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. 2018 Feb;16(2):367-380.
doi: 10.1111/pbi.12777. Epub 2017 Jul 26.

Production of red-flowered oilseed rape via the ectopic expression of Orychophragmus violaceus OvPAP2

Wenqin Fu  1 , Daozong Chen  1 , Qi Pan  1 , Fengfeng Li  1 , Zhigang Zhao  2 , Xianhong Ge  1 , Zaiyun Li  1
Affiliations

Production of red-flowered oilseed rape via the ectopic expression of Orychophragmus violaceus OvPAP2

Wenqin Fu et al. Plant Biotechnol J. 2018 Feb.

Abstract

Oilseed rape (Brassica napus L.), which has yellow flowers, is both an important oil crop and a traditional tourism resource in China, whereas the Orychophragmus violaceus, which has purple flowers, likely possesses a candidate gene or genes to alter the flower colour of oilseed rape. A previously established B. napus line has a particular pair of O. violaceus chromosomes (M4) and exhibits slightly red petals. In this study, the transcriptomic analysis of M4, B. napus (H3), and O. violaceus with purple petals (OvP) and with white petals (OvW) revealed that most anthocyanin biosynthesis genes were up-regulated in both M4 and OvP. Read assembly and sequence alignment identified a homolog of AtPAP2 in M4, which produced the O. violaceus transcript (OvPAP2). The overexpression of OvPAP2 via the CaMV35S promoter in Arabidopsis thaliana led to different levels of anthocyanin accumulation in most organs, including the petals. However, the B. napus overexpression plants showed anthocyanin accumulation primarily in the anthers, but not the petals. However, when OvPAP2 was driven by the petal-specific promoter XY355, the transgenic B. napus plants produced red anthers and red petals. The results of metabolomic experiments showed that specific anthocyanins accumulated to high levels in the red petals. This study illustrates the feasibility of producing red-flowered oilseed rape, thereby enhancing its ornamental value, via the ectopic expression of the OvPAP2 gene. Moreover, the practical application of this study for insect pest management in the crop is discussed.

Keywords: Brassica napus; Orychophragmus violaceus; PAP2; anthocyanin; flower colour; transcriptome.

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Figures

Figure 1
Figure 1
Floral phenotypes of Brassica napus and Orychophragmus violaceus of different colours. (a, left to right) Flowers of Brassica napus, monosomic and disomic addition lines. (b, c) Purple and white flowers of Orychophragmus violaceus. (d) White (top) and purple (bottom) flower buds and petals of Orychophragmus violaceus.
Figure 2
Figure 2
Phenotypes of OvPAP2‐overexpressing Arabidopsis plants. (a) and (b) Flowers. (c) Pistils. (d) Leaves. (e) Inflorescences. (f) When flowering declined, the flowers of the transgenic plants showed obvious purple petals and sepals. The wild‐type is on the left, and transgenic plants are on the right in all the panels.
Figure 3
Figure 3
Phenotypes of CaMV35S::OvPAP2 Brasica napus plant and the offspring. (a) and (c) Inflorescence and young inflorescence of CaMV35S::OvPAP2 T0 plant. (b) Flowers of Westar (left) and T0 plant (right). (d) Inflorescence of the T1 plant. (e) Inflorescence of an F1 plant of a cross between the CaMV35S::OvPAP2 T0 plant and Brassica napus cv. G1300, which has white flowers.
Figure 4
Figure 4
The relative expression levels of ten ABGs in the petals (P) and anthers (A) of Brassica napus cv. Westar and P6 based on qRT‐PCR. ABGs: anthocyanin biosynthesis genes; P6: CaMV35S::OvPAP2 plant with red anthers.
Figure 5
Figure 5
Phenotypes of the XY355::OvPAP2 Brassica napus plant. (a) Inflorescence. (b) Flowers of J9709 (left) and transgenic Brassica napus (right). (c) Flower buds after removing the sepals. (d) and (e) Adaxial and abaxial sides, respectively, of petals on the flower bud in c. (c–e) From left to right: J9709, transgenic Brassica napus and M4.
Figure 6
Figure 6
Phenotypes of individual T1 XY355::OvPAP2 Brassica napus plants. (a) and (d) Adaxial and abaxial sides of the flowers, respectively. (b) and (c) Flower buds after removing the sepals. (e) and (f) Flower buds after removing the sepals and petals. (a) (left), (b), (d) (left) and (e) T1 plant without red petals and anthers. (a) (right), (c), (d) (right) and (f) T1 plant with red petals and anthers.
Figure 7
Figure 7
The relative expression levels of ten ABGs in the petals (P) of Brassica napus (J9709 and XYP3) based on qRT‐PCR. ABGs: anthocyanin biosynthesis genes; XYP3: XY355::OvPAP2 plant.
Figure 8
Figure 8
Phenotypes of F1 individuals from a cross between a XY355::OvPAP2 Brassica napus plant and Brassica napus cv. G1300, which has white flowers. (a) Inflorescence. (b) and (c) Flower buds after removing the sepals. (d) and (e) Flowers. (f) and (g) Flower buds after removing the sepals and petals. (a), (c), (e) and (g) F1 plant with red petals and anthers. (b), (d) and (f) F1 plant without red petals and anthers.
Figure 9
Figure 9
The relative expression levels of ten ABGs in the petals (P) or anthers (A) of different Brassica napus samples. P6: CaMV35S::OvPAP2 plant with red anthers; XYP3: XY355::OvPAP2 plant; P4: CaMV35S::OvPAP2 plant without a phenotype; ABGs: anthocyanin biosynthesis genes.
Figure 10
Figure 10
Principal component analysis (PCA) and orthogonal projection to latent structure‐discriminant analysis (OPLS‐DA) of petals of Brassica napus (M4, XYP3, H3 and P6). (a, b) Unsupervised PCA score plots and loading plot of four samples. (c, d) OPLS‐DA score plots and loading S‐plots of red petals (M4 and XYP3) vs. yellow petals (H3 and P6). Potential biomarkers for grouping were identified by analysing the S‐plot, which was plotted using covariance (p) and correlation (pcorr). Biomarkers indicated by molecular weight represent compounds with the most positive or negative VIP values of identified anthocyanins. XYP3: XY355::OvPAP2 plant; P6: CaMV35S::OvPAP2 plant with red anthers; VIP: variable importance in the projection.
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