Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves.
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.
Gutha
Linga R
LR
Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA.
Casassa
Luis F
LF
Harbertson
James F
JF
Naidu
Rayapati A
RA
D000872:Anthocyanins / Q000096:biosynthesis* / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D029381:Closteroviridae; D005419:Flavonoids / Q000096:biosynthesis*; D044948:Flavonols / Q000378:metabolism; D005638:Fruit / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005960:Glucosides / Q000378:metabolism; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000821:virology; D044945:Proanthocyanidins / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation; D027843:Vitis / Q000235:genetics / Q000378:metabolism / Q000821:virology*
D000872:Anthocyanins; D005419:Flavonoids; D044948:Flavonols; D005960:Glucosides; D044945:Proanthocyanidins; C462279:cyanidin-3-O-beta-glucopyranoside; C458419:malvidin-3-glucoside; C040015:myricetin