Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves
Identifieur interne : 000587 ( Pmc/Corpus ); précédent : 000586; suivant : 000588Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves
Auteurs : Linga R. Gutha ; Luis F. Casassa ; James F. Harbertson ; Rayapati A. NaiduSource :
- BMC Plant Biology [ 1471-2229 ] ; 2010.
Abstract
Symptoms of grapevine leafroll disease (GLRD) in red-fruited wine grape (
We examined six putative constitutively expressed genes,
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
Url:
DOI: 10.1186/1471-2229-10-187
PubMed: 20731850
PubMed Central: 2956537
Links to Exploration step
PMC:2956537Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (<italic>Vitis vinifera </italic>L.) leaves</title><author><name sortKey="Gutha, Linga R" sort="Gutha, Linga R" uniqKey="Gutha L" first="Linga R" last="Gutha">Linga R. Gutha</name><affiliation><nlm:aff id="I1">Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</nlm:aff></affiliation></author><author><name sortKey="Casassa, Luis F" sort="Casassa, Luis F" uniqKey="Casassa L" first="Luis F" last="Casassa">Luis F. Casassa</name><affiliation><nlm:aff id="I2">School of Food Science, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</nlm:aff></affiliation></author><author><name sortKey="Harbertson, James F" sort="Harbertson, James F" uniqKey="Harbertson J" first="James F" last="Harbertson">James F. Harbertson</name><affiliation><nlm:aff id="I2">School of Food Science, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</nlm:aff></affiliation></author><author><name sortKey="Naidu, Rayapati A" sort="Naidu, Rayapati A" uniqKey="Naidu R" first="Rayapati A" last="Naidu">Rayapati A. Naidu</name><affiliation><nlm:aff id="I1">Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">20731850</idno><idno type="pmc">2956537</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956537</idno><idno type="RBID">PMC:2956537</idno><idno type="doi">10.1186/1471-2229-10-187</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">000587</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000587</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (<italic>Vitis vinifera </italic>L.) leaves</title><author><name sortKey="Gutha, Linga R" sort="Gutha, Linga R" uniqKey="Gutha L" first="Linga R" last="Gutha">Linga R. Gutha</name><affiliation><nlm:aff id="I1">Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</nlm:aff></affiliation></author><author><name sortKey="Casassa, Luis F" sort="Casassa, Luis F" uniqKey="Casassa L" first="Luis F" last="Casassa">Luis F. Casassa</name><affiliation><nlm:aff id="I2">School of Food Science, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</nlm:aff></affiliation></author><author><name sortKey="Harbertson, James F" sort="Harbertson, James F" uniqKey="Harbertson J" first="James F" last="Harbertson">James F. Harbertson</name><affiliation><nlm:aff id="I2">School of Food Science, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</nlm:aff></affiliation></author><author><name sortKey="Naidu, Rayapati A" sort="Naidu, Rayapati A" uniqKey="Naidu R" first="Rayapati A" last="Naidu">Rayapati A. Naidu</name><affiliation><nlm:aff id="I1">Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</nlm:aff></affiliation></author></analytic><series><title level="j">BMC Plant Biology</title><idno type="eISSN">1471-2229</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Symptoms of grapevine leafroll disease (GLRD) in red-fruited wine grape (<italic>Vitis vinifera </italic>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.</p></sec><sec><title>Results</title><p>We examined six putative constitutively expressed genes, <italic>Ubiquitin, Actin</italic>, <italic>GAPDH</italic>, <italic>EF1-a, SAND </italic>and <italic>NAD5</italic>, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using the <italic>geNorm </italic>program, a combination of two genes (<italic>Actin </italic>and <italic>NAD5</italic>) 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 <italic>Grapevine leafroll-associated virus 3 </italic>(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, <italic>CHS3</italic>, <italic>F3'5'H</italic>, <italic>F3H1</italic>, <italic>LDOX</italic>, <italic>LAR1 </italic>and <italic>MybA1 </italic>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.</p></sec><sec><title>Conclusions</title><p>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 <italic>de novo </italic>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.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Holton, Ta" uniqKey="Holton T">TA Holton</name></author><author><name sortKey="Cornish, Ec" uniqKey="Cornish E">EC Cornish</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Winkel Shirley, B" uniqKey="Winkel Shirley B">B Winkel-Shirley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Koes, R" uniqKey="Koes R">R Koes</name></author><author><name sortKey="Verweij, W" uniqKey="Verweij W">W Verweij</name></author><author><name sortKey="Quattrocchio, F" uniqKey="Quattrocchio F">F Quattrocchio</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peters, Dj" uniqKey="Peters D">DJ Peters</name></author><author><name sortKey="Constabel, Cp" uniqKey="Constabel C">CP Constabel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gould, Ks" uniqKey="Gould K">KS Gould</name></author><author><name sortKey="Lister, C" uniqKey="Lister C">C Lister</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dixon, Ra" uniqKey="Dixon R">RA Dixon</name></author><author><name sortKey="Paiva, Nl" uniqKey="Paiva N">NL Paiva</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chalker Scott, L" uniqKey="Chalker Scott L">L Chalker-Scott</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hernandez, I" uniqKey="Hernandez I">I Hernández</name></author><author><name sortKey="Alegre, L" uniqKey="Alegre L">L Alegre</name></author><author><name sortKey="Van Breusegem, F" uniqKey="Van Breusegem F">F Van Breusegem</name></author><author><name sortKey="Munne Bosch, S" uniqKey="Munne Bosch S">S Munné-Bosch</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Korkina, Lg" uniqKey="Korkina L">LG Korkina</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ververidis, F" uniqKey="Ververidis F">F Ververidis</name></author><author><name sortKey="Trantas, E" uniqKey="Trantas E">E Trantas</name></author><author><name sortKey="Douglas, C" uniqKey="Douglas C">C Douglas</name></author><author><name sortKey="Vollmer, G" uniqKey="Vollmer G">G Vollmer</name></author><author><name sortKey="Kretzschmar, G" uniqKey="Kretzschmar G">G Kretzschmar</name></author><author><name sortKey="Panopoulos, N" uniqKey="Panopoulos N">N Panopoulos</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Crozier, A" uniqKey="Crozier A">A Crozier</name></author><author><name sortKey="Jaganath, Ib" uniqKey="Jaganath I">IB Jaganath</name></author><author><name sortKey="Clifford, Mn" uniqKey="Clifford M">MN Clifford</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Espley, Rv" uniqKey="Espley R">RV Espley</name></author><author><name sortKey="Hellens, Rp" uniqKey="Hellens R">RP Hellens</name></author><author><name sortKey="Putterill, J" uniqKey="Putterill J">J Putterill</name></author><author><name sortKey="Stevenson, De" uniqKey="Stevenson D">DE Stevenson</name></author><author><name sortKey="Kutty Amma, S" uniqKey="Kutty Amma S">S Kutty-Amma</name></author><author><name sortKey="Allan, Ac" uniqKey="Allan A">AC Allan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shirley, Bw" uniqKey="Shirley B">BW Shirley</name></author><author><name sortKey="Kubasek, Wl" uniqKey="Kubasek W">WL Kubasek</name></author><author><name sortKey="Storz, G" uniqKey="Storz G">G Storz</name></author><author><name sortKey="Bruggemann, E" uniqKey="Bruggemann E">E Bruggemann</name></author><author><name sortKey="Koornneef, M" uniqKey="Koornneef M">M Koornneef</name></author><author><name sortKey="Ausubel, Fm" uniqKey="Ausubel F">FM Ausubel</name></author><author><name sortKey="Goodman, Hm" uniqKey="Goodman H">HM Goodman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bogs, J" uniqKey="Bogs J">J Bogs</name></author><author><name sortKey="Ebadi, A" uniqKey="Ebadi A">A Ebadi</name></author><author><name sortKey="Mcdavid, D" uniqKey="Mcdavid D">D McDavid</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Castellarin, Sd" uniqKey="Castellarin S">SD Castellarin</name></author><author><name sortKey="Di Gaspero, G" uniqKey="Di Gaspero G">G Di Gaspero</name></author><author><name sortKey="Marconi, R" uniqKey="Marconi R">R Marconi</name></author><author><name sortKey="Nonis, A" uniqKey="Nonis A">A Nonis</name></author><author><name sortKey="Peterlunger, E" uniqKey="Peterlunger E">E Peterlunger</name></author><author><name sortKey="Paillard, S" uniqKey="Paillard S">S Paillard</name></author><author><name sortKey="Adam Blondon, Af" uniqKey="Adam Blondon A">AF Adam-Blondon</name></author><author><name sortKey="Testolin, R" uniqKey="Testolin R">R Testolin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Havsteen, Bh" uniqKey="Havsteen B">BH Havsteen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kennedy, Ja" uniqKey="Kennedy J">JA Kennedy</name></author><author><name sortKey="Hayasaka, Y" uniqKey="Hayasaka Y">Y Hayasaka</name></author><author><name sortKey="Vidal, S" uniqKey="Vidal S">S Vidal</name></author><author><name sortKey="Waters, Ej" uniqKey="Waters E">EJ Waters</name></author><author><name sortKey="Jones, Gp" uniqKey="Jones G">GP Jones</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Adams, Do" uniqKey="Adams D">DO Adams</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Grimplet, J" uniqKey="Grimplet J">J Grimplet</name></author><author><name sortKey="Deluc, Lg" uniqKey="Deluc L">LG Deluc</name></author><author><name sortKey="Tillett, Rl" uniqKey="Tillett R">RL Tillett</name></author><author><name sortKey="Wheatley, Md" uniqKey="Wheatley M">MD Wheatley</name></author><author><name sortKey="Schlauch, Ka" uniqKey="Schlauch K">KA Schlauch</name></author><author><name sortKey="Cramer, Gr" uniqKey="Cramer G">GR Cramer</name></author><author><name sortKey="Cushman, Jc" uniqKey="Cushman J">JC Cushman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Deluc, L" uniqKey="Deluc L">L Deluc</name></author><author><name sortKey="Bogs, J" uniqKey="Bogs J">J Bogs</name></author><author><name sortKey="Walker, Ar" uniqKey="Walker A">AR Walker</name></author><author><name sortKey="Ferrier, T" uniqKey="Ferrier T">T Ferrier</name></author><author><name sortKey="Decendit, A" uniqKey="Decendit A">A Decendit</name></author><author><name sortKey="Merillon, Jm" uniqKey="Merillon J">JM Merillon</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author><author><name sortKey="Barrieu, F" uniqKey="Barrieu F">F Barrieu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kobayashi, S" uniqKey="Kobayashi S">S Kobayashi</name></author><author><name sortKey="Goto Yamamoto, N" uniqKey="Goto Yamamoto N">N Goto-Yamamoto</name></author><author><name sortKey="Hirochika, H" uniqKey="Hirochika H">H Hirochika</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Walker, Ar" uniqKey="Walker A">AR Walker</name></author><author><name sortKey="Lee, E" uniqKey="Lee E">E Lee</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Walker, Ar" uniqKey="Walker A">AR Walker</name></author><author><name sortKey="Lee, E" uniqKey="Lee E">E Lee</name></author><author><name sortKey="Bogs, J" uniqKey="Bogs J">J Bogs</name></author><author><name sortKey="Mcdavid, Da" uniqKey="Mcdavid D">DA McDavid</name></author><author><name sortKey="Thomas, Mr" uniqKey="Thomas M">MR Thomas</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="This, P" uniqKey="This P">P This</name></author><author><name sortKey="Lacombe, T" uniqKey="Lacombe T">T Lacombe</name></author><author><name sortKey="Cadle Davidson, M" uniqKey="Cadle Davidson M">M Cadle-Davidson</name></author><author><name sortKey="Owens, Cl" uniqKey="Owens C">CL Owens</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Boss, Pk" uniqKey="Boss P">PK Boss</name></author><author><name sortKey="Davies, C" uniqKey="Davies C">C Davies</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kobayashi, S" uniqKey="Kobayashi S">S Kobayashi</name></author><author><name sortKey="Ishimaru, M" uniqKey="Ishimaru M">M Ishimaru</name></author><author><name sortKey="Ding, Ck" uniqKey="Ding C">CK Ding</name></author><author><name sortKey="Yakushiji, H" uniqKey="Yakushiji H">H Yakushiji</name></author><author><name sortKey="Goto, N" uniqKey="Goto N">N Goto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Boss, Pk" uniqKey="Boss P">PK Boss</name></author><author><name sortKey="Davies, C" uniqKey="Davies C">C Davies</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Verries, C" uniqKey="Verries C">C Verries</name></author><author><name sortKey="Guiraud, Jl" uniqKey="Guiraud J">JL Guiraud</name></author><author><name sortKey="Souquet, Jm" uniqKey="Souquet J">JM Souquet</name></author><author><name sortKey="Vialet, S" uniqKey="Vialet S">S Vialet</name></author><author><name sortKey="Terrier, N" uniqKey="Terrier N">N Terrier</name></author><author><name sortKey="Olle, D" uniqKey="Olle D">D Olle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liakopoulos, G" uniqKey="Liakopoulos G">G Liakopoulos</name></author><author><name sortKey="Nikolopoulos, D" uniqKey="Nikolopoulos D">D Nikolopoulos</name></author><author><name sortKey="Klouvatou, A" uniqKey="Klouvatou A">A Klouvatou</name></author><author><name sortKey="Vekkos, Ka" uniqKey="Vekkos K">KA Vekkos</name></author><author><name sortKey="Manetas, Y" uniqKey="Manetas Y">Y Manetas</name></author><author><name sortKey="Karabourniotis, G" uniqKey="Karabourniotis G">G Karabourniotis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rayapati, An" uniqKey="Rayapati A">AN Rayapati</name></author><author><name sortKey="O Neil, S" uniqKey="O Neil S">S O'Neil</name></author><author><name sortKey="Walsh, D" uniqKey="Walsh D">D Walsh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Martelli, Gp" uniqKey="Martelli G">GP Martelli</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jarugula, S" uniqKey="Jarugula S">S Jarugula</name></author><author><name sortKey="Gowda, S" uniqKey="Gowda S">S Gowda</name></author><author><name sortKey="Dawson, Wo" uniqKey="Dawson W">WO Dawson</name></author><author><name sortKey="Naidu, Ra" uniqKey="Naidu R">RA Naidu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cabaleiro, C" uniqKey="Cabaleiro C">C Cabaleiro</name></author><author><name sortKey="Segura, A" uniqKey="Segura A">A Segura</name></author><author><name sortKey="Garcia Berrios, Jj" uniqKey="Garcia Berrios J">JJ Garcia-berrios</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Golino, Da" uniqKey="Golino