Overexpression of LAR1 Suppresses Anthocyanin Biosynthesis by Enhancing Catechin Competition Leading to Promotion of Proanthocyanidin Pathway in Spine Grape (Vitis davidii) Cells

Abstract

Proanthocyanidins (PAs) are a class of polyphenolic compounds recognized for their potent antioxidant, anti-cancer, anti-inflammatory, and cardioprotective properties. However, the production of PAs from natural sources is often limited by high costs, resource wastage, and environmental damage. In this study, we investigated the overexpression of VdLAR1, along with phenotypic observation, metabolite determination, light quality treatment, and RT-qPCR analysis, in spine grape cells. The results demonstrated a significant increase in the contents of proanthocyanidins and flavonoids in pVdLAR1-overexpressing transgenic cell lines, while anthocyanin levels showed a decreasing trend. Furthermore, the treatment with white and blue light on the T5 cell line resulted in enhanced accumulation of proanthocyanidins, catechins, and flavonoids, whereas anthocyanins and epicatechins exhibited a declining pattern. Thus, short-wavelength light promoted the accumulation of metabolites, with the proanthocyanidin content in the T5 transformed cell line reaching 2512.0 μg/g (FW) during blue light incubation. RT-qPCR analysis revealed that the key genes involved in the biosynthesis of proanthocyanidin and anthocyanin were upregulated in the transgenic spine grape cell lines, with VdLAR1 expression increasing by several hundredfold, far surpassing the expression levels of LDOX and ANR. The VdLAR1 overexpression markedly improved substrate competitiveness within the metabolic pathway, promoting catechin biosynthesis while inhibiting the production of epicatechins and anthocyanins. This finding provides compelling evidence that LAR1 is a crucial gene for catechin biosynthesis. This research establishes both theoretical and practical foundations for the regulation and development of natural proanthocyanidins, addressing issues related to high costs, safety concerns, resource wastage, and environmental damage associated with their production.

Keywords: leucoanthocyanin reductase (LAR); light quality; overexpression; proanthocyanidins; spine grape (Vitis davidii).

Figure 1

Bioinformatic analysis of VdLAR1 protein. (A) Hydrophilicity of VdLAR1. (B) Signal peptide of VdLAR1. (C) Prediction of protein secondary structure. (D) Conserved domain analysis. (E) The phylogenetic tree was generated using the maximum likelihood method in MEGA 6.0 software. The bar indicates a genetic distance of 0.05. The red dot indicates the VdLAR1 protein examined in this study. Plant species and GenBank accession numbers of their LAR1 proteins used for phylogenetic analysis are as follows: Vitis davidii (VvLAR1, XFC73052.1), Vitis vinifera (VvLAR1, NM_001280958.1), Vitis bellula (VbLAR1, JQ308626.1), Diospyros kaki (DkLAR1, AB472681.1), Actinidia eriantha (AeLARL, XM_057630487.1), Vaccinium myrtillus (VmLAR1b, MT013358.1), Camellia nitidissima (CnLAR, KR045740.1), Populus trichocarpa (PtLAR, XM_024611241.2), Cornus florida (CfLARL, XM_059799574.1), Quercus suber (QsLARL, XM_024069347.2), Pyrus communis (PcLAR1, DQ251190.1), Alnus glutinosa (AgLARL, XM_062303224.1), Paeonia delavayi (PdLAR, ON226996.1), Malus sylvestris (MsLARL, XM_050275045.1), Corylus avellana (CaLARL, XM_059598379.1), Carya illinoinensis (CiLARL, XM_043109036.1), Hevea brasiliensis (HbLAR, XM_021826359.2), Rhododendron vialii (RvLARL, XM_058336992.1), Coffea arabica (CaLARL, XM_027241678.1), Nicotiana tomentosiformis (NtLARL, XM_009616017.3), and Oryza brachyantha (ObLARL, XM_006649713.3).

Figure 2

Construction of the expression vector and identification of transformed cell lines. (A) Schematic diagram of the expression vector. (B) Subcellular localization of VdLAR1 in spine grape protoplasts. Fluorescence signals were detected in the cytoplasm of the cells expressing the pBI121-VdLAR1-EGFP fusion gene. (C) PCR amplification of VdLAR1. (D) GFP fluorescence assay of GFP, non-transgenic, and transgenic spine grape cell lines.

Figure 3

Phenotype and contents of proanthocyanidin, flavonoid, and anthocyanin in WT and transgenic cell lines. (A) Phenotype of WT and transgenic cell lines (T1, T2, T3, T4, and T5). (B) Contents of proanthocyanidin, flavonoid, and anthocyanin in WT and transgenic cell lines; different letters indicate significant differences by one-way ANOVA and Duncan’s test p < 0.05.

Figure 4

Phenotype and contents of proanthocyanidin, flavonoid, and anthocyanin in WT and T5 transgenic cell lines under different light treatments. (A) Phenotype of WT and T5 cell line. (B) Contents of proanthocyanidin, flavonoid, and anthocyanin; * for significance of p < 0.05 and ** for significance of p < 0.01. (C) Contents of catechin and epicatechin; * for significance of p < 0.05 and ** for significance of p < 0.01.

Figure 5

Relative gene expression involved in the proanthocyanidin pathway. (A) The gene expression model in the VdLAR1-transformed cell line under different light treatments; * for significance of p < 0.05 and ** for significance of p < 0.01. (B) Gene expression and metabolite accumulation related to proanthocyanidin and anthocyanin biosynthesis. Overexpression of VdLAR1 promoted catechin biosynthesis, enhanced proanthocyanidin levels, and inhibited anthocyanin production.