A large insertion in bHLH transcription factor BrTT8 resulting in yellow seed coat in Brassica rapa

Abstract

Yellow seed is a desirable quality trait of the Brassica oilseed species. Previously, several seed coat color genes have been mapped in the Brassica species, but the molecular mechanism is still unknown. In the present investigation, map-based cloning method was used to identify a seed coat color gene, located on A9 in B. rapa. Blast analysis with the Arabidopsis genome showed that there were 22 Arabidopsis genes in this region including at4g09820 to at4g10620. Functional complementation test exhibited a phenotype reversion in the Arabidopsis thaliana tt8-1 mutant and yellow-seeded plant. These results suggested that the candidate gene was a homolog of TRANSPARENT TESTA8 (TT8) locus. BrTT8 regulated the accumulation of proanthocyanidins (PAs) in the seed coat. Sequence analysis of two alleles revealed a large insertion of a new class of transposable elements, Helitron in yellow sarson. In addition, no mRNA expression of BrTT8 was detected in the yellow-seeded line. It indicated that the natural transposon might have caused the loss in function of BrTT8. BrTT8 encodes a basic/helix-loop-helix (bHLH) protein that shares a high degree of similarity with other bHLH proteins in the Brassica. Further expression analysis also revealed that BrTT8 was involved in controlling the late biosynthetic genes (LBGs) of the flavonoid pathway. Our present findings provided with further studies could assist in understanding the molecular mechanism involved in seed coat color formation in Brassica species, which is an important oil yielding quality trait.

Figure 1. Seed coat structure of the B. rapa.

(A) The black seeds of the B. rapa. (B) The whole black seed treatment with Safranine O and Fast Green. Arrowheads show the Phenolic compounds are localised in the seed coat of immature seed (20 days after flower). (C) Magnified image of (B). Phenolic compounds stain red, formed granules are localised in the endothelium cell of the ii1 layer (circle in C). (D) The yellow seeds of the B. rapa. (E) The whole yellow seed treatment with Safranine O and Fast Green (20 days after flower). Phenolic compounds are absent in the endothelium of the yellow seed (arrowhead in E). (F) Magnified image of (E). There are only unknown fragmented structures in the region of the endothelium cell layer (circle in F). EM, embryo; EN, endosperm; ii, inner integument; Oi, outer integument. Bar(N) = 100 µm for (B) and (E); 50 µm for (C) and (F).

Figure 2. Mapping of the BrTT8 gene.

(A) The genetic linkage map of the BrTT8 gene. The markers gsr23, gsr44, and gsr29 are derived from Scaffold000135. (B) BLAST analysis with the Arabidopsis genome showed that Scaffold000135 shared similarity with a region on chromosome 4. The rectangles containing Arabidopsis genes present several representative genes (E<10⁻³⁰) in this region.

Figure 3. Differences in DNA and mRNA expression.

(A) The insertion location is shown in gray rectangles in the ORF of the BrTT8. The black rectangles represent the exons, and TL1, TL2, TR1 and YCR1 are the primers that were developed from the corresponding exon sequences and insert sequence. The arrows are used to indicate the directions of the primers. The insertion sequences and flanking BrTT8 intron 2 are shown in red and black, respectively. The conserved sequences at the termini of the element are underlined. Palindromic sequences that are capable of forming a hairpin are shown in blue. (B) The amplification products of the genomic DNA of the yellow-seeded (1) and black-seeded line (2) using the primers TL1 and TR1. (C) The primers TL2, TR1 and YCR1 amplified the genomic DNA from the three genotypes: (1) the homozygous yellow-seeded, (2) heterozygous black-seeded, (3) homozygous black-seeded plants. (D) mRNA levels in the immature seeds of the yellow-seeded line (three on the left) and black-seeded line (three on the right). The numbers 10, 20, and 30 signify the number of days after pollination. (E) 18S control.

Figure 4. BrTT8 shows features of a bHLH DNA-Binding domain protein.

(A) Amino acid comparison of the bHLH domain of BrTT8, B. napus (ABY59772.1), B. rapa (AEA03281), B. oleracea (ADP76654.1), Arabidopsis (NP_192720.2) and Populus (XP_0023067). (B) Dendrogram of the relationships among the bHLH domains from several bHLH-related proteins. For the construction of the tree, the BrTT8 protein sequence and other selected bHLH-related proteins were used. The sequence similarity was calculated using the MEGA programme to generate a branching pattern. The numbers below the branches indicate the percentages of bootstrap support after 1000 replicates. The sequences used are Brassica rapa AEA03281, Arabidopsis NP_192720.2(AtTT8), Brassica napus ABY59772.1, Brassica oleracea ADP76654.1, Populus XP_002306769.1, Vitis vinifera CBI32369.3, Lotus BAH28881.1(LjTT8), Raphanus AEO53065.1, Pisum sativum ADO13282.1, Perilla BAC56998.1(F3G1), Ricinus XP_002520758.1, Malus AEI84807.1, Petunia AAG25927.1(AN1), Nicotiana AEE99260.1, Nicotiana AEE99258.1(NtAN1b), Hordeum vulgare BAJ92594.1, Lilium BAE20058.1, Sorghum XP_002448313.1, Dahlia BAJ33515.1, Oryza NP_001053530.2, Zea mays NP_001105706.1, Cornus AAR21675.1, Cornus AAS86268.1, Oryza EEC77782.1, Mimulus ACA04013.1, and Gynura bicolor BAJ17663(GbMYC1).

Figure 5. Complementary test and PAs localization in T₂ seeds.

(A) Seeds of the TT8 wild-type genotype (left), mutant (middle) and T₂ progeny of a tt8 homozygous-transformed Arabidopsis with the BrTT8 genomic region (right). (B–D) Detection of PAs and their precursors in immature seed (heart stage) treated with vanillin HCl. The vanillin test stains the PAs and their precursors (leucoanthocyanidins and catechins) red in the endothelium of the wild type (B); the complete absence of these compounds in tt8-1 immature seed (C); the recovery of these compounds in T₂ immature seeds (D). Bar (N) = 100 µm for the (B), (C) and (D).

Figure 6. Expression of flavonoid biosynthetic genes in developing seed.

Seeds were obtained from the yellow-seeded and black-seeded plants 10 days after pollination. The expression of the different genes was detected by quantitative Real-time PCR. Transcripts for two flavonoid EBGs BrTT6 (encode F3H, flavanone 3-hydroxylase) and BrTT7 (encode F3′H, flavanone 3′-hydroxylase), three flavonoid LBGs BrDFR (encode dihydroflavonol reductase), BrBAN (encode anthocyanidin reductase) and BrLDOX (encode LDOX, leucoanthocyanidin dioxygenase) in B. rapa. The comparative Ct method was used to calculate the levels of transcripts relative to black-seeded plant. (“B” in the legend is the black seed and “Y” is the yellow seed).