D">DA Golino</name></author><author><name sortKey="Wolpert, J" uniqKey="Wolpert J">J Wolpert</name></author><author><name sortKey="Sim, St" uniqKey="Sim S">ST Sim</name></author><author><name sortKey="Benz, J" uniqKey="Benz J">J Benz</name></author><author><name sortKey="Anderson, M" uniqKey="Anderson M">M Anderson</name></author><author><name sortKey="Rowhani, A" uniqKey="Rowhani A">A Rowhani</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Komar, V" uniqKey="Komar V">V Komar</name></author><author><name sortKey="Vigne, E" uniqKey="Vigne E">E Vigne</name></author><author><name sortKey="Demangeat, G" uniqKey="Demangeat G">G Demangeat</name></author><author><name sortKey="Lemaire, O" uniqKey="Lemaire O">O Lemaire</name></author><author><name sortKey="Fuchs, M" uniqKey="Fuchs M">M Fuchs</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huggett, J" uniqKey="Huggett J">J Huggett</name></author><author><name sortKey="Dheda, K" uniqKey="Dheda K">K Dheda</name></author><author><name sortKey="Bustin, S" uniqKey="Bustin S">S Bustin</name></author><author><name sortKey="Zumla, A" uniqKey="Zumla A">A Zumla</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nicot, N" uniqKey="Nicot N">N Nicot</name></author><author><name sortKey="Hausman, Jf" uniqKey="Hausman J">JF Hausman</name></author><author><name sortKey="Hoffmann, L" uniqKey="Hoffmann L">L Hoffmann</name></author><author><name sortKey="Evers, D" uniqKey="Evers D">D Evers</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mackay, Im" uniqKey="Mackay I">IM Mackay</name></author><author><name sortKey="Arden, Ke" uniqKey="Arden K">KE Arden</name></author><author><name sortKey="Nitsche, A" uniqKey="Nitsche A">A Nitsche</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bustin, S" uniqKey="Bustin S">S Bustin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Czechowski, T" uniqKey="Czechowski T">T Czechowski</name></author><author><name sortKey="Bari, Rp" uniqKey="Bari R">RP Bari</name></author><author><name sortKey="Stitt, M" uniqKey="Stitt M">M Stitt</name></author><author><name sortKey="Scheible, Wr" uniqKey="Scheible W">WR Scheible</name></author><author><name sortKey="Udvardi, Mk" uniqKey="Udvardi M">MK Udvardi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gachon, C" uniqKey="Gachon C">C Gachon</name></author><author><name sortKey="Mingam, A" uniqKey="Mingam A">A Mingam</name></author><author><name sortKey="Charrier, B" uniqKey="Charrier B">B Charrier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bustin, Sa" uniqKey="Bustin S">SA Bustin</name></author><author><name sortKey="Benes, V" uniqKey="Benes V">V Benes</name></author><author><name sortKey="Nolan, T" uniqKey="Nolan T">T Nolan</name></author><author><name sortKey="Pfaffl, Mw" uniqKey="Pfaffl M">MW Pfaffl</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vandesompele, J" uniqKey="Vandesompele J">J Vandesompele</name></author><author><name sortKey="De Preter, K" uniqKey="De Preter K">K De Preter</name></author><author><name sortKey="Pattyn, F" uniqKey="Pattyn F">F Pattyn</name></author><author><name sortKey="Poppe, B" uniqKey="Poppe B">B Poppe</name></author><author><name sortKey="Van Roy, N" uniqKey="Van Roy N">N Van Roy</name></author><author><name sortKey="De Paepe, A" uniqKey="De Paepe A">A De Paepe</name></author><author><name sortKey="Speleman, F" uniqKey="Speleman F">F Speleman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rieu, I" uniqKey="Rieu I">I Rieu</name></author><author><name sortKey="Powers, Sj" uniqKey="Powers S">SJ Powers</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Radonic, A" uniqKey="Radonic A">A Radonic</name></author><author><name sortKey="Thulke, S" uniqKey="Thulke S">S Thulke</name></author><author><name sortKey="Mackay, Im" uniqKey="Mackay I">IM Mackay</name></author><author><name sortKey="Landt, O" uniqKey="Landt O">O Landt</name></author><author><name sortKey="Siegert, W" uniqKey="Siegert W">W Siegert</name></author><author><name sortKey="Nitsche, A" uniqKey="Nitsche A">A Nitsche</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Remans, T" uniqKey="Remans T">T Remans</name></author><author><name sortKey="Smeets, K" uniqKey="Smeets K">K Smeets</name></author><author><name sortKey="Opdenakker, K" uniqKey="Opdenakker K">K Opdenakker</name></author><author><name sortKey="Mathijsen, D" uniqKey="Mathijsen D">D Mathijsen</name></author><author><name sortKey="Vangronsveld, J" uniqKey="Vangronsveld J">J Vangronsveld</name></author><author><name sortKey="Cuypers, A" uniqKey="Cuypers A">A Cuypers</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gutierrez, L" uniqKey="Gutierrez L">L Gutierrez</name></author><author><name sortKey="Mauriat, M" uniqKey="Mauriat M">M Mauriat</name></author><author><name sortKey="Guenin, S" uniqKey="Guenin S">S Guenin</name></author><author><name sortKey="Pelloux, J" uniqKey="Pelloux J">J Pelloux</name></author><author><name sortKey="Lefebvre, Jf" uniqKey="Lefebvre J">JF Lefebvre</name></author><author><name sortKey="Louvet, R" uniqKey="Louvet R">R Louvet</name></author><author><name sortKey="Rusterucci, C" uniqKey="Rusterucci C">C Rusterucci</name></author><author><name sortKey="Moritz, T" uniqKey="Moritz T">T Moritz</name></author><author><name sortKey="Guerineau, F" uniqKey="Guerineau F">F Guerineau</name></author><author><name sortKey="Bellini, C" uniqKey="Bellini C">C Bellini</name></author><author><name sortKey="Van Wuytswinkel" uniqKey="Van Wuytswinkel">Van Wuytswinkel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gutierrez, L" uniqKey="Gutierrez L">L Gutierrez</name></author><author><name sortKey="Mauriat, M" uniqKey="Mauriat M">M Mauriat</name></author><author><name sortKey="Pelloux, J" uniqKey="Pelloux J">J Pelloux</name></author><author><name sortKey="Bellini, C" uniqKey="Bellini C">C Bellini</name></author><author><name sortKey="Van Wuytswinkel, O" uniqKey="Van Wuytswinkel O">O Van Wuytswinkel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bustin, Sa" uniqKey="Bustin S">SA Bustin</name></author><author><name sortKey="Benes, V" uniqKey="Benes V">V Benes</name></author><author><name sortKey="Garson, Ja" uniqKey="Garson J">JA Garson</name></author><author><name sortKey="Hellemans, J" uniqKey="Hellemans J">J Hellemans</name></author><author><name sortKey="Huggett, J" uniqKey="Huggett J">J Huggett</name></author><author><name sortKey="Kubista, M" uniqKey="Kubista M">M Kubista</name></author><author><name sortKey="Mueller, R" uniqKey="Mueller R">R Mueller</name></author><author><name sortKey="Nolan, T" uniqKey="Nolan T">T Nolan</name></author><author><name sortKey="Pfaffl, Mw" uniqKey="Pfaffl M">MW Pfaffl</name></author><author><name sortKey="Shipley, Gl" uniqKey="Shipley G">GL Shipley</name></author><author><name sortKey="Vandesompele, J" uniqKey="Vandesompele J">J Vandesompele</name></author><author><name sortKey="Wittwer, Ct" uniqKey="Wittwer C">CT Wittwer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hu, R" uniqKey="Hu R">R Hu</name></author><author><name sortKey="Fan, C" uniqKey="Fan C">C Fan</name></author><author><name sortKey="Li, H" uniqKey="Li H">H Li</name></author><author><name sortKey="Zhang, Q" uniqKey="Zhang Q">Q Zhang</name></author><author><name sortKey="Fu, Yf" uniqKey="Fu Y">YF Fu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Maule, A" uniqKey="Maule A">A Maule</name></author><author><name sortKey="Leh, V" uniqKey="Leh V">V Leh</name></author><author><name sortKey="Lederer, C" uniqKey="Lederer C">C Lederer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Culver, Jn" uniqKey="Culver J">JN Culver</name></author><author><name sortKey="Padmanabhan, Ms" uniqKey="Padmanabhan M">MS Padmanabhan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dunoyer, P" uniqKey="Dunoyer P">P Dunoyer</name></author><author><name sortKey="Voinnet, O" uniqKey="Voinnet O">O Voinnet</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Whitham, Sa" uniqKey="Whitham S">SA Whitham</name></author><author><name sortKey="Yang, C" uniqKey="Yang C">C Yang</name></author><author><name sortKey="Goodin, Mm" uniqKey="Goodin M">MM Goodin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lovdal, T" uniqKey="Lovdal T">T Lovdal</name></author><author><name sortKey="Lillo, C" uniqKey="Lillo C">C Lillo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Reid, Ke" uniqKey="Reid K">KE Reid</name></author><author><name sortKey="Olsson, N" uniqKey="Olsson N">N Olsson</name></author><author><name sortKey="Schlosser, J" uniqKey="Schlosser J">J Schlosser</name></author><author><name sortKey="Peng, F" uniqKey="Peng F">F Peng</name></author><author><name sortKey="Lund, St" uniqKey="Lund S">ST Lund</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kobayashi, H" uniqKey="Kobayashi H">H Kobayashi</name></author><author><name sortKey="Suzuki, S" uniqKey="Suzuki S">S Suzuki</name></author><author><name sortKey="Tanzawa, F" uniqKey="Tanzawa F">F Tanzawa</name></author><author><name sortKey="Takayanagi, T" uniqKey="Takayanagi T">T Takayanagi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bogs, J" uniqKey="Bogs J">J Bogs</name></author><author><name sortKey="Downey, Mo" uniqKey="Downey M">MO Downey</name></author><author><name sortKey="Harvey, Js" uniqKey="Harvey J">JS Harvey</name></author><author><name sortKey="Ashton, Ar" uniqKey="Ashton A">AR Ashton</name></author><author><name sortKey="Tanner, Gj" uniqKey="Tanner G">GJ Tanner</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hummer, W" uniqKey="Hummer W">W Hummer</name></author><author><name sortKey="Schreier, P" uniqKey="Schreier P">P Schreier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Treutter, D" uniqKey="Treutter D">D Treutter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goto Yamamoto, N" uniqKey="Goto Yamamoto N">N Goto-Yamamoto</name></author><author><name sortKey="Wan, Gh" uniqKey="Wan G">GH Wan</name></author><author><name sortKey="Masaki, K" uniqKey="Masaki K">K Masaki</name></author><author><name sortKey="Kobayashi, S" uniqKey="Kobayashi S">S Kobayashi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ageorges, A" uniqKey="Ageorges A">A Ageorges</name></author><author><name sortKey="Fernandez, L" uniqKey="Fernandez L">L Fernandez</name></author><author><name sortKey="Vialet, S" uniqKey="Vialet S">S Vialet</name></author><author><name sortKey="Merdinoglu, D" uniqKey="Merdinoglu D">D Merdinoglu</name></author><author><name sortKey="Terrier, N" uniqKey="Terrier N">N Terrier</name></author><author><name sortKey="Romieu, C" uniqKey="Romieu C">C Romieu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Griesbach, Rj" uniqKey="Griesbach R">RJ Griesbach</name></author><author><name sortKey="Beck, Rm" uniqKey="Beck R">RM Beck</name></author><author><name sortKey="Hammond, J" uniqKey="Hammond J">J Hammond</name></author><author><name sortKey="Stommel, Jr" uniqKey="Stommel J">JR Stommel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Teycheney, Py" uniqKey="Teycheney P">PY Teycheney</name></author><author><name sortKey="Tepfer, M" uniqKey="Tepfer M">M Tepfer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Senda, M" uniqKey="Senda M">M Senda</name></author><author><name sortKey="Masuta, C" uniqKey="Masuta C">C Masuta</name></author><author><name sortKey="Ohnishi, S" uniqKey="Ohnishi S">S Ohnishi</name></author><author><name sortKey="Goto, K" uniqKey="Goto K">K Goto</name></author><author><name sortKey="Kasai, A" uniqKey="Kasai A">A Kasai</name></author><author><name sortKey="Sano, T" uniqKey="Sano T">T Sano</name></author><author><name sortKey="Hong, Js" uniqKey="Hong J">JS Hong</name></author><author><name sortKey="Macfarlane, S" uniqKey="Macfarlane S">S MacFarlane</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Koseki, M" uniqKey="Koseki M">M Koseki</name></author><author><name sortKey="Goto, K" uniqKey="Goto K">K Goto</name></author><author><name sortKey="Masuta, C" uniqKey="Masuta C">C Masuta</name></author><author><name sortKey="Kanazawa, A" uniqKey="Kanazawa A">A Kanazawa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kortekamp, A" uniqKey="Kortekamp A">A Kortekamp</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hren, M" uniqKey="Hren M">M Hren</name></author><author><name sortKey="Nikolic, P" uniqKey="Nikolic P">P Nikolic</name></author><author><name sortKey="Rotter, A" uniqKey="Rotter A">A Rotter</name></author><author><name sortKey="Blejec, A" uniqKey="Blejec A">A Blejec</name></author><author><name sortKey="Terrier, N" uniqKey="Terrier N">N Terrier</name></author><author><name sortKey="Ravnikar, M" uniqKey="Ravnikar M">M Ravnikar</name></author><author><name sortKey="Dermastia, M" uniqKey="Dermastia M">M Dermastia</name></author><author><name sortKey="Gruden, K" uniqKey="Gruden K">K Gruden</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rotter, A" uniqKey="Rotter A">A Rotter</name></author><author><name sortKey="Camps, C" uniqKey="Camps C">C Camps</name></author><author><name sortKey="Lohse, M" uniqKey="Lohse M">M Lohse</name></author><author><name sortKey="Kappel, C" uniqKey="Kappel C">C Kappel</name></author><author><name sortKey="Pilati, S" uniqKey="Pilati S">S Pilati</name></author><author><name sortKey="Hren, M" uniqKey="Hren M">M Hren</name></author><author><name sortKey="Stitt, M" uniqKey="Stitt M">M Stitt</name></author><author><name sortKey="Coutos Thevenot, P" uniqKey="Coutos Thevenot P">P Coutos-Thevenot</name></author><author><name sortKey="Moser, C" uniqKey="Moser C">C Moser</name></author><author><name sortKey="Usadel, B" uniqKey="Usadel B">B Usadel</name></author><author><name sortKey="Delrot, S" uniqKey="Delrot S">S Delrot</name></author><author><name sortKey="Gruden, K" uniqKey="Gruden K">K Gruden</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Miranda, M" uniqKey="Miranda M">M Miranda</name></author><author><name sortKey="Ralph, Sg" uniqKey="Ralph S">SG Ralph</name></author><author><name sortKey="Mellway, R" uniqKey="Mellway R">R Mellway</name></author><author><name sortKey="White, R" uniqKey="White R">R White</name></author><author><name sortKey="Heath, Mc" uniqKey="Heath M">MC Heath</name></author><author><name sortKey="Bohlmann, J" uniqKey="Bohlmann J">J Bohlmann</name></author><author><name sortKey="Constabel, Cp" uniqKey="Constabel C">CP Constabel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kobayashi, S" uniqKey="Kobayashi S">S Kobayashi</name></author><author><name sortKey="Ishimaru, M" uniqKey="Ishimaru M">M Ishimaru</name></author><author><name sortKey="Hiraoka, K" uniqKey="Hiraoka K">K Hiraoka</name></author><author><name sortKey="Honda, C" uniqKey="Honda C">C Honda</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ramsay, Na" uniqKey="Ramsay N">NA Ramsay</name></author><author><name