Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves

Abstract

Background: Symptoms of grapevine leafroll disease (GLRD) in red-fruited wine grape (Vitis vinifera L.) cultivars consist of green veins and red and reddish-purple discoloration of inter-veinal areas of leaves. The reddish-purple color of symptomatic leaves may be due to the accumulation of anthocyanins and could reflect an up-regulation of genes involved in their biosynthesis.

Results: We examined six putative constitutively expressed genes, Ubiquitin, Actin, GAPDH, EF1-a, SAND and NAD5, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using the geNorm program, a combination of two genes (Actin and NAD5) was identified as the stable set of reference genes for normalization of gene expression data obtained from grapevine leaves. By using gene-specific RT-qPCR in combination with a reliable normalization factor, we compared relative expression of the flavonoid biosynthetic pathway genes between leaves infected with Grapevine leafroll-associated virus 3 (GLRaV-3) and exhibiting GLRD symptoms and virus-free green leaves obtained from a red-fruited wine grape cultivar (cv. Merlot). The expression levels of these different genes ranged from two- to fifty-fold increase in virus-infected leaves. Among them, CHS3, F3'5'H, F3H1, LDOX, LAR1 and MybA1 showed greater than 10-fold increase suggesting that they were expressed at significantly higher levels in virus-infected symptomatic leaves. HPLC profiling of anthocyanins extracted from leaves indicated the presence of cyanidin-3-glucoside and malvidin-3-glucoside only in virus-infected symptomatic leaves. The results also showed 24% higher levels of flavonols in virus-infected symptomatic leaves than in virus-free green leaves, with quercetin followed by myricetin being the predominant compounds. Proanthocyanidins, estimated as total tannins by protein precipitation method, were 36% higher in virus-infected symptomatic leaves when compared to virus-free green leaves.

Conclusions: The results, the first example to our knowledge, showed that modulation of the flavonoid biosynthetic pathway occurred in GLRaV-3-infected leaves of a red-fruited wine grape cultivar (cv. Merlot) leading to de novo synthesis of two classes of anthocyanins. These anthocyanins have contributed to the expression of reddish-purple color of virus-infected grapevine leaves exhibiting GLRD symptoms.

Figure 1

Schematic representation of the flavonoid biosynthetic pathway. The pathway is drawn based on information from Hummer and Schreier and Boss et al. [59,81]. PAL, phenylalanine ammonia-lyase; CHS1, CHS2, and CHS3, chalcone synthase 1, 2, and 3, respectively; CHI1 and CHI2, chalcone isomerase 1 and 2, respectively; F3'H -flavonoid-3'-hydroxylase; F3'5'H - flavonoid-3', 5'-hydroxylase; F3H1 and F3H2, flavanone-3-hydroxylase 1 and 2, respectively; DFR- dihydroflavonol reductase; LDOX- leucoanthocyanidin dioxygenase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase, FLS1, flavonol synthase 1, LAR1 and LAR2, leucoanthocyanidin reductase 1 and 2, respectively; ANR, anthocyanidin reductase; MT, methyl transferase; MybA1, MYB transcription factor gene.

Figure 2

GLRD symptoms in GLRaV-3-infected red-fruited wine grape cv. Merlot. Picture on the left shows leaves from GLRaV-3-infected grapevine showing green veins and red and reddish-purple discoloration between inter-veinal areas and downward rolling of leaf margins and picture on the right shows green leaves from an adjacent virus-free grapevine.

Figure 3

Box plot representation of raw C_q values obtained from amplification curves for reference genes. Lower and upper boundaries of each box indicate the 25^th and the 75^th percentile, respectively. Ranges are represented as bars (whiskers) below and above the box and indicate the 10^th and 90^th percentiles, respectively. The horizontal line in each box represents mean and outliers by (•). SAND: SAND family protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; EF1-a: elongation factor1-alpha; Ubiquitin: ubiquitin-60S ribosomal L40 fusion protein; Actin, NAD5: NADH dehydrogenase subunit 5.

Figure 4

Stability of reference genes in grapevine leaves. Stability value (M) for a set of reference genes is analyzed with geNorm algorithm in (a) GLRaV-3-infected (designated as virus-infected), (b) virus-free and (c) combined (virus-free and virus-infected) samples. Reference genes in the x-axis are ranked from left to right based on average expression stability. The GAPDH gene in the extreme left in all graphs with the highest M value denotes lowest expression stability among the reference genes in all samples. Genes at the extreme right in each graph shows the highest expression stability among the reference genes. See legend for Figure 3 for names of reference genes.

Figure 5

Expression patterns of flavonoid biosynthetic pathway genes in GLRaV-3-infected symptomatic and virus-free green leaves. The relative expression levels of (a) the flavonoid biosynthetic pathway genes and (b) the MybA1 gene in GLRaV-3-infected (designated as virus-infected) and virus-free leaves are shown as arbitrary units on the y-axis. The raw C_q values for each gene was normalized using two reference genes (NF_{[Actin and NAD5]}). Columns represent mean value from five biological replicates, except in case of MybA1 that represents only four biological replicates and vertical bars indicate standard errors. Significant differences between virus-infected and virus-free leaves was determined by one-way ANOVA, using the SigmaPlot 11 software and indicated by asterisks (* = p < 0.05 and ** = p < 0.001). See legend for Figure 1 for names of genes.

Figure 6

Estimation of flavonoids in GLRaV-3-infected symptomatic and virus-free green leaves. Total amounts of (a) anthocyanins, (b) flavonols and (c) proanthocyanidins from GLRaV-3-infected (designated as virus-infected) and virus-free samples are shown. Columns represent mean value from five biological replicates and vertical bars indicate standard errors. NONE in (a) indicates no anthocyanins detected in virus-free leaves. Significant differences between virus-infected and virus-free leaves were determined by one-way ANOVA using the SigmaPlot 11 software and indicated by asterisks (* = p < 0.05). C.E. = catechin equivalent.

Figure 7

HPLC profiling of anthocyanins and flavonols in GLRaV-3-infected symptomatic and virus-free green leaves. The chromatograms show profile of anthocyanins from (a) virus-free and (b) GLRaV-3-infected leaves and profile of flavonols from (c) virus-free and (d) virus-infected leaves. None in (a) indicates no anthocyanins detected in virus-free, green leaves. Anthocyanins identified in (b) are: 1 = Cyanidin-3-glucoside; 2 = Malvidin-3-glucoside; 3 = Peonidin-3-O-6-coumarilated. Flavonols identified in (c) and (d) are: 1 = Myricetin-3-glucoside; 2 = Unknown; 3 = Unknown; 4 = Unknown; 5 = Quercetin-3-glucoside; 6 = Quercetin-3-glucuronide; 7 = Unknown; 8 = Unknown.