Genome-Wide Identification of PAP1 Direct Targets in Regulating Seed Anthocyanin Biosynthesis in Arabidopsis

Abstract

Anthocyanins are widespread water-soluble pigments in the plant kingdom. Anthocyanin accumulation is activated by the MYB-bHLH-WD40 (MBW) protein complex. In Arabidopsis, the R2R3-MYB transcription factor PAP1 activates anthocyanin biosynthesis. While prior research primarily focused on seedlings, seeds received limited attention. This study explores PAP1's genome-wide target genes in anthocyanin biosynthesis in seeds. Our findings confirm that PAP1 is a positive regulator of anthocyanin biosynthesis in Arabidopsis seeds. PAP1 significantly increased anthocyanin content in developing and mature seeds in Arabidopsis. Transcriptome analysis at 12 days after pollination reveals the upregulation of numerous genes involved in anthocyanin accumulation in 35S:PAP1 developing seeds. Chromatin immunoprecipitation and dual luciferase reporter assays demonstrate PAP1's direct promotion of ten key genes and indirect upregulation of TT8, TTG1, and eight key genes during seed maturation, thus enhancing seed anthocyanin accumulation. These findings enhance our understanding of PAP1's novel role in regulating anthocyanin accumulation in Arabidopsis seeds.

Keywords: Arabidopsis; MBW complex; PAP1; anthocyanin biosynthesis; seeds.

Figure 1

Characterization of Col-0 35S:PAP1 lines: (A) Schematic diagram of the constitutive expression cassette of the PAP1 gene in the binary vector pGreen-35S-6HA used for plant transformation. RB, right border; LB, left border; NOS-pro, nopaline synthase promoter; NOS-ter, nopaline synthase terminator; Basta, glyphosate; 35S-pro, CaMV 35S promoter. (B) PCR-based DNA genotyping of Col-0 35S:PAP1 transgenic plants using specific primers for the 35S_P/PAP1_R. Cas, cassette. (C) Expression analysis of PAP1 in wild-type (Col-0) and three independent Col-0 35S:PAP1 transgenic plants using RT-qPCR. RNA samples were extracted from rosette leaves. Results were normalized against the expression of Arabidopsis EF1aA4 as the internal control. Values are means ± SD (n = 3). Asterisks (*) indicate a significant difference in gene expression in the transgenic plants of PAP1 compared with Col-0 plants (two-tailed paired Student’s t-test, p ≤ 0.05).

Figure 2

Effect of PAP1 on the accumulation of anthocyanin and PAs in seeds: (A–C) Phenotypes of wild type (Col-0) and Col-0 35S:AtPAP1 immature seeds at 10 DAP (A), 12 DAP (B), and mature seeds (C). Scale bars = 1 mm. (D) Total anthocyanin contents in mature seeds of wild-type (Col-0) and Col-0 35S:PAP1. Asterisks (*) denote the statistically significant differences between the indicated samples (Student’s t-test, p ≤ 0.05). Values are means ± SD (n = 3). DW, dry weight. (E) Total PAs contents in mature seeds of wild-type (Col-0) and Col-0 35S:PAP1. Values are means ± SD (n = 3). DW, dry weight.

Figure 3

RT-qPCR analysis of the expression of anthocyanin biosynthesis-related genes in the wild-type (Col-0) and Col-0 35S:PAP1 #5 developing seeds at 12 DAP. Results were normalized against the expression of Arabidopsis ACTIN7 as the internal control. Values are means ± SD (n = 3). ** p ≤ 0.01 and * p ≤ 0.05 indicate highly significant or significant differences in gene expression levels between wild-type (Col-0) and Col-0 35S:PAP1 #5 plants (two-tailed paired Student’s t-test).

Figure 4

PAP1 targets ADT5, CHS, F3H, DFR, ANS, 3GT, UGT79B2, UGT79B3, 5MAT, and GST26 promoters and directly promotes their expressions in developing Arabidopsis seeds. Schematic diagrams show the promoter regions of ADT5, CHS, F3H, DFR, ANS, 3GT, UGT79B2, UGT79B3, 5MAT, and GST26, while ChIP-qPCR assays show PAP1 binding to their promoter regions in the developing Arabidopsis siliques at 12 DAP. The transcriptional start site (TSS) and exons are represented by black boxes, whereas promoter regions are represented by white boxes. The triangle represents the MYB-recognizing element (MRE) site ANCNNCC and the black lines represented the DNA fragments amplified in ChIP assays for each gene. The enrichment fold of each fragment was calculated first by normalizing the amount of a target DNA fragment against a genomic fragment of Arabidopsis EF1aA4 as the internal control. Then, the value for Col-0 35S:PAP1 #5 was normalized against it for wild-type (Col-0) plants. The Arabidopsis ACTIN7 fragment was amplified as the negative control. Values are means ± SD (n = 3). Significant differences in comparison with the ACTIN7 fragment enrichment are indicated with asterisks (*) (two-tailed paired Student’s t-test, p ≤ 0.05).

Figure 5

PAP1 directly activates ADT5, CHS, F3H, DFR, ANS, 3GT, UGT79B2, UGT79B3, 5MAT, and GST26 transcription in N. benthamiana leaves: (A) Schematic diagrams show the effectors with and without PAP1 and the reporters containing ADT5, CHS, F3H, DFR, ANS, 3GT, UGT79B2, UGT79B3, 5MAT, and GST26 promoters. (B) Transient dual-luciferase reporter assay. The reporter constructs were transiently expressed in N. benthamiana leaf cells together with empty or PAP1 effector constructs. The expression level of Renilla (REN) was used as an internal control, and the LUC/REN represents the relative activity of ADT5, CHS, F3H, DFR, ANS, 3GT, UGT79B2, UGT79B3, 5MAT, and GST26 promoters. Values are means ± SD (n = 6). Double asterisks (**) and asterisk (*) separately indicate highly significant (p ≤ 0.01) and significant differences (p ≤ 0.05), respectively, in the LUC/REN compared to PAP1 effector with an empty effector (two-tailed paired Student’s t-test).

Figure 6

A simplified scheme shows that PAP1 directly regulates the expression of structural genes that control the accumulation of seed anthocyanin in Arabidopsis. ①: Shikimic pathway; ADT: arogenate dehydratase; CHS: chalcone synthase; F3H: flavanone 3-hydroxylase; DFR: dihydroflavonol-4-reducatse; ANS: anthocyanidin synthase; 3GT: flavonoid 3-O-glycosyltransferase; UGTs: UDP-glycosyltransferases; 5MAT: anthocyanidin 5-O-glucoside-6″-O-malonyltransferase; GST: glutathione S-transferase; ER: endoplasmic reticulum.