sortKey="Glover, Bj" uniqKey="Glover B">BJ Glover</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lovdal, T" uniqKey="Lovdal T">T Lovdal</name></author><author><name sortKey="Olsen, Km" uniqKey="Olsen K">KM Olsen</name></author><author><name sortKey="Slimestad, R" uniqKey="Slimestad R">R Slimestad</name></author><author><name sortKey="Verheul, M" uniqKey="Verheul M">M Verheul</name></author><author><name sortKey="Lillo, C" uniqKey="Lillo C">C Lillo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Field, Ts" uniqKey="Field T">TS Field</name></author><author><name sortKey="Lee, Dw" uniqKey="Lee D">DW Lee</name></author><author><name sortKey="Holbrook, Nm" uniqKey="Holbrook N">NM Holbrook</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mekuria, Ta" uniqKey="Mekuria T">TA Mekuria</name></author><author><name sortKey="Soule, Mj" uniqKey="Soule M">MJ Soule</name></author><author><name sortKey="Jarugula, S" uniqKey="Jarugula S">S Jarugula</name></author><author><name sortKey="Naidu, Ra" uniqKey="Naidu R">RA Naidu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Porra, Rj" uniqKey="Porra R">RJ Porra</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lichtenthaler, Hk" uniqKey="Lichtenthaler H">HK Lichtenthaler</name></author><author><name sortKey="Welburn" uniqKey="Welburn">Welburn</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lefever, S" uniqKey="Lefever S">S Lefever</name></author><author><name sortKey="Hellemans, J" uniqKey="Hellemans J">J Hellemans</name></author><author><name sortKey="Pattyn, F" uniqKey="Pattyn F">F Pattyn</name></author><author><name sortKey="Przybylski, Dr" uniqKey="Przybylski D">DR Przybylski</name></author><author><name sortKey="Taylor, C" uniqKey="Taylor C">C Taylor</name></author><author><name sortKey="Geurts, R" uniqKey="Geurts R">R Geurts</name></author><author><name sortKey="Untergasser, A" uniqKey="Untergasser A">A Untergasser</name></author><author><name sortKey="Vandesompele, J" uniqKey="Vandesompele J">J Vandesompele</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Downey, Mo" uniqKey="Downey M">MO Downey</name></author><author><name sortKey="Rochfort, S" uniqKey="Rochfort S">S Rochfort</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Harbertson, Jf" uniqKey="Harbertson J">JF Harbertson</name></author><author><name sortKey="Kennedy, Ja" uniqKey="Kennedy J">JA Kennedy</name></author><author><name sortKey="Adams, Do" uniqKey="Adams D">DO Adams</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Boss, Pk" uniqKey="Boss P">PK Boss</name></author><author><name sortKey="Davies, C" uniqKey="Davies C">C Davies</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fujita, A" uniqKey="Fujita A">A Fujita</name></author><author><name sortKey="Soma, N" uniqKey="Soma N">N Soma</name></author><author><name sortKey="Goto Yamamoto, N" uniqKey="Goto Yamamoto N">N Goto-Yamamoto</name></author><author><name sortKey="Mizuno, A" uniqKey="Mizuno A">A Mizuno</name></author><author><name sortKey="Kiso, K" uniqKey="Kiso K">K Kiso</name></author><author><name sortKey="Hashizume, K" uniqKey="Hashizume K">K Hashizume</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Menzel, W" uniqKey="Menzel W">W Menzel</name></author><author><name sortKey="Jelkmann, W" uniqKey="Jelkmann W">W Jelkmann</name></author><author><name sortKey="Maiss, E" uniqKey="Maiss E">E Maiss</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jeong, St" uniqKey="Jeong S">ST Jeong</name></author><author><name sortKey="Goto Yamamoto, N" uniqKey="Goto Yamamoto N">N Goto-Yamamoto</name></author><author><name sortKey="Kobayashi, S" uniqKey="Kobayashi S">S Kobayashi</name></author><author><name sortKey="Esaka, M" uniqKey="Esaka M">M Esaka</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Downey, Mo" uniqKey="Downey M">MO Downey</name></author><author><name sortKey="Harvey, Js" uniqKey="Harvey J">JS Harvey</name></author><author><name sortKey="Robinson, Sp" uniqKey="Robinson S">SP Robinson</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">BMC Plant Biol</journal-id><journal-title-group><journal-title>BMC Plant Biology</journal-title></journal-title-group><issn pub-type="epub">1471-2229</issn><publisher><publisher-name>BioMed Central</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">20731850</article-id><article-id pub-id-type="pmc">2956537</article-id><article-id pub-id-type="publisher-id">1471-2229-10-187</article-id><article-id pub-id-type="doi">10.1186/1471-2229-10-187</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (<italic>Vitis vinifera </italic>L.) leaves</article-title></title-group><contrib-group><contrib contrib-type="author" id="A1"><name><surname>Gutha</surname><given-names>Linga R</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>glreddy@wsu.edu</email></contrib><contrib contrib-type="author" id="A2"><name><surname>Casassa</surname><given-names>Luis F</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>luis.casassa@email.wsu.edu</email></contrib><contrib contrib-type="author" id="A3"><name><surname>Harbertson</surname><given-names>James F</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>jfharbertson@wsu.edu</email></contrib><contrib contrib-type="author" corresp="yes" id="A4"><name><surname>Naidu</surname><given-names>Rayapati A</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>naidu@wsu.edu</email></contrib></contrib-group><aff id="I1"><label>1</label>Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</aff><aff id="I2"><label>2</label>School of Food Science, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA</aff><pub-date pub-type="collection"><year>2010</year></pub-date><pub-date pub-type="epub"><day>23</day><month>8</month><year>2010</year></pub-date><volume>10</volume><fpage>187</fpage><lpage>187</lpage><history><date date-type="received"><day>9</day><month>3</month><year>2010</year></date><date date-type="accepted"><day>23</day><month>8</month><year>2010</year></date></history><permissions><copyright-statement>Copyright ©2010 Gutha et al; licensee BioMed Central Ltd.</copyright-statement><copyright-year>2010</copyright-year><copyright-holder>Gutha et al; licensee BioMed Central Ltd.</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0">http://creativecommons.org/licenses/by/2.0</ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href="http://www.biomedcentral.com/1471-2229/10/187"></self-uri><abstract><sec><title>Background</title><p>Symptoms of grapevine leafroll disease (GLRD) in red-fruited wine grape (<italic>Vitis vinifera </italic>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.</p></sec><sec><title>Results</title><p>We examined six putative constitutively expressed genes, <italic>Ubiquitin, Actin</italic>, <italic>GAPDH</italic>, <italic>EF1-a, SAND </italic>and <italic>NAD5</italic>, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using the <italic>geNorm </italic>program, a combination of two genes (<italic>Actin </italic>and <italic>NAD5</italic>) 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 <italic>Grapevine leafroll-associated virus 3 </italic>(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, <italic>CHS3</italic>, <italic>F3'5'H</italic>, <italic>F3H1</italic>, <italic>LDOX</italic>, <italic>LAR1 </italic>and <italic>MybA1 </italic>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.</p></sec><sec><title>Conclusions</title><p>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 <italic>de novo </italic>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.</p></sec></abstract></article-meta></front><body><sec><title>Background</title><p>In plants, three major classes of flavonoids (anthocyanins, proanthocyanidins and flavonols) are synthesized via the branched flavonoid biosynthetic pathway [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>]. These secondary metabolites contribute to the 'colorful' pigmentation of flowers, fruits, seeds and leaves and are involved in several physiological and biochemical processes in plants such as UV protection, insect attraction, herbivore defense and symbiosis [<xref ref-type="bibr" rid="B3">3</xref>-<xref ref-type="bibr" rid="B5">5</xref>]. Plants also utilize various colors conferred by anthocyanins to recruit pollinators and attract animals to disperse seeds [<xref ref-type="bibr" rid="B2">2</xref>]. The flavonoids are often produced in vegetative tissues as well under stress conditions, such as high light intensity, cold temperature, nutrient deficiency and pathogen attack or senescence [<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B8">8</xref>]. Due to a multitude of biological and agricultural importance and favorable health benefits, the genetics and biochemistry of the flavonoid biosynthetic pathway has been intensively studied in several plant species [<xref ref-type="bibr" rid="B9">9</xref>-<xref ref-type="bibr" rid="B11">11</xref>]. These studies indicated that flavonoid composition among plant species and even different tissues of a plant can be remarkably different [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B12">12</xref>-<xref ref-type="bibr" rid="B15">15</xref>]. Further details about the flavonoid biosynthetic pathway are available in many publications [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B5">5</xref>]. A generalized scheme of the pathway is shown in Figure <xref ref-type="fig" rid="F1">1</xref>.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>Schematic representation of the flavonoid biosynthetic pathway</bold>. The pathway is drawn based on information from Hummer and Schreier and Boss <italic>et al</italic>. [<xref ref-type="bibr" rid="B59">59</xref>,<xref ref-type="bibr" rid="B81">81</xref>]. <italic>PAL</italic>, phenylalanine ammonia-lyase; <italic>CHS1, CHS2</italic>, and <italic>CHS3</italic>, chalcone synthase 1, 2, and 3, respectively; <italic>CHI1 </italic>and <italic>CHI2</italic>, chalcone isomerase 1 and 2, respectively; <italic>F3'H </italic>-flavonoid-3'-hydroxylase; <italic>F3'5'H </italic>- flavonoid-3', 5'-hydroxylase; <italic>F3H1 </italic>and <italic>F3H2</italic>, flavanone-3-hydroxylase 1 and 2, respectively; <italic>DFR</italic>- dihydroflavonol reductase; <italic>LDOX</italic>- leucoanthocyanidin dioxygenase; <italic>UFGT</italic>, UDP-glucose:flavonoid 3-<italic>O</italic>-glucosyltransferase, <italic>FLS1</italic>, flavonol synthase 1, <italic>LAR1 </italic>and <italic>LAR2</italic>, leucoanthocyanidin reductase 1 and 2, respectively; <italic>ANR</italic>, anthocyanidin reductase; <italic>MT</italic>, methyl transferase; <italic>MybA1</italic>, MYB transcription factor gene.</p></caption><graphic xlink:href="1471-2229-10-187-1"></graphic></fig><p>For many years, berries of the grapevine (<italic>Vitis vinifera </italic>L.) have received more attention due to their significance as an important edible source of flavonoid compounds with nutrient and health benefits for humans [<xref ref-type="bibr" rid="B16">16</xref>]. Different flavonoid compounds are largely localized in berry skin and play a critical role in the quality of wine by contributing to its astringency and color [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>]. The major flavonoid classes accumulated in red-fruited grapevine berries are flavonols, proanthocyanidins (also called condensed tannins) and anthocyanins, with anthocyanins accumulating mostly in berry skin and the tannins in seed [<xref ref-type="bibr" rid="B17">17</xref>,<xref ref-type="bibr" rid="B18">18</xref>]. Thus, the flavonoid biosynthetic pathway in berries is regulated in a temporal and tissue-specific manner and the expression pattern of the pathway genes correlates to the synthesis of flavonoids in different grapevine berry tissues during fruit development [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. The synthesis of flavonoids via the flavonoid biosynthetic pathway requires two classes of genes: structural genes that encode enzymes for synthesis of anthocyanins and other flavonoids, and the regulatory genes involved in spatial and temporal regulation of these structural genes [<xref ref-type="bibr" rid="B20">20</xref>]. Although these two classes of genes are present in both red- and white-fruited grapevine cultivars, the color pigments are not expressed in white-fruited cultivars due to multiallelic mutations in the regulatory genes called <italic>MybA1 </italic>and <italic>MybA2 </italic>[<xref ref-type="bibr" rid="B21">21</xref>-<xref ref-type="bibr" rid="B24">24</xref>]. These two genes regulate expression of the UDP-glucose:flavonoid 3-<italic>O</italic>-glucosyltransferase (<italic>UFGT</italic>) gene, which mediates the conversion of anthocyanidins to anthocyanins by glycosylation [<xref ref-type="bibr" rid="B25">25</xref>,<xref ref-type="bibr" rid="B26">26</xref>]. Thus, the last biosynthetic step of <italic>UFGT</italic>-mediated anthocyanin synthesis does not occur in white-fruited grapevine cultivars and hence these cultivars do not express color in their berries. In the case of red-fruited berries, anthocyanins are transported into vacuoles and ultimately accumulated in berry skin cells [<xref ref-type="bibr" rid="B25">25</xref>,<xref ref-type="bibr" rid="B27">27</xref>]. In general, berries from red-fruited cultivars show various grades of color depending on the quantity and composition of anthocyanins in the berry skin. Proanthocyanidins are synthesized mainly at the green stage of berry development, whereas synthesis of anthocyanins begins at <italic>véraison </italic>(a transitional phase of grapevine berry development representing the beginning of berry ripening) and continue to accumulate in berry skins during ripening [<xref ref-type="bibr" rid="B17">17</xref>,<xref ref-type="bibr" rid="B27">27</xref>,<xref ref-type="bibr" rid="B28">28</xref>]. Although anthocyanins are present largely in berry skins of red-fruited grapevine cultivars, they can also accumulate in some cases in various plant organs such as leaves, flowers, stems, tendrils and berry flesh [<xref ref-type="bibr" rid="B29">29</xref>].</p><p>Grapevine leafroll disease (GLRD) is the most serious and complex virus disease known to infect grapevines worldwide [<xref ref-type="bibr" rid="B30">30</xref>]. Up to ten serologically distinct viruses, termed grapevine leafroll-associated viruses (GLRaVs) and numbered sequentially GLRaV-1 to -10 in the order of their discovery, have thus far been documented in grapevines infected with GLRD [<xref ref-type="bibr" rid="B31">31</xref>]. GLRaVs are flexuous rods, 1400-2200 nm long and 10-12 nm diameter with a monopartite, positive sense, single-stranded RNA genome. They are phloem-limited and predominantly dispersed long distances via clonally propagated vegetative planting materials. Some of the currently documented GLRaVs have been shown to be spread by different species of mealybugs and scale insects [<xref ref-type="bibr" rid="B30">30</xref>]. Among them, GLRaV-3 (genus <italic>Ampelovirus</italic>, family <italic>Closteroviridae</italic>) is the most economically important and widely prevalent. The virus has the largest genome size (18,498 nucleotides) encoding 13 open reading frames and represents the most complex gene organization among the currently known closteroviruses infecting grapevines [<xref ref-type="bibr" rid="B32">32</xref>].