Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA.
BMC Plant Biol. 2010 Aug 23;10:187. doi: 10.1186/1471-2229-10-187.
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.
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.
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.
葡萄卷叶病（GLRD）在红色果实酿酒葡萄（Vitis vinifera L.）品种中的症状包括绿色叶脉和叶脉间区域的红色和紫红色变色。症状叶片的紫红色可能是由于花青素的积累所致，这可能反映了参与其生物合成的基因的上调。
我们检查了六个假定的组成型表达基因，泛素、肌动蛋白、GAPDH、EF1-a、SAND 和 NAD5，以确定它们作为逆转录定量实时聚合酶链反应（RT-qPCR）中基因表达归一化的参考基因的潜力。使用 geNorm 程序，确定了两个基因（肌动蛋白和 NAD5）的组合作为葡萄叶片基因表达数据归一化的稳定参考基因集。通过使用基因特异性 RT-qPCR 结合可靠的归一化因子，我们比较了感染葡萄卷叶相关病毒 3（GLRaV-3）并表现出 GLRD 症状的叶片与来自红色果实酿酒葡萄品种（品种。梅洛）的无病毒绿叶之间黄酮类生物合成途径基因的相对表达。这些不同基因的表达水平在病毒感染的叶片中增加了 2 到 50 倍。其中，CHS3、F3'5'H、F3H1、LDOX、LAR1 和 MybA1 的表达水平增加了 10 倍以上，表明它们在病毒感染的症状叶片中表达水平显著升高。从叶片中提取的花青素的 HPLC 分析表明，只有在感染病毒的症状叶片中才存在矢车菊素-3-葡萄糖苷和飞燕草素-3-葡萄糖苷。结果还表明，与无病毒的绿叶相比，感染病毒的症状叶片中的类黄酮含量高 24%，其中槲皮素其次是杨梅素是主要化合物。用蛋白质沉淀法估计的原花青素作为总单宁，与无病毒的绿叶相比，感染病毒的症状叶片中的原花青素含量高 36%。
结果是我们所知的第一个例子，表明在红色果实酿酒葡萄品种（品种。梅洛）感染 GLRaV-3 的叶片中，黄酮类生物合成途径发生了调节，导致两种类黄酮的从头合成。这些花青素导致了表现出 GLRD 症状的感染葡萄叶片的红色变。
