepper reference genome Dempsey and identified 6,672 significant single nucleotide polymorphisms (SNPs) by calculating the Δ(SNP-index) between the two pools. We then conducted molecular mapping to delimit the target region of CaAN3 to the interval 184.6–186.4 Mbp on chromosome 10. We focused on Dem.v1.00043895, encoding an R2R3 MYB transcription factor, as the strongest candidate gene. Sequence analysis revealed four insertion/deletion polymorphisms in the promoter region of the green CaAN3 allele. We employed virus-induced gene silencing and transient overexpression assays to characterize the function of the candidate gene. When Dem.v1.00043895 was silenced in pepper, anthocyanin accumulation decreased in the pericarp, while the transient overexpression of Dem.v1.00043895 in Nicotiana benthamiana leaves resulted in the accumulation of anthocyanins around the infiltration sites. These results showed that Dem.v1.00043895 is CaAN3, an activator of anthocyanin biosynthesis in pepper fruits.

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Data availability

Transcriptome analysis of immature fruit color in Capsicum annuum was deposited into Sequence read archive (SRA) database under the accession numbers of SAMN25289192 and SAMN25289193, respectively.

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Acknowledgements

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Technology Commercialization Support Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (821012-03). This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ015881)" Rural Development Administration, Republic of Korea.

Funding

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Technology Commercialization Support Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (821012–03). This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ015881)" Rural Development Administration, Republic of Korea.

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Authors and Affiliations

  1. Department of Agriculture, Forestry, and Bioresources, Plant Genomics Breeding Institute, College of Agriculture and Life Sciences, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea

    Jinyoung Byun, Joung-Ho Lee, Soyoung Jung & Byoung-Cheorl Kang

  2. Department of Agriculture, Forestry, and Bioresources, Plant Genomics Breeding Institute, College of Agriculture and Life Sciences, Interdisciplinary Program in Agricultural GenomicsResearch Institute of Agriculture and Life SciencesSeoul National University, Seoul, South Korea

    Tae-Gun Kim

  3. Cash Crops Research Institute, Hubei Academy of Agricultural Sciences, Wuhan City, 430064, Hubei Province, China

    Ning Li

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  1. Jinyoung Byun
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Contributions

JB participated in population generation, phenotype analysis, development of markers, gene functional studies and manuscript writing. NL and TGK participated in the fine-mapping of the gene. JHL participated in the BSR-seq analysis. SYJ participated in initial experiment. BCK participated in the design of the study and editing of the manuscript.

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Correspondence to Byoung-Cheorl Kang.

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Supplementary Information

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122_2022_4106_MOESM1_ESM.jpg

Fig. S1 Transcriptome analysis using fruit-specific DEGs. a GO term enrichment analysis. GO analysis was performed for the three categories biological process, molecular function, and cellular component. b KEGG pathway enrichment analysis, using 2175 DEGs between green and purple immature fruits. The red box highlights pathways known to be related to anthocyanin biosynthesis. (JPG 126 kb)

122_2022_4106_MOESM2_ESM.jpg

Fig. S2 Expression of anthocyanin biosynthetic genes in the pericarp of green and purple pools from the F2 population derived from the hybrid Salad Piment Purple. a Core anthocyanin biosynthesis pathway in pepper. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl CoA; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavonoid 3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; 3GT, flavonoid 3-O-glucosyltransferase. b Expression levels of structural genes in the anthocyanin biosynthetic pathway. The numbers indicate gene expression levels as Log2(mean FPKM + 1), of each pool. (JPG 85 kb)

122_2022_4106_MOESM3_ESM.jpg

Fig. S3 Synteny of homologs of Dem.v1.00043895 in other reference genomes. The number in parentheses is the % identity of the annotated gene to Dem.v1.00043895 (JPG 74 kb)

122_2022_4106_MOESM4_ESM.jpg

Fig. S4 Alignment of the promoter sequences of CaAN3 of MAB1 (green), MAB2 (purple), two reference genomes (green), and two accessions (green). The pink boxes indicate deletion regions. (JPG 16633 kb)

122_2022_4106_MOESM5_ESM.jpg

Fig. S5 Physiological changes of MAB2 fruits during development. a Phenotypic changes of fruit pigmentation from 10 to 50 DPA. b Relative Dem.v1.00043895 expression levels as a function of fruit development. c Changes in concentration of delphinidins in the pericarp. Most of the anthocyanidins measured are delphinidins. d Relative Dem.v1.00043895 expression levels in different tissues. Dem.v1.00043895 showed higher expression levels immature fruit compared to leaves, stems, and flowers at 20 DPA. (JPG 108 kb)

122_2022_4106_MOESM6_ESM.jpg

Fig. S6 Transient overexpression of CaAN3 in N. benthamiana leaves. a Schematic diagram of the CaAN3 effector construct, consisting of the CaAN3 coding sequence placed under the control of the cauliflower mosaic virus 35S promoter. b Phenotypic alterations caused by CaAN3 overexpression in N. benthamiana leaves. GFP was overexpressed as a negative control. c Anthocyanin extracts and total anthocyanin contents for each extract measured by spectrophotometry. Each transiently infiltrated leaf sector with 35S:CaAN3 showed some accumulation of anthocyanins over levels detected in 35S:GFP leaves and control non-infiltrated leaves. (JPG 77 kb)

122_2022_4106_MOESM7_ESM.jpg

Fig. S7 Phenotypes of immature fruits and flowers from 24 pepper accessions. Four phenotypic patterns were observed. a–h have both purple fruits and purple pigmented flowers. i–l exhibit fruit-specific purple pigmentation. m–p display pale purple pigmentation with a yellowish background. q–x have no purple pigmentation in either fruits or flowers. a IT218962 b KC00134 c AC08-181 d IT223782 e IT286136 f IT218615 g IT301140 h IT158844 i MAB2 j Mavis k Mavras l IT305471 m IT286162 n IT158637 o IT229203 p AC09-003 q KC01237 r IT218898 s Chilbok No.2 t Jeju u IT238052 v Yuwolcho w IT238056 x MAB1. (JPG 149 kb)

122_2022_4106_MOESM8_ESM.jpg

Fig. S8 Testing the CaAN3 SCAR marker on 24 pepper accessions. Although there are different expression patterns of CaAN3 among 24 pepper lines, genotypic variation was not correlated. Except for MAB1, most other pepper lines have the identical structure as MAB2 with fruit-specific anthocyanin accumulation. IT229203 might harbor another type of promoter structure, since we failed to amplify a PCR product despite several attempts. (JPG 76 kb)

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Byun, J., Kim, TG., Lee, JH. et al. Identification of CaAN3 as a fruit-specific regulator of anthocyanin biosynthesis in pepper (Capsicum annuum). Theor Appl Genet 135, 2197–2211 (2022). https://doi.org/10.1007/s00122-022-04106-y

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