</p><p>It has been documented in several grape-growing regions that GLRaV-3 can cause reduced plant vigor and longevity, and significant losses in both yield and quality of berries [<xref ref-type="bibr" rid="B33">33</xref>-<xref ref-type="bibr" rid="B35">35</xref>]. In red-fruited wine grape cultivars infected with GLRaV-3, mature leaves at the bottom portions of canes begin to show GLRD symptoms at or soon after <italic>véraison</italic>. As the season progresses, the symptoms extend upward to other leaves and the foliar discolorations expand and coalesce to form a reddish-purple color within the inter-veinal areas of the leaf; a narrow strip of leaf tissue often remains green on either side of the main veins (hence called green veins). By the later part of the season (August-October), a typical infection in a red-fruited cultivar will consist of green veins and red and reddish-purple coloration of inter-veinal areas [<xref ref-type="bibr" rid="B30">30</xref>]. In advanced stages, the margins of infected leaves roll downward, expressing the symptom that gives the disease its common name. White-fruited cultivars may express GLRD symptoms as mild yellowing or chlorotic mottling and, in some cases, leaf margins may roll downward toward the end of the season. Unlike white-fruited cultivars, the phenotypic expression of reddish-purple coloration of leaves in red-fruited cultivars due to GLRD may be an indication of the accumulation of anthocyanins and could reflect the up-regulation of genes involved in their biosynthesis in GLRaV-3-infected symptomatic leaves. However, no studies have been conducted to elucidate the expression pattern of flavonoid biosynthetic pathway genes or analyze different flavonoids in grapevine leaves showing GLRD symptoms.</p><p>In recent years, reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) has been widely employed as a powerful tool for investigating the expression of cellular genes in response to biotic and abiotic stresses [<xref ref-type="bibr" rid="B36">36</xref>,<xref ref-type="bibr" rid="B37">37</xref>]. Throughout the manuscript, we used the abbreviation qPCR for quantitative real-time polymerase chain reaction, RT-qPCR for reverse transcription-qPCR and RT-PCR for 'traditional' RT-PCR. Due to its high throughput nature, sensitivity and accuracy in quantifying target genes, RT-qPCR is capable of the relative or absolute quantification of target genes in a given sample over a large dynamic range of conditions [<xref ref-type="bibr" rid="B38">38</xref>]. Considering its ability to discriminate between the expression of closely related genes and to quantify very weekly expressed genes, RT-qPCR is considered particularly useful for elucidating molecular mechanisms that underlie changes in gene expression [<xref ref-type="bibr" rid="B39">39</xref>,<xref ref-type="bibr" rid="B40">40</xref>]. Even though RT-qPCR is a method of choice, the reliability and reproducibility of experimental results for quantitative gene expression is dependent on the quality of RNA template and cDNA, primer specificity, assay efficiency, experimental conditions and rigorous analysis of the data using appropriate quality controls [<xref ref-type="bibr" rid="B41">41</xref>,<xref ref-type="bibr" rid="B42">42</xref>]. To circumvent bias, normalization of relative quantities of the target genes is carried out widely using appropriate endogenous reference genes, also referred in earlier studies as housekeeping genes [<xref ref-type="bibr" rid="B43">43</xref>,<xref ref-type="bibr" rid="B44">44</xref>]. One of the most critical issues in RT-qPCR is the choice of reference genes used for gene expression analysis, since the expression of a number of such reference genes varies considerably under each experimental condition in different lab settings [<xref ref-type="bibr" rid="B45">45</xref>-<xref ref-type="bibr" rid="B48">48</xref>]. Validation of reference genes is necessary, since the use of a single non-validated reference gene has been shown to significantly increase bias in experimental validation of gene expression changes ranging from more than 3-fold in 25% of the results up to 6-fold in 10% of the results [<xref ref-type="bibr" rid="B43">43</xref>].</p><p>In this study, we evaluated six reference genes for their use in gene expression studies in virus-free green leaves and virus-infected leaves exhibiting GLRD symptoms. Using RT-qPCR assay, based on SYBR green detection, we analyzed expression stability of these reference genes in grapevine leaves using the <italic>geNorm </italic>algorithm [<xref ref-type="bibr" rid="B43">43</xref>]. A combination of two genes was identified as suitable candidates for normalization of gene expression data in both virus-free and virus-infected leaves. By using gene-specific RT-qPCR, in combination with a reliable normalization factor, we present evidence that up-regulation of flavonoid biosynthetic pathway genes occurred in symptomatic leaves of a red-fruited wine grape cultivar infected with GLRaV-3. Together with estimation of anthocyanins, flavonols and proanthocyanidins, these results indicated modulation of the flavonoid biosynthetic pathway genes towards accumulation of certain classes of end-products in grapevine leaves exhibiting GLRD symptoms.</p></sec><sec><title>Results</title><sec><title>GLRD symptoms in grapevine leaves</title><p>At the time of sampling in mid September, representing post-<italic>véraison </italic>stage of berry development, mature leaves at the bottom portion of canes in GLRD affected Merlot grapevines showed green veins and red and reddish-purple color in the inter-veinal areas (Figure <xref ref-type="fig" rid="F2">2</xref>, left). The margins of some of these leaves showed downward rolling. GLRD symptoms were not observed in adjacent grapevines (Figure <xref ref-type="fig" rid="F2">2</xref>, right). Symptomatic leaves from GLRD affected grapevines and comparable leaves from adjacent grapevines not affected by GLRD were tested by single tube-one step RT-PCR for the presence of different grapevine viruses. Symptomatic leaves from GLRD affected grapevines were tested positive for GLRaV-3 but not for other viruses (data not shown). GLRaV-3 was detected in green veins as well as in reddish-purple inter-veinal areas (since minor veins and veinlets are present in these areas) of symptomatic leaves from GLRD affected grapevines (Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>, Figure S1). Green leaves from adjacent grapevines were tested negative for these viruses.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>GLRD symptoms in GLRaV-3-infected red-fruited wine grape cv. Merlot</bold>. 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.</p></caption><graphic xlink:href="1471-2229-10-187-2"></graphic></fig></sec><sec><title>Chlorophyll and carotenoid pigments in symptomatic leaves</title><p>Total chlorophylls and carotenoids were estimated in GLRaV-3-infected symptomatic and virus-free green leaves (Table <xref ref-type="table" rid="T1">1</xref>). Total chlorophyll content in symptomatic leaves was less by 20.1% when compared to green leaves. Similarly, total carotenoids were less by 19.8% in virus-infected symptomatic leaves. These results indicate reduced levels of both chrolophylls and carotenoids in virus-infected leaves exhibiting GLRD symptoms.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Total chlorophylls and carotenoids in GLRaV-3-infected symptomatic and virus-free green leaves</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Pigments (mg/g fresh wt)</th><th align="center">GLRaV-3-infected*</th><th align="center">Virus-free*</th></tr></thead><tbody><tr><td align="left"><bold>Total chlorophylls</bold></td><td align="center">4.81 ± 0.45</td><td align="center">6.02 ± 0.16</td></tr><tr><td align="left"><bold>Total carotenoids</bold></td><td align="center">2.19 ± 0.19</td><td align="center">2.73 ± 0.08</td></tr></tbody></table><table-wrap-foot><p>*Values are mean ± SE. Asterisk indicates significant difference between GLRaV-3-infected and virus-free leaves using one way ANOVA test (*<italic>P </italic>< 0.05)</p></table-wrap-foot></table-wrap></sec><sec><title>Sequence specificity and amplification efficiency analysis of target genes</title><p>In initial experiments using total RNA isolated from grapevine leaves, gene-specific sequences amplified by RT-PCR were cloned and nucleotide sequence determined (Table <xref ref-type="table" rid="T2">2</xref>). Nucleotide sequence obtained for each gene showed high level of similarity (97 to 100%) with corresponding gene sequence available in GenBank, confirming the specificity of each amplicon to the respective gene. The amplification efficiency (E) of each gene-specific primer pair in RT-qPCR shown in Table <xref ref-type="table" rid="T2">2</xref> indicated the suitability of primer pairs for RT-qPCR-based amplification and quantification of target genes. Melting curve analysis for each amplicon showed a single peak (Additional file <xref ref-type="supplementary-material" rid="S2">2</xref>, Figure S2), further confirming the homogeneity and specificity of amplicons produced in qPCR for all target genes. Agarose gel electrophoretic separation of each amplicon showed a single DNA fragment of the expected size with no visible primer-dimer products (data not shown). No amplifications were observed in all control assays. All these results indicated that the total RNA and the derived cDNA template were free of contaminating genomic DNA, demonstrating high quality of nucleic acid preparations obtained for gene expression level analyses by RT-qPCR.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Genes, primers, length of amplicons and amplification efficiency</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Gene<sup>1</sup></th><th align="left">Primer sequence 5'-3' (forward/reverse)</th><th align="left">Amplicon length (bp)</th><th align="left">qPCR efficiency</th><th align="left">Reference</th><th align="left">GenBank accession number*</th></tr></thead><tbody><tr><td align="left" colspan="6"><bold>a) Reference genes</bold></td></tr><tr><td align="left"><italic>Ubiquitin</italic></td><td align="left">TCTGAGGCTTCGTGGTGGTA/AGGCGTGCATAACATTTGCG</td><td align="center">99</td><td align="center">2.16</td><td align="center">[<xref ref-type="bibr" rid="B82">82</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585868">GU585868</ext-link></td></tr><tr><td align="left"><italic>Actin</italic></td><td align="left">CTTGCATCCCTCAGCACCTT/TCCTGTGGACAATGGATGGA</td><td align="center">82</td><td align="center">2.11</td><td align="center">[<xref ref-type="bibr" rid="B56">56</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585869">GU585869</ext-link></td></tr><tr><td align="left"><italic>GAPDH</italic></td><td align="left">TTCTCGTTGAGGGCTATTCCA/CCACAGACTTCATCGGTGACA</td><td align="center">70</td><td align="center">1.84</td><td align="center">[<xref ref-type="bibr" rid="B56">56</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585870">GU585870</ext-link></td></tr><tr><td align="left"><italic>EF1-a</italic></td><td align="left">GAACTGGGTGCTTGATAGGC/AACCAAAATATCCGGAGTAAAAGA</td><td align="center">164</td><td align="center">1.90</td><td align="center">[<xref ref-type="bibr" rid="B56">56</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585871">GU585871</ext-link></td></tr><tr><td align="left"><italic>SAND</italic></td><td align="left">CAACATCCTTTACCCATTGACAGA/GCATTTGATCCACTTGCAGATAAG</td><td align="center">76</td><td align="center">1.88</td><td align="center">[<xref ref-type="bibr" rid="B56">56</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585872">GU585872</ext-link></td></tr><tr><td align="left"><italic>NAD5</italic></td><td align="left">GATGCTTCTTGGGGCTTCTTGTT/CTCCAGTCACCAACATTGGCATAA</td><td align="center">181</td><td align="center">1.82</td><td align="center">[<xref ref-type="bibr" rid="B83">83</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585873">GU585873</ext-link></td></tr><tr><td align="left" colspan="6"><bold>b) Candidate genes</bold></td></tr><tr><td align="left"><italic>PAL</italic></td><td align="left">TCTGGTGGAAGGAATCCAAG/CAAAGTGCCACCAGGTAGGT</td><td align="center">230</td><td align="center">1.77</td><td align="center">[<xref ref-type="bibr" rid="B62">62</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585850">GU585850</ext-link></td></tr><tr><td align="left"><italic>CHS1</italic></td><td align="left">AGCCAGTGAAGCAGGTAGCC/GTGATCCGGAAGTAGTAAT</td><td align="center">155</td><td align="center">1.74</td><td align="center">[<xref ref-type="bibr" rid="B61">61</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585851">GU585851</ext-link></td></tr><tr><td align="left"><italic>CHS2</italic></td><td align="left">TCTGAGCGAGTATGGGAACA/AGGGTAGCTGCGTAGGTTGG</td><td align="center">294</td><td align="center">1.80</td><td align="center">[<xref ref-type="bibr" rid="B61">61</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585852">GU585852</ext-link></td></tr><tr><td align="left"><italic>CHS3</italic></td><td align="left">TCACTTGGACAGCCTTGTTG/CAATTCGAACATGGGCTTCT</td><td align="center">106</td><td align="center">1.87</td><td align="center">$</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585853">GU585853</ext-link></td></tr><tr><td align="left"><italic>CHI1</italic></td><td align="left">CAGGCAACTCCATTCTTTTC/TTCTCTATCACTGCATTCCC</td><td align="center">103</td><td align="center">1.69</td><td align="center">[<xref ref-type="bibr" rid="B84">84</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585854">GU585854</ext-link></td></tr><tr><td align="left"><italic>CHI2</italic></td><td align="left">TCCAGATCAAGTTCACAGCA/GAAACAAGAGCCTCAAAGAA</td><td align="center">127</td><td align="center">1.60</td><td align="center">[<xref ref-type="bibr" rid="B84">84</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585855">GU585855</ext-link></td></tr><tr><td align="left"><italic>F3'H</italic></td><td align="left">ATTCGCCACCCTGAAATGAT/AGCCGTTGATCTCACAGCTC</td><td align="center">196</td><td align="center">1.82</td><td