Overexpression of BraLTP2, a Lipid Transfer Protein of Brassica napus, Results in Increased Trichome Density and Altered Concentration of Secondary Metabolites

Abstract

Plant non-specific lipid transfer proteins (nsLTPs) belong to a large multigene family that possesses complex physiological functions. Trichomes are present on the aerial surfaces of most plants and include both glandular secretory hairs and non-glandular hairs. In this study, BraLTP2 was isolated from Brassica rapa (B. rapa) and its function was characterized in the important oilseed crop Brassica napus (B. napus). B. rapa lipid transfer protein 2 (BraLTP2) belongs to the little-known Y class of nsLTPs and encodes a predicted secretory protein. In Pro_BraLTP2::GUS (β-glucuronidase) transgenic plants, strong GUS activity was observed in young leaves and roots, while low activity was observed in the anther. It is noteworthy that strong GUS activity was observed in trichomes of the first four leaves of 4-week-old and 8-week-old seedings, however, it disappeared in 12-week-old seedings. In transgenic plants expressing a BraLTP2::GFP (green fluorescent protein) fusion protein, GFP fluorescence localized in the extracellular space of epidermal cells and trichomes. Overexpression of BraLTP2 in B. napus caused an increase in trichome number and altered the accumulation of secondary metabolites in leaves, including 43 upregulated secondary metabolites. Moreover, transgenic plants showed significantly increased activities of antioxidant enzymes. These results suggest that BraLTP2, a new nsLTP gene, may play a role in trichome development and the accumulation of secondary metabolites.

Keywords: BraLTP2; Brassica napus; antioxidant enzymes; overexpression; secondary metabolites; trichome development.

Figure 1

Analysis of the deduced amino acid sequences of B. rapa lipid transfer protein 2 (BraLTP2) with homologous sequences in other Cruciferae. Variable sites (dark grey), the nsLTP-like conserved 8 CM domain (light gray) with conserved cysteine residues (asterisks), and putative extracellular secretory signals (underlined) have been displayed. The sequences are from Arabidopsis thaliana AtLTP1 (AT1g52415.1), B. oleracea BolLTP1 (Bol019670), B. napus BnaLTP1 (BnaAnng31360D), and B. rapa BraLTP2 (Bra040156).

Figure 2

Histochemical β-glucuronidase (GUS) staining in tissues of Pro_BraLTP2::GUS plants. (A): Four-week-old Pro_BraLTP2::GUS seedling; (B): the first four lotus leaves from the apex of an 8-week-old Pro_BraLTP2::GUS seedling; (C): the front part of the first four lotus leaves from the apex of a 12-week-old Pro_BraLTP2::GUS seedling; (D,E,F): stem leaf and bud of a 15-week old plant during bud stage; (G,H): 18-week old Pro_BraLTP2::GUS plant during flowering stage; (I–P): negative control groups in corresponding periods; (Q,T,W): Trichome of lotus leaves of 4-week old seedling; (R,U,X): trichome of lotus leaves of 8-week old seedling; (S,V,Y): trichome of lotus leaves of 12-week old seedling; (Q,R,S,W,X,Y): the first leaves; (T,U,V): the second leaves; (Q–V): trichome of Pro_BraLTP2::GUS leaves; and (W–Y): trichome of negative controls. (A–E,G–M,O,P): Scale bars = 1 cm; (F,N): scale bars = 0.5 cm; and (I–Y): scale bars = 5 µm.

Figure 3

Localization of the fusion protein. (A–D): BraLTP2::GFP; and (E–H): negative controls. (A,C,E,G): B. napus petioles epidermis cells were transformed with construct and visualized with a fluorescence microscope; and (B,D,F,H): Trichomes on the leaf apex edge, visualized with a confocal laser scanning microscopy system. Scale bars = 50 µm.

Figure 4

Analysis of BraLTP2 mRNA levels in the wild type (WT) and BraLTP2 overexpression lines. BraLTP2 mRNA levels in 10-week-old wild type (WT) and 35S::BraLTP2 transgenic plants by real-time polymerase chain reaction (PCR). Actin was used as an internal loading control in the y-axis. Standard errors were derived from three biological repeated experiments for the expression levels of each T₀ plant. * Statistically significant difference from wild type (* p < 0.05).

Figure 5

Phenotype identification, scanning electron microscopy (SEM) observation, and the number of epidermal trichome statistics. (A): Leaf epidermal trichomes imaged by light microscopy. (B): Leaf epidermal trichomes imaged by SEM. (C): Trichome number from 1 cm² leaf area from apex edge. * Statistically significant difference from wild type (* p < 0.05).

Figure 6

Differential classes of up-regulated secondary metabolites clustering heatmap. Seven classes of up-regulated secondary metabolites between 35S::BraLTP2-3 rep 1–3 group and WT1–3 group in the clustering heatmap. The abscissa corresponds to the group number, the right ordinate corresponds to the secondary metabolites number, and the left ordinate of different color modules corresponds to the seven classes of metabolites. The color indicates abundance of changes in secondary metabolites from −1 to 1. Green corresponds to a higher correlation, the intensity of green is an indication of a higher content of secondary metabolites in the corresponding group.

Figure 7

Changes in activities of antioxidant enzymes of WT, 35S::BraLTP2-2, and 35S::BraLTP2-3 leaves. The activity of antioxidant enzymes was determined after treatment with methyl viologen (MV) for four hours. * Statistically significant difference from wild type (* p < 0.05) in each different treatment. Experiments were performed in triplicate. (A): CAT—Catalase; (B): APX—Ascorbate peroxidase; (C): GR—Glutathione reductase; (D): POD—Peroxidase; and (E): SOD—Super oxide dimutese.

Figure 8

The relationship of different metabolic pathways. Important primary metabolic precursors and intermediate secondary metabolites are shown in black font, the final secondary metabolites are shown in red font. Pathways are boxed as follows: red box, mevalonic acid pathway; purple box, shikimic acid/cinnamic acid pathway; green box, amino acid pathway; yellow box, fatty acid pathway. The red callout boxes refer to the index number of up-regulated secondary metabolites, as detailed in Table S2.