align="center">[<xref ref-type="bibr" rid="B15">15</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585856">GU585856</ext-link></td></tr><tr><td align="left"><italic>F3'5'H</italic></td><td align="left">GAAGTTCGACTGGTTATTAACAAAGAT/<break></break>AGGAGGAGTGCTTTAATGTTGGTA</td><td align="center">156</td><td align="center">1.68</td><td align="center">[<xref ref-type="bibr" rid="B15">15</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585857">GU585857</ext-link></td></tr><tr><td align="left"><italic>F3H1</italic></td><td align="left">CCAATCATAGCAGACTGTCC/TCAGAGGATACACGGTTGCC</td><td align="center">69</td><td align="center">1.83</td><td align="center">[<xref ref-type="bibr" rid="B84">84</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585858">GU585858</ext-link></td></tr><tr><td align="left"><italic>F3H2</italic></td><td align="left">CTGTGGTGAACTCCGACTGC/CAAATGTTATGGGCTCCTCC</td><td align="center">129</td><td align="center">1.70</td><td align="center">[<xref ref-type="bibr" rid="B84">84</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585859">GU585859</ext-link></td></tr><tr><td align="left"><italic>DFR</italic></td><td align="left">GAAACCTGTAGATGGCAAGA/GGCCAAATCAAACTACCAGA</td><td align="center">114</td><td align="center">1.85</td><td align="center">[<xref ref-type="bibr" rid="B84">84</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585860">GU585860</ext-link></td></tr><tr><td align="left"><italic>LDOX</italic></td><td align="left">AGGGAAGGGAAAACAAGTAG/ACTCTTTGGGGATTGACTGG</td><td align="center">109</td><td align="center">1.76</td><td align="center">[<xref ref-type="bibr" rid="B84">84</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585861">GU585861</ext-link></td></tr><tr><td align="left"><italic>UFGT</italic></td><td align="left">GGGATGGTAATGGCTGTGG/ACATGGGTGGAGAGTGAGTT</td><td align="center">152</td><td align="center">1.74</td><td align="center">[<xref ref-type="bibr" rid="B84">84</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585862">GU585862</ext-link></td></tr><tr><td align="left"><italic>MybA1</italic></td><td align="left">TAGTCACCACTTCAAAAAGG/GAATGTGTTTGGGGTTTATC</td><td align="center">66</td><td align="center">1.67</td><td align="center">[<xref ref-type="bibr" rid="B84">84</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585863">GU585863</ext-link></td></tr><tr><td align="left"><italic>FLS1</italic></td><td align="left">CAGGGCTTGCAGGTTTTTAG/GGGTCTTCTCCTTGTTCACG</td><td align="center">154</td><td align="center">1.82</td><td align="center">[<xref ref-type="bibr" rid="B85">85</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585864">GU585864</ext-link></td></tr><tr><td align="left"><italic>LAR1</italic></td><td align="left">AAATGAACTCGCATCTGTGT/CTGTGGGATGATGTTTTCTC</td><td align="center">109</td><td align="center">1.75</td><td align="center">[<xref ref-type="bibr" rid="B82">82</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585865">GU585865</ext-link></td></tr><tr><td align="left"><italic>LAR2</italic></td><td align="left">TGATATCAGCTGTGGGTGGA/CCCAAATTCTGATGGAAGGA</td><td align="center">104</td><td align="center">1.74</td><td align="center">$</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585866">GU585866</ext-link></td></tr><tr><td align="left"><italic>ANR</italic></td><td align="left">GCTGCTGTTACCATCAATCA/GCAGGATAGCCCCAAGTAGG</td><td align="center">113</td><td align="center">1.62</td><td align="center">[<xref ref-type="bibr" rid="B82">82</xref>]</td><td align="left"><ext-link ext-link-type="gen" xlink:href="GU585867">GU585867</ext-link></td></tr></tbody></table><table-wrap-foot><p><sup>1</sup>See legends for Figure 1 and Figure 3 for names of genes</p><p>*Accession numbers indicate sequences generated from this study.</p><p><sup>$</sup>Primers were designed based on sequence available in GenBank.</p></table-wrap-foot></table-wrap></sec><sec><title>Expression stability analysis of candidate reference genes</title><p>A total of six putative reference genes (Table <xref ref-type="table" rid="T2">2</xref>) were evaluated for their expression stability under our experimental conditions. Since all RT-qPCR reactions were performed with cDNA derived from equal quantity of total RNA, transcript abundance of these six genes were analyzed by direct comparison of C<sub>q </sub>values, assuming equal C<sub>q </sub>for equal transcript number. As shown in Figure <xref ref-type="fig" rid="F3">3</xref>, the six reference genes were grouped into two arbitrary categories based on their C<sub>q </sub>values combined from both GLRaV-3-infected symptomatic and virus-free green leaf samples. Four genes (<italic>GAPDH</italic>, <italic>EF1-a</italic>, <italic>Ubiquitin </italic>and <italic>Actin</italic>) showed higher transcript levels, since they presented C<sub>q </sub>values with a median between 15 and 20 cycles. The other two (<italic>SAND </italic>and <italic>NAD5</italic>) were categorized as genes with relatively low transcript levels, since their median C<sub>q </sub>values were between 20 and 25 cycles.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Box plot representation of raw C<sub>q </sub>values obtained from amplification curves for reference genes</bold>. Lower and upper boundaries of each box indicate the 25<sup>th </sup>and the 75<sup>th </sup>percentile, respectively. Ranges are represented as bars (whiskers) below and above the box and indicate the 10<sup>th </sup>and 90<sup>th </sup>percentiles, respectively. The horizontal line in each box represents mean and outliers by (•). <italic>SAND</italic>: SAND family protein; <italic>GAPDH</italic>: glyceraldehyde 3-phosphate dehydrogenase; <italic>EF1-a</italic>: elongation factor1-alpha; <italic>Ubiquitin</italic>: ubiquitin-60S ribosomal L40 fusion protein; <italic>Actin</italic>, <italic>NAD5</italic>: NADH dehydrogenase subunit 5.</p></caption><graphic xlink:href="1471-2229-10-187-3"></graphic></fig><p>The raw C<sub>q </sub>data for each reference gene was subsequently analyzed using <italic>geNorm </italic>algorithm to evaluate their expression stability in virus-infected and virus-free samples and ranked according to their expression stability measure "M" (Figure <xref ref-type="fig" rid="F4">4a-c</xref>). All six genes showed high expression stability and had M values lower than 0.8, below the default limit of 1.5 suggested by the <italic>geNorm </italic>program. From this analysis, <italic>Actin </italic>and <italic>NAD5 </italic>genes were estimated to have the lowest M values, indicating that these two genes showed high expression stability in both virus-infected and virus-free samples (Figure <xref ref-type="fig" rid="F4">4a</xref>&<xref ref-type="fig" rid="F4">4b</xref>). <italic>GAPDH </italic>gene gave the highest M value and hence considered as having lowest stability in both types of samples under our experimental conditions. However, the M values for <italic>SAND</italic>, <italic>EF1-a </italic>and <italic>Ubiquitin </italic>were variable between the two types of samples, indicating differences in their expression stability due to virus infection. The expression stability of the six reference genes differed when M values were calculated by combining raw C<sub>q </sub>data of each gene from both virus-infected and virus-free samples (Figure <xref ref-type="fig" rid="F4">4c</xref>). In this case, <italic>EF1-a </italic>and <italic>Ubiquitin </italic>were estimated to have the lowest M values, followed by <italic>Actin</italic>, <italic>SAND </italic>and <italic>NAD5 </italic>genes. Based on these results, a subset of two genes (<italic>Actin </italic>and <italic>NAD5</italic>) was used to calculate normalization factor (NF) through the geometrical averaging of their raw C<sub>q </sub>values. The resulting NF was used to normalize raw C<sub>q </sub>data generated in RT-qPCR for flavonoid biosynthetic pathway genes in virus-infected and virus-free grapevine leaves.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p><bold>Stability of reference genes in grapevine leaves</bold>. Stability value (M) for a set of reference genes is analyzed with <italic>geNorm </italic>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 <italic>GAPDH </italic>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.</p></caption><graphic xlink:href="1471-2229-10-187-4"></graphic></fig></sec><sec><title>Expression patterns of flavonoid biosynthetic pathway genes</title><p>The expression patterns of flavonoid upstream pathway gene (<italic>PAL</italic>), genes involved in the biosynthesis of different flavonoids (<italic>CHS1, CHS2, CHS3, CHI1, CHI2</italic>, <italic>F3'H</italic>, <italic>F3'5'H</italic>, <italic>F3H1, F3H2</italic>, <italic>DFR</italic>, <italic>LDOX</italic>, <italic>UFGT, FLS1</italic>, <italic>LAR1, LAR2 </italic>and <italic>ANR</italic>) and a regulatory gene (<italic>MybA1</italic>) were examined in GLRaV-3-infected symptomatic and virus-free green leaves. The distribution overview of expression levels of gene transcripts showed (Additional file <xref ref-type="supplementary-material" rid="S3">3</xref>, Figure S3) that many of the flavonoid biosynthetic pathway genes from virus-infected samples presented lower median C<sub>q </sub>values. This indicated higher transcript levels for these genes in virus-infected symptomatic leaves, assuming equal C<sub>q </sub>for equal transcript number, since all RT-qPCR reactions were performed with equal amount of cDNA derived from equal quantity of total RNA. Using the two best reference genes identified (NF<sub>[<italic>Actin </italic>and <italic>NAD5</italic>]</sub>) from gene expression stability analyses described above, we normalized the raw C<sub>q </sub>data for each gene from virus-infected and virus-free samples and their relative expression levels are shown in Figure <xref ref-type="fig" rid="F5">5a</xref>&<xref ref-type="fig" rid="F5">5b</xref>. In general, flavonoid biosynthetic pathway genes analyzed in this study showed higher expression levels in virus-infected symptomatic leaves when compared with expression levels of corresponding genes from virus-free green leaves. The expression levels of these genes as fold increase in virus-infected symptomatic leaves over the corresponding values from virus-free green leaves is shown in Table <xref ref-type="table" rid="T3">3</xref>. Their expression levels ranged from two- to fifty-fold increase in virus-infected samples. Among them, <italic>CHS3</italic>, <italic>F3'5'H</italic>, <italic>F3H1</italic>, <italic>LDOX </italic>and <italic>LAR1 </italic>showed greater than 10-fold increase suggesting that these genes were expressed at higher levels in virus-infected leaves. <italic>MybA1</italic>, which regulates anthocyanin biosynthesis in grapevines via expression of the <italic>UFGT </italic>gene, was expressed by about 19-fold higher in virus-infected symptomatic than in virus-free green leaves (Figure <xref ref-type="fig" rid="F5">5b</xref>, Table <xref ref-type="table" rid="T3">3</xref>). Similar trend in expression levels of flavonoid biosynthetic pathway genes and <italic>MybA1 </italic>was obtained when the two best reference genes (NF<sub>[<italic>EF1-a </italic>and <italic>Ubiquitin</italic>]</sub>), identified when expression stability of reference genes was calculated by combining raw C<sub>q </sub>data from both virus-infected and virus-free samples (Figure <xref ref-type="fig" rid="F4">4c</xref>), were considered for data normalization (Table <xref ref-type="table" rid="T3">3</xref>). However, the values were slightly lower than those obtained when <italic>Actin </italic>and <italic>NAD5 </italic>were used as reference genes for data normalization. Based on these results, it can be concluded that some of the flavonoid biosynthetic pathway genes are significantly up-regulated in virus-infected symptomatic leaves when compared to expression levels of corresponding genes in virus-free green leaves.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold>Expression patterns of flavonoid biosynthetic pathway genes in GLRaV-3-infected symptomatic and virus-free green leaves</bold>. The relative expression levels of (a) the flavonoid biosynthetic pathway genes and (b) the <italic>MybA1 </italic>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<sub>q </sub>values for each gene was normalized using two reference genes (NF<sub>[<italic>Actin </italic>and <italic>NAD5</italic>]</sub>). Columns represent mean value from five biological replicates, except in case of <italic>MybA1 </italic>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 (* = <italic>p </italic>< 0.05 and ** = <italic>p </italic>< 0.001). See legend for Figure 1 for names of genes.</p></caption><graphic xlink:href="1471-2229-10-187-5"></graphic></fig><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Relative fold increase of flavonoid biosynthetic pathway genes in GLRaV-3-infected, symptomatic leaves over virus-free green leaves</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Gene<sup>1</sup></th><th align="center"><italic>Actin </italic>+ <italic>NAD5</italic>*</th><th align="center"><italic>EF1-a </italic>+ <italic>Ubiquitin</italic>*</th></tr></thead><tbody><tr><td align="left"><italic>PAL</italic></td><td align="center">4.99 ± 2.99</td><td align="center">3.00 ± 1.36</td></tr><tr><td align="left"><italic>CHS1</italic></td><td align="center">4.23 ± 1.34</td><td align="center">2.93 ± 0.65</td></tr><tr><td align="left"><italic>CHS2</italic></td><td align="center">3.02 ± 0.97</td><td align="center">2.05 ± 0.41</td></tr><tr><td align="left"><italic>CHS3</italic></td><td align="center">37.43 ± 5.09</td><td align="center">28.91 ± 6.97</td></tr><tr><td align="left"><italic>CHI1</italic></td><td align="center">3.42 ± 0.24</td><td align="center">2.62 ± 0.52</td></tr><tr><td align="left"><italic>CHI2</italic></td><td align="center">4.40 ± 1.35</td><td align="center">3.40 ± 1.24</td></tr><tr><td align="left"><italic>F3'H</italic></td><td align="center">4.60 ± 1.69</td><td align="center">3.14 ± 0.80</td></tr><tr><td align="left"><italic>F3'5'H</italic></td><td align="center">11.33 ± 2.91</td><td align="center">8.52 ± 2.62</td></tr><tr><td align="left"><italic>F3H1</italic></td><td align="center">23.62 ± 11.47</td><td align="center">15.45 ± 5.46</td></tr><tr><td align="left"><italic>F3H2</italic></td><td align="center">4.83 ± 1.30</td><td align="center">3.32 ± 0.62</td></tr><tr><td align="left"><italic>DFR</italic></td><td align="center">5.73 ± 1.13</td><td align="center">3.82 ± 0.41</td></tr><tr><td align="left"><italic>LDOX</italic></td><td align="center">40.75 ± 15.59</td><td align="center">25.56 ± 6.36</td></tr><tr><td align="left"><italic>UFGT</italic></td><td align="center">9.22 ± 1.53</td><td align="center">6.77 ± 1.11</td></tr><tr><td align="left"><italic>MybA1</italic></td><td align="center">19.03 ± 5.56</td><td align="center">12.04 ± 1.92</td></tr><tr><td align="left"><italic>FLS1</italic></td><td align="center">3.77 ± 1.33</td><td align="center">2.54 ± 0.70</td></tr><tr><td align="left"><italic>LAR1</italic></td><td align="center">58.35 ± 18.54</td><td align="center">36.90 ± 6.91</td></tr><tr><td align="left"><italic>LAR2</italic></td><td align="center">2.51 ± 0.29</td><td align="center">1.82 ± 0.28</td></tr><tr><td align="left"><italic>ANR</italic></td><td align="center">3.84 ± 1.14</td><td align="center">3.18 ± 1.50</td></tr></tbody></table><table-wrap-foot><p><sup>1</sup>See legend for Figure 1 for names of genes</p><p>*Values are mean ± SE.</p></table-wrap-foot></table-wrap><p>Among the three isogenes of chalcone synthase (<italic>CHS1</italic>, <italic>CHS2 </italic>and <italic>CHS3</italic>) that are involved in recruitment of flavonoid precursors to enter the flavonoid biosynthetic pathway, <italic>CHS1 </italic>and <italic>CHS2 </italic>showed about 4- and 3-folds higher expression levels, respectively, while <italic>CHS3 </italic>exhibited about 37-fold increase in virus-infected leaves. These results indicate preferential up-regulation of <italic>CHS3 </italic>in virus-infected symptomatic leaves when compared with virus-free green leaves. The two flavonoid hydroxylases, <italic>F3'H</italic>, which regulates the synthesis of cyanidin-based anthocyanins, and <italic>F3'5'H</italic>, which regulates the synthesis of delphinidin-based anthocyanins, were expressed at about 5- and 11-fold higher, respectively, in virus-infected symptomatic leaves compared to virus-free green leaves. Higher expression levels of the flavonoid pathway genes like <italic>F3H1 (~23-fold)</italic>, <italic>DFR (~6-fold)</italic>, <italic>LDOX (~40-fold)</italic>, and <italic>UFGT (~9-fold) </italic>and <italic>LAR1 </italic>(~<italic>58-fold</italic>) genes specific to anthocyanins and proanthocyanidins, respectively, in virus-infected symptomatic leaves indicate enhanced synthesis of anthocyanins and proanthocyanidins in these leaves. It is likely that the synthesis of more flavonols was also favored in virus-infected leaves due to ~4-fold higher expression levels of the <italic>FLS1 </italic>gene. Higher expression levels of <italic>LAR1</italic>, <italic>LAR2 </italic>and <italic>ANR </italic>indicate that these genes were contributing to the enhanced synthesis of proanthocyanidins in virus-infected leaves.</p></sec><sec><title>Estimation of anthocyanins, flavonols and proanthocyanidins</title><p>To be able to correlate gene expression data with the accumulation of different flavonoid compounds, we analyzed the secondary metabolite constituents of GLRaV-3-infected symptomatic and virus-free green leaves. Anthocyanins and flavonols were analyzed by HPLC and proanthocyanidins were estimated by protein precipitation method as total tannins. Figure <xref ref-type="fig" rid="F6">6</xref> shows total amounts of anthocyanins, flavonols and proanthocyanidins and Figure <xref ref-type="fig" rid="F7">7</xref> shows HPLC profiles of anthocyanins and flavonols from virus-infected and virus-free leaves. Anthocyanins were not detected in virus-free green leaves (Figure <xref ref-type="fig" rid="F6">6a</xref> and <xref ref-type="fig" rid="F7">7a</xref>), whereas two clearly discernible peaks (numbered 1 and 2 with increasing retention times) were observed in virus-infected leaves (Figure <xref ref-type="fig" rid="F7">7b</xref>) with no corresponding peaks in virus-free green leaf samples. Based on their retention times and spectral data, the two major peaks in virus-infected leaves were identified as cyanidin-3-glucoside and malvidin-3-glucoside. Further analysis indicated that cyanidin-3-glucoside accounted for 61% and malvidin-3-glucoside accounted for 39% of total anthocyanins detected in virus-infected leaves. A minor peak, designated as #3 in Figure <xref ref-type="fig" rid="F7">7b</xref>, was tentatively identified as Peonidin-3-O-6-coumarilated. Although total flavonols were detected in both virus-infected and virus-free leaves (Figure <xref ref-type="fig" rid="F6">6b</xref>), they were 24% higher in virus-infected leaves than in virus-free leaves. As shown in Figure <xref ref-type="fig" rid="F7">7c</xref>&<xref ref-type="fig" rid="F7">7d</xref>, HPLC analysis showed three clear peaks in both virus-infected and virus-free leaves. Based on retention times, they corresponded to putative myricetin (peak 1) and quercetin (peak 5 & 6) derivatives, respectively, with quercetin derivatives accounting for about 69% and myricetin derivatives accounting for about 20% of total flavonols and the rest accounting for other unidentified flavonols. Estimation of proanthocyanidins in leaves as total tannins showed that their concentration was 36% higher in virus-infected than virus-free leaves (Figure <xref ref-type="fig" rid="F6">6c</xref>). It is important to note that the method we used to measure proanthocyanidins is limited to estimating total amount of these compounds rather than a method that provides structural information. Potentially both delphinidin and cyandin sub-units are present in both types of leaf tissues. Taken together, the above results (Figure <xref ref-type="fig" rid="F6">6</xref>) indicated that the three classes of flavonoids (anthocyanins, flavonols and proanthocyanidins) are present in significantly higher amounts in virus-infected leaves and correlate with up-regulation of the flavonoid biosynthetic pathway genes shown in Figure <xref ref-type="fig" rid="F5">5</xref> and Table <xref ref-type="table" rid="T3">3</xref>.</p><fig id="F6" position="float"><label>Figure 6</label><caption><p><bold>Estimation of flavonoids in GLRaV-3-infected symptomatic and virus-free green leaves</bold>. 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 (* = <italic>p </italic>< 0.05). C.E. = catechin equivalent.</p></caption><graphic xlink:href="1471-2229-10-187-6"></graphic></fig><fig id="F7" position="float"><label>Figure 7</label><caption><p><bold>HPLC profiling of anthocyanins and flavonols in GLRaV-3-infected symptomatic and virus-free green leaves</bold>. 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.</p></caption><graphic xlink:href="1471-2229-10-187-7"></graphic></fig></sec></sec><sec><title>Discussion</title><p>The necessity for ensuring quality-assurance measures in RT-qPCR analysis of gene expression is well recognized and a set of guidelines have been outlined for appropriate normalization strategy to control for non-specific variation between samples [<xref ref-type="bibr" rid="B49">49</xref>]. Although a range of endogenous reference genes have been listed as good candidates for normalization of gene expression, identification of the most suitable reference genes for the given experimental conditions, rather than using reference genes published in the literature, is extremely important in functional genomics studies [<xref ref-type="bibr" rid="B47">47</xref>,<xref ref-type="bibr" rid="B48">48</xref>]. In addition, certain reference genes may be stably expressed in one plant species but are not be well suited for use in other species [<xref ref-type="bibr" rid="B50">50</xref>]. Apart from other fields of research, this knowledge is highly relevant to studies in plant host-virus interactions, as viruses are known to modulate key cellular processes in plants which may involve changes in the expression of endogenous host genes normally used as reference genes in RT-qPCR [<xref ref-type="bibr" rid="B51">51</xref>,<xref ref-type="bibr" rid="B52">52</xref>]. Moreover, viruses manipulate different host cellular transcription pathways and the extent to which these pathways are affected will be dependent on the specific virus-host combination [<xref ref-type="bibr" rid="B53">53</xref>,<xref ref-type="bibr" rid="B54">54</xref>]. Consequently, we evaluated geometric averaging of multiple reference genes as a means to avoid experimental bias in gene expression data.</p><p>In this study, we analyzed a set of six putative reference genes (<italic>Ubiquitin, Actin</italic>, <italic>GAPDH</italic>, <italic>EF1-a, SAND </italic>and <italic>NAD5</italic>) for their expression stability in leaf samples collected from a red-fruited wine grape cultivar (cv. Merlot) grown under field-conditions. Since expression stability of reference genes is known to vary with environmental conditions under which plants are grown, the type of plant tissue used and under a diverse set of biotic and abiotic stress conditions, we validated the expression stability of these six genes under our experimental conditions using the <italic>geNorm </italic>software and selected <italic>Actin </italic>and <italic>NAD5 </italic>to normalize RT-qPCR data obtained for the flavonoid biosynthetic pathway genes in virus-infected and virus-free grapevine leaves [<xref ref-type="bibr" rid="B55">55</xref>]. In a previous study, <italic>GAPDH </italic>was ranked as one of the top three reference genes (<italic>GAPDH </italic><<italic>Actin </italic><<italic>EF1-a</italic>/<italic>SAND</italic>) for gene expression studies in grape berry development [<xref ref-type="bibr" rid="B56">56</xref>]. However, we found that <italic>GAPDH </italic>is the least reliable in the context of our investigations on relative expression of the flavonoid biosynthetic pathway genes in grapevine leaf samples (Figure <xref ref-type="fig" rid="F4">4</xref>). These results clearly highlight the importance of validating reference genes as the most invariant internal controls for a particular experimental condition prior to investigating the relative expression of target genes by RT-qPCR.</p><p>By using gene-specific RT-qPCR, we present evidence in this study, the first example to our knowledge, that overall up-regulation of <italic>PAL</italic>, an enzyme that commits the flux of primary metabolism into the flavonoid biosynthetic pathway, and both "early" (<italic>CHS</italic>, <italic>CHI</italic>, <italic>F3'H</italic>, <italic>F3'5'H</italic>, <italic>F3H </italic>and <italic>FLS</italic>) and "late" genes (<italic>DFR, LDOX, UFGT </italic>and <italic>LAR</italic>) of the pathway occurred in GLRaV-3-infected symptomatic grapevine leaves (Figure <xref ref-type="fig" rid="F5">5</xref>). In red-fruited cultivars of wine grapes, anthocyanin pigments accumulate predominantly in berry skins displaying various shades of colors ranging from brick red to dark blue and their biosynthesis is developmentally triggered at the onset of <italic>véraison </italic>via the activation of flavonoid biosynthetic pathway genes [<xref ref-type="bibr" rid="B25">25</xref>]. Under normal circumstances, these cultivars do not exhibit such coloration in their foliage during the growing season. Thus, changes in leaf color (Figure <xref ref-type="fig" rid="F2">2</xref>) and accumulation of specific classes of anthocyanins (Figure <xref ref-type="fig" rid="F6">6</xref> and <xref ref-type="fig" rid="F7">7</xref>) only in GLRaV-3-infected symptomatic leaves supported our hypothesis that expression of the flavonoid biosynthetic pathway genes was activated in virus-infected leaves. Although this study was based on the expression analysis of flavonoid biosynthetic pathway genes and qualitative and quantitative variation of anthocyanins, flavonols and proanthocyanidins, it should be noted that mRNA expression is only one aspect of functional gene regulation of the pathway that result in changes in color of leaves in virus-infected plants. Since changes in leaf coloration begins to occur soon after <italic>véraison</italic>, even though GLRaV-3 can be detected in leaves of infected grapevines during the entire season including pre-<italic>véraison</italic>, it remains to be studied if the specific induction of anthocyanins in virus-infected leaves during post-<italic>véraison </italic>is tightly coupled with a cascade of physiological and/or molecular events triggered as a consequence of virus-host interactions during <italic>véraison</italic>.</p><p>In plants, delphinidin- and cyanidin-based anthocyanins exhibit blue and reddish color, respectively, under the acidic conditions of plant vacuoles [<xref ref-type="bibr" rid="B17">17</xref>]. HPLC profiling of total anthocyanins showed that both cyanidin-3-glucoside and malvidin-3-glucoside accumulated in virus-infected symptomatic leaves and they are virtually undetected in virus-free green leaves (Figure <xref ref-type="fig" rid="F6">6a</xref> and Figure <xref ref-type="fig" rid="F7">7a</xref>&<xref ref-type="fig" rid="F7">7b</xref>). We believe that presence of these two classes of anthocyanins, although cyanidin-3-glucoside is slightly but not significantly higher than malvidin-3-glucoside in virus-infected leaves, contributes to red and reddish-purple discoloration of virus-infected leaves. Since <italic>F3'5'H </italic>regulates the synthesis of delphinidin-based anthocyanins and <italic>F3'H </italic>regulates the synthesis of cyanidin-based anthocyanins, expression profiles of these two genes in concert with increased expression of anthocyanin-specific gene <italic>UFGT </italic>and its transcription factor gene <italic>MybA1 </italic>would ensure the flux of flavonoid intermediates towards the synthesis of these two classes of anthocyanins in virus-infected leaves. The levels of <italic>F3'H </italic>and <italic>F3'5'H </italic>gene transcripts observed in virus-free green leaves is in agreement with recent reports that <italic>F3'H </italic>gene was only slightly detectable and <italic>F3'5'H </italic>gene was expressed at non-detectable levels in green, fully expanded grapevine leaves [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B57">57</xref>]. Our results also showed significantly higher levels of flavonols in virus-infected leaves than in virus-free leaves, and the predominant flavonols were quercetin followed by myricetin (Figure <xref ref-type="fig" rid="F6">6b</xref> and <xref ref-type="fig" rid="F7">7c</xref>&<xref ref-type="fig" rid="F7">7d</xref>). Bogs <italic>et al</italic>. showed that total amounts of proanthocyanidins decline with leaf maturity and the two LAR isogenes have different patterns of expression with <italic>LAR1 </italic>showing seed-specific expression and insignificant levels in mature leaves and <italic>LAR2 </italic>readily present in different tissues, including leaves [<xref ref-type="bibr" rid="B58">58</xref>]. Hummer and Schreier reported that proanthocyanidins as condensed tannins can precipitate proteins and several methods using protein precipitation have been used to estimate proanthocyanidins in various agricultural products [<xref ref-type="bibr" rid="B59">59</xref>]. Using this approach, we showed that higher amounts of proanthocyanidins are present in virus-infected leaves than in virus-free leaves (Figure <xref ref-type="fig" rid="F6">6c</xref>) and the data correlate with strong induction of proanthocyanidin-specific genes; namely, <italic>LAR1</italic>, <italic>LAR2 </italic>and <italic>ANR</italic>. Since <italic>LAR </italic>and <italic>ANR </italic>genes provide two separate pathways for the synthesis of the terminal units of proanthocyanidin polymers, specific induction of <italic>LAR1 </italic>in virus-infected leaves (Figure <xref ref-type="fig" rid="F5">5a</xref>) would suggest that this gene may be contributing towards higher amounts of proanthocyanidins [<xref ref-type="bibr" rid="B58">58</xref>]. Overall, these results are compatible with our hypothesis that activation of the flavonoid biosynthetic pathway genes occurred in GLRaV-3-infected symptomatic leaves during post-<italic>véraison </italic>period resulting in <italic>de novo </italic>synthesis of specific flavonoid classes and leading to phenotypic expression of GLRD symptoms. It is also likely that these flavonoid compounds confer protection from oxidative damage and/or against attack by opportunistic pathogens due to their antioxidant and free radical scavenging properties [<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B60">60</xref>].</p><p>The use of more sensitive and gene-specific RT-qPCR technique enabled us to study relative abundance of the three highly homologous CHS gene family transcripts in virus-infected grapevine leaves. The results showed that three members of the CHS family (<italic>CHS1</italic>, <italic>CHS2 </italic>and <italic>CHS3</italic>) identified to date in grapevine, accumulated to varying levels, with <italic>CHS3 </italic>expression being significantly higher than the other two isogenes indicating its important role in color development in virus-infected leaves. This result is consistent with previous studies that <italic>CHS3</italic>, which is phylogenetically divergent from a cluster formed together by <italic>CHS1 </italic>and <italic>CHS2</italic>, was predominant in grape berry skins of red-fruited cultivars during coloration [<xref ref-type="bibr" rid="B61">61</xref>,<xref ref-type="bibr" rid="B62">62</xref>]. The exact role of <italic>CHS1 </italic>and <italic>CHS2 </italic>in the biosynthesis of flavonoids may be insignificant, although their expression was implicated in the production of proanthocyanidins in unpigmented tissues of both red- and white-fruited grapevine cultivars [<xref ref-type="bibr" rid="B61">61</xref>,<xref ref-type="bibr" rid="B62">62</xref>]. Among the two flavanone-3-hydroxylase isogenes, <italic>F3H1 </italic>showed higher expression levels than <italic>F3H2</italic>, and <italic>LAR1 </italic>of the two LAR isogenes of leucoanthocyanidin reductase was expressed at higher levels in virus-infected leaves. No such differential expression was observed in CHI isogenes. Thus, members of multigenic families appear to be induced differentially during the biosynthesis of flavonoids in virus-infected leaves of cv. Merlot showing GLRD symptoms.</p><p>Induced accumulation of anthocyanins and development of reddish-purple coloration in GLRaV-3 infected grapevine leaves appears to be analogous in some ways with stimulation of pigmentation in other plant species infected with taxonomically disparate viruses [<xref ref-type="bibr" rid="B63">63</xref>]. It has been shown that mottling symptoms present on the seed coats of virus-infected soybean plants or induction of floral anthocyanin pigmentation in petunias can be caused by suppression of CHS posttranscriptional gene silencing (PTGS) via the expression of a virus-encoded silencing suppressor protein and that the reversion to pigmentation in virus-infected tissues is correlated with an increase in the CHS mRNA level [<xref ref-type="bibr" rid="B64">64</xref>-<xref ref-type="bibr" rid="B66">66</xref>]. Since CHS is the first committed enzyme in the flavonoid biosynthetic pathway, it is tempting to speculate that modulation of PTGS suppression of CHS isogenes by GLRaV-3-encoded silencing suppressor protein(s) occurs during post-<italic>véraison </italic>in virus-infected grapevine leaves leading to a cascade of molecular events resulting in up-regulation of <italic>CHS3 </italic>and the ensuing production of secondary metabolites conferring color to otherwise green leaves. However, identification of silencing suppressors of GLRaV-3 awaits further validation of this possibility.</p><p>An alternative explanation would be that, since grapevine leaves begin to show GLRD symptoms only during post-<italic>véraison </italic>even though GLRaV-3 can be detected in infected plants throughout the season (i.e. both during pre- and post-<italic>véraison</italic>) and the virus is phloem-limited, appearance of reddish-purple coloration in symptomatic leaves could be due to a consequence of changes occurring in host metabolism and altered phloem translocation during <italic>véraison</italic>. In this context, up-regulation of the flavonoid biosynthetic pathway genes in GLRaV-3-infected Merlot leaves may not entirely represent a host defense response to pathogen infection and, therefore, our results differ somewhat from other compatible plant-pathogen interactions in grapevine leaves and hybrid poplar, where genes encoding key enzymes of the flavonoid biosynthetic pathway were strongly induced after infection with phytoplasma or fungal pathogens [<xref ref-type="bibr" rid="B67">67</xref>-<xref ref-type="bibr" rid="B70">70</xref>]. Nevertheless, the present study contributes towards a better understanding of virus-host interactions leading to the development of GLRD symptoms in red-fruited wine grape cultivars.</p><p>In the present study, we observed higher transcript levels of <italic>MybA1 </italic>gene that encodes a MYB transcription factor in virus-infected leaves (Figure <xref ref-type="fig" rid="F5">5b</xref>). Although other MYB transcription factors have recently been reported in grapevines, our rationale for analyzing only <italic>MybA1 </italic>was because of its main role in the regulation of anthocyanin biosynthesis via expression of the <italic>UFGT </italic>gene [<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B71">71</xref>]. However, further research is necessary to determine whether fine regulation of the flavonoid biosynthetic pathway genes in virus-infected leaves involves a combinatorial action(s) of different R2R3-MYB transcription factors, including basic helix-loop-helix (bHLH) and WD40 factors expressed in a spatially and temporally controlled manner [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B72">72</xref>].</p><p>It has been documented that the flavonoid biosynthetic pathway in fruits and vegetative tissues of plants is up-regulated by different environmental stress factors and in response to nutritional status [<xref ref-type="bibr" rid="B73">73</xref>]. It has also been suggested that in woody perennials like red-osier dogwood, anthocyanins accumulate during senescence to provide optical masking of chlorophyll in order to reduce the risk of photo-oxidative damage to leaf cells [<xref ref-type="bibr" rid="B74">74</xref>]. However, reduced levels of chlorophylls and carotenoids and higher amounts of specific classes of anthocyanins and the resulting changes in coloration of GLRaV-3-infected grapevine leaves during post-<italic>véraison </italic>may represent specific host-virus interactions as discussed above rather than a generalized abiotic stress response to environmental and/or nutritional imbalances. An integrated approach involving proteomic and metabolomic analyses combined with studies on modulation of cellular transcriptome would provide additional data for a comprehensive understanding of events that underlie changing colors of virus-infected grapevine leaves in red-fruited cultivars during post-<italic>véraison </italic>stage of berry development. Such information would also help to delineate grapevine's response to compatible virus infections from generic stress responses stimulated by a variety of abiotic and environmental factors.</p><p>Since berries in many red-fruited wine grape cultivars infected with GLRD show uneven ripening with reduced levels of extractable anthocyanins from berry skins (Naidu <italic>et al</italic>., unpublished results), the methodologies and results described in this study is providing leads for a deeper exploration of impacts of GLRD on berry skin pigments at the molecular level. In addition, there are several outstanding questions in GLRD-grapevine interactions that need to be addressed. They include: Do other red-fruited wine grape cultivars exhibit similar responses in the expression of flavonoid biosynthetic pathway genes and the profile of flavonoids to infection with GLRaV-3? Do genetically different GLRaVs trigger homologous responses in different red-fruited wine grape cultivars? Is the absence of dramatic symptoms in white-fruited wine grape cultivars an indication of non-responsiveness of the flavonoid biosynthetic pathway to virus infection? Indeed, GLRD-grapevine offers an excellent model system to address these questions.</p></sec><sec><title>Conclusions</title><p>In summary, we compared the relative expression of the flavonoid biosynthetic pathway genes between GLRaV-3-infected symptomatic and virus-free green leaves in a red-fruited wine grape cultivar (cv. Merlot) using RT-qPCR. The results showed up-regulation of genes in virus-infected symptomatic leaves suggesting modulation of the pathway towards <italic>de novo </italic>synthesis of certain classes of end-products and laid a foundation for deeper exploration of molecular mechanisms of biosynthesis and accumulation of flavonoids in virus-infected wine grape cultivars. The information on evaluation of reference genes suggests that validation of a set of reference genes as the most invariant internal controls for a particular experimental condition is essential for exploring genomics of plant-virus interactions in ecologically relevant, agriculturally important non-model perennial crops like grapevine under field conditions.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Plant samples</title><p>Leaf samples used in this study came from 10 year-old, own-rooted grapevines (cv. Merlot). The block is located near Prosser in Washington State, USA (46.2°N latitude, 119.8°W longitude), and the grapevines are grown under standard viticultural practices with drip irrigation. The grapevines were spaced 6 ft within rows and 8 ft between rows and the rows are in North-South orientation. The vineyard soil was classified as sandy loam. Plants for sampling were selected in such a way that individual grapevines exhibiting typical GLRD symptoms are adjacent to disease-free grapevines in a given row to minimize error in sampling and experimental results due to variations in growing conditions. Each pair of symptomatic and adjacent non-symptomatic grapevines was tested for different grapevine viruses by RT-PCR [<xref ref-type="bibr" rid="B75">75</xref>]. Mature leaves at the 4<sup>th </sup>and 5<sup>th </sup>node from the basal portion of primary canes showing typical symptoms of GLRD from virus-infected vines and comparable leaves from adjacent virus-free vines (Figure <xref ref-type="fig" rid="F2">2</xref>) were collected at the same time in mid September (representing post-<italic>véraison </italic>stage of berry development) to minimize variation due to developmental stage of leaves. The leaves were frozen immediately in liquid N<sub>2 </sub>upon collection in the field, transported to the lab in liquid N<sub>2 </sub>and stored at -80°C until required for RNA extraction. Leaves from individual grapevines were pooled and a pair of adjacent virus-infected and virus-free grapevines constituted one biological replicate. A total of five biological replicates (i.e. five virus-infected and five virus-free grapevines) were used for this study. Anecdotal evidence suggested that GLRD was introduced into the vineyard block via planting virus-infected cuttings. Hence, there is no bias in the age of virus-infected and virus-free grapevines used in this study.</p></sec><sec><title>Estimation of chlorophylls and carotenoids</title><p>Frozen leaf tissue (100 mg) was extracted in 80% acetone and total chlorophylls and carotenoids were estimated using a spectrophotometer [<xref ref-type="bibr" rid="B76">76</xref>,<xref ref-type="bibr" rid="B77">77</xref>]. Leaves from five virus-infected grapevines along with their respective controls were used separately and pigments estimated by two independent times using separate batches of tissue.</p></sec><sec><title>RNA isolation</title><p>Total RNA was isolated from leaves using Spectrum Plant Total RNA kit (Sigma-Aldrich, St Louis, MO, USA) following the manufacturer's instructions. Any contaminating genomic DNA was removed by on-column DNase I digestion (Qiagen Inc., Valencia, CA, USA). The integrity of RNA was verified by resolving in 1% formaldehyde-agarose gels and subsequent ethidium bromide staining. RNA purity was assessed based on absorbance ratio of 1.8 to 2.0 at 260/280 nm using Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA).</p></sec><sec><title>Primers, RT-PCR and analysis of gene sequences</title><p>Sequences of primers used in this study were retrieved from literature and used for amplifying partial gene-specific sequences. A list of primer pairs and amplicon lengths are provided in Table <xref ref-type="table" rid="T2">2</xref>. One μg of total RNA was reverse transcribed in 25 μl reaction mixture containing gene-specific complementary primer using Superscript III reverse transcriptase kit (Roche Diagnostics, Mannheim, Germany) by following the manufacturer's instructions. Reverse transcription (RT) was carried out at 50°C for 30 min followed by thirty five consecutive cycles of PCR amplification (denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s), with a final extension at 72°C for 5 min using 1 μM each of gene-specific forward and reverse primers. Amplified fragments specific to each gene were cloned separately into pCR 2.1-TOPO vector (Invitorgen, Carlsbad, CA) and recombinant clones purified using QIAGEN plasmid mini-prep kit (Qiagen Inc., Valencia, CA, USA). Two independent clones were sequenced in both orientations by automated DNA sequencing at Molecular Biology Core facility at the Center for Reproductive Biology, Washington State University, Pullman, WA, USA. The sequences were compared with corresponding sequences in GenBank with BLAST 2 sequences software (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/Blast.cgi">http://www.ncbi.nlm.nih.gov/Blast.cgi</ext-link>). The partial sequences of genes obtained in this study were deposited in GenBank with accession numbers <ext-link ext-link-type="gen" xlink:href="GU585850">GU585850</ext-link> to <ext-link ext-link-type="gen" xlink:href="GU585873">GU585873</ext-link>.</p></sec><sec><title>Reverse transcription-quantitative real-time PCR</title><p>One μg of total RNA was reverse transcribed in 20 μl reaction mixture containing oligo d(T)<sub>18 </sub>primer using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) by following the manufacturer's instructions. Quantitative real-time PCR (qPCR) reactions were performed in 384-well plates with LightCycler<sup>® </sup>480 real-time PCR instrument (Roche Diagnostics, Mannheim, Germany) using SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany) as described in the manufacturer's manual. All qPCR assays were performed with proper controls according to Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [<xref ref-type="bibr" rid="B49">49</xref>]. Each reaction was carried out in 20 μl reaction mixture containing 2 μl of cDNA, 0.5 μM each of gene-specific forward and reverse primer (Table <xref ref-type="table" rid="T2">2</xref>) and 10 μl of 2 × SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany). The following conditions were used for each qPCR assay: denaturation for 5 min at 95°C, followed by 45 cycles of PCR (10 s at 95°C for template denaturation, 10 s at 56°C for annealing and 30 s at 72°C for extension). All assays included no-RT and no-template controls to verify non-specific amplification. At the end of each qPCR, a melting curve analysis was performed over the range 65-97°C to determine the specificity of amplicons (Additional file <xref ref-type="supplementary-material" rid="S2">2</xref>, Figure S2). The amplicons were also resolved in 1.2% agarose gels, stained with ethidium bromide and visualized under UV light. cDNA from five biological replicates (virus-infected and virus-free leaves collected from five individual grapevines for each category) were used for qPCR analysis, and three technical replicates were analyzed for each biological replicate. Aliquots from the same cDNA were used in all technical replications.</p><p>LightCycler<sup>® </sup>480 Software (version 1.5; Roche Diagnostics) was used to analyze the data. We used the term <italic>quantification cycle </italic>(C<sub>q</sub>), instead of <italic>threshold cycle </italic>(C<sub>t</sub>), <italic>crossing point </italic>(C<sub>p</sub>) or <italic>take-off point </italic>(TOP) currently used in the literature, to describe the fractional qPCR cycle used for quantification according to the Real-Time PCR Data Markup Language (RDML) data standard [<xref ref-type="bibr" rid="B78">78</xref>]. The C<sub>q </sub>is defined as the number of cycles at which the fluorescence signal exceeds a specific threshold level of detection and is inversely correlated with the amount of target nucleic acid present in the reaction. qPCR efficiencies (E) were calculated using the equation E = 10<sup>-1/slope </sup>on a standard curve generated based on 10-fold dilution of gene-specific plasmid DNA (five dilution points, starting with 10 pg of respective plasmid DNA of each gene). The LightCycler<sup>® </sup>480 Software automatically calculates the efficiency and displays it on the analysis window.</p></sec><sec><title>Expression stability analysis of reference genes</title><p>Six candidate reference genes were selected for this study (Table <xref ref-type="table" rid="T1">1</xref>). Reference gene stability analyses were performed with the Microsoft excel-based <italic>geNorm </italic>software program available at <ext-link ext-link-type="uri" xlink:href="http://medgen.ugent.be/genorm/">http://medgen.ugent.be/genorm/</ext-link>[<xref ref-type="bibr" rid="B43">43</xref>]. The <italic>geNorm </italic>software uses pairwise comparison method to calculate gene expression stability measure "M" for a potential reference gene in a given cDNA sample panel. This measure was demonstrated in many studies to be valuable for selecting appropriate reference genes across several experimental conditions and treatments [<xref ref-type="bibr" rid="B45">45</xref>]. Using this program, the average expression stability value M (defined as the constancy of the expression ratio between two reference genes across samples) for each gene was obtained in a stepwise fashion excluding the gene with the highest M for the next calculation round. This process was repeated until only two genes remained. Genes with an M value below the default limit of M = 1.5 were considered as having acceptable expression stability (or suitability as normalizing gene) and genes with the lowest M values were taken as having the most stable expression [<xref ref-type="bibr" rid="B43">43</xref>].</p><p>The relative expression level of each candidate gene in a virus-infected sample (target) was analyzed over the virus-free sample (calibrator) using the <italic>geNorm </italic>software [<xref ref-type="bibr" rid="B43">43</xref>]. Briefly, the sample with the lowest C<sub>q </sub>value was assigned the value 1, and raw C<sub>q </sub>values were calculated using the delta-C<sub>q </sub>formula <italic>Q </italic>= <italic>E<sup>ΔCq</sup></italic>, where <italic>E </italic>is the primer efficiency and ΔC<sub>q </sub>is the sample with the highest expression (minimum C<sub>q </sub>value) from the data set minus C<sub>q </sub>value of the sample in question. The raw C<sub>q </sub>value (i.e. non-normalized) for each candidate gene in each sample was divided by the normalization factor (NF). Subsequently, the normalized value for each candidate gene in the target was divided by the normalized value for the corresponding gene in the calibrator to generate relative expression of flavonoid biosynthetic pathway genes in virus-infected leaves. The relative expression value for each gene represents mean of five biological replicates, with each replicate, in turn, representing a mean of three technical replicates. Each technical replicate, in turn, is a mean of duplicate values.</p></sec><sec><title>Extraction and HPLC analysis of anthocyanins and flavonols</title><p>Anthocyanins and flavonols were extracted and subsequently analyzed by reverse-phase high performance liquid chromatography (HPLC) as described by Downey and Rochfort with slight modifications [<xref ref-type="bibr" rid="B79">79</xref>]. Frozen leaf samples were ground into fine powder in a mortar using liquid N<sub>2 </sub>and 100 mg powder per sample was added separately to 1 ml of 50% methanol. The samples were sonicated for 20 min, clarified by centrifugation at 13,000 g for 10 min and the supernatant filtered through a 0.22 uM Nylon Costar Spin-X Centrifuge Tube Filters (Corning Incorporated, Corning, NY, USA). The filtrate was directly transferred to 1.5 ml brown vials and analyzed for anthocyanins and flavonols. The HPLC system consisted of an Agilent 1100 series with a quaternary pump, coupled with diode array and multiple wavelength detectors (Palo Alto, CA). Column temperature was maintained at 40°C and separation occurred under the following conditions and gradients: solvent A, water/formic acid (90:10); solvent B, methanol/formic acid (90:10); flow rate at 1.0 ml/min; column: C-18 SS Wakosil (150 mm×4.6 mm, 3 m packing; SGE, Ringwood, Australia) protected by an SGE C-18 guard column of the same packing material; gradient program: 0 min 6% B, 10 min 12% B, 15 min 18% B, 20 min 24% B, 30 min 30% B and 45 min 45% B. Anthocyanins and flavonols were monitored by photodiode array detection (DAD) with the detection wavelength set at 520 nm and 353 nm, respectively. Malvidin-3-glucoside (Extrasynthese Co., Genay, France), cyanidin-3-glucoside (Extrasynthese Co., Genay, France) and quercetin-3-glucuronide (Sigma-Aldrich, St Louis, MO, USA) were quantified with their respective standard curves over three orders of magnitudes, with linear correlation coefficients greater than 0.999. Myricetin-3-glucoside, quercetin-3-glucoside and peonidin-3-glucoside-<italic>p</italic>-coumarate were putatively identified according to spectra and retention time. Five biological replicates (virus-infected and virus-free leaves collected from five individual grapevines for each category) were used for these analyses and measurements for each sample were carried out in duplicate.</p></sec><sec><title>Estimation of proanthocyanidins</title><p>Proanthocyanidins (PAs) were extracted from leaves (collected from five virus-infected and five virus-free grapevines) as described in Harbertson <italic>et al</italic>. with some modifications and estimated as total tannins [<xref ref-type="bibr" rid="B80">80</xref>]. Briefly, 100 mg of frozen leaf tissue was extracted in 5 ml of 70% aqueous acetone (v/v) for 12 hours and filtered using Whatman No. 1 filter paper. Aqueous extract containing PAs was collected after removal of acetone using a rotary evaporator (Buchi Syncore, Buchi Switzerland) at 40°C and 525 mm Hg pressure. PAs were precipitated and resuspended in an alkaline detergent buffer and reacted subsequently with ferric chloride. The resulting reaction was monitored after 10 min at 510 nm using a Beckman DU 640 spectrophotometer (Beckman Instruments, St. Louis USA). A standard curve was developed using known amounts of (+)-catechin (a PA sub-unit) reacted with ferric chloride in an alkaline detergent buffer to interpret PA values. Concentration of PAs in leaf samples were reported in catechin equivalents (C.E.).</p></sec><sec><title>Statistical analysis</title><p>Differences in total chlorophylls and carotenoids, total anthocyanins, total flavonols, total proanthocyanidns and relative gene expression values between virus-infected and virus-free leaves were analyzed by one-way ANOVA, using the SigmaPlot 11 software. The confidence level of all analyses was set at 95% and values with <italic>p </italic>≤ 0.05 were considered significant.</p></sec></sec><sec><title>Authors' contributions</title><p>RAN conceived and coordinated the study. RAN and LRG designed the research. LRG collected samples, performed experiments related to gene expression by RT-qPCR and extraction of pigments, and analyzed the data. LFC and LRG performed HPLC analysis, and LFC and JFH analyzed the HPLC results. RAN and LRG wrote the manuscript with contributions from LFC and JFH. All authors read and approved the final manuscript.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="S1"><caption><title>Additional file 1</title><p><bold>Figure S1. Detection of GLRaV-3 in green veins and reddish-purple inter-veinal areas of virus-infected grapevine leaves by single tube RT-PCR</bold>. L and V represent reddish-purple inter-veinal areas and green veins, respectively, and 1408, 1508, 1409, 1509, 3109 are code numbers for virus-infected grapevines. Lanes N and P represent negative and positive controls, respectively, for GLRaV-3. Lane M represents DNA molecular weight markers used to estimate the size of virus-specific DNA fragment amplified by RT-PCR. The 546 nucleotide DNA band amplified in test samples (indicated by arrow on the right) represents a portion of the 70-kDa heat-shock protein homolog of GLRaV-3 [<xref ref-type="bibr" rid="B32">32</xref>,<xref ref-type="bibr" rid="B75">75</xref>].</p></caption><media xlink:href="1471-2229-10-187-S1.DOC" mimetype="application" mime-subtype="msword"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S2"><caption><title>Additional file 2</title><p><bold>Figure S2. Melting curve analysis of gene-specific amplicons</bold>. The blue colored horizontal line indicates base line generated with no template control and the red colored curve indicates dissociation curve for each gene. See legends for Figure 1 and 3 for names of genes.</p></caption><media xlink:href="1471-2229-10-187-S2.DOC" mimetype="application" mime-subtype="msword"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S3"><caption><title>Additional file 3</title><p><bold>Figure S3. Box plot representation of raw C<sub>q </sub>values obtained from amplification curves for the flavonoid biosynthetic pathway genes in GLRaV-3-infected and virus-free leaves</bold>. Lower and upper boundaries of each box indicate the 25<sup>th </sup>and the 75<sup>th </sup>percentile, respectively. Ranges are represented as bars (whiskers) below and above the box and indicate the 10<sup>th </sup>and 90<sup>th </sup>percentiles, respectively. The horizontal line in each box represents mean and outliers by (·). Suffix -D and -H for each gene denotes virus-infected and virus-free samples, respectively. See legend for Figure 1 for names of genes.</p></caption><media xlink:href="1471-2229-10-187-S3.DOC" mimetype="application" mime-subtype="msword"><caption><p>Click here for file</p></caption></media></supplementary-material></sec></body><back><sec><title>Acknowledgements</title><p>This work was supported in part by Agricultural Research Center of the College of Agricultural, Human, and Natural Resource Sciences, Washington State University, Washington Wine Commission's Wine Advisory Committee, and USDA-NIFA Specialty Crop Research Initiative Award No. 2009-51181-06027. PPNS # 0540, Department of Plant Pathology, College of Agricultural, Human, and Natural Resource Sciences, Agricultural Research Center Project No. WNPO 0616, Washington State University, Pullman, WA 99164-6240, USA. We sincerely thank Dr. Nnadozie Oraguzie for access to LightCycler<sup>® </sup>480 real-time PCR instrument, and Drs. Pat Okubara and Roy Navarre for helpful comments and suggestions.</p></sec><ref-list><ref id="B1"><mixed-citation publication-type="journal"><name><surname>Holton</surname><given-names>TA</given-names></name><name><surname>Cornish</surname><given-names>EC</given-names></name><article-title>Genetics and biochemistry of anthocyanin biosynthesis</article-title><source>Plant Cell</source><year>1995</year><volume>7</volume><issue>7</issue><fpage>1071</fpage><lpage>1083</lpage><pub-id pub-id-type="doi">10.1105/tpc.7.7.1071</pub-id><pub-id pub-id-type="pmid">12242398</pub-id></mixed-citation></ref><ref id="B2"><mixed-citation publication-type="journal"><name><surname>Winkel-Shirley</surname><given-names>B</given-names></name><article-title>Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology</article-title><source>Plant Physiol</source><year>2001</year><volume>126</volume><issue>2</issue><fpage>485</fpage><lpage>493</lpage><pub-id pub-id-type="doi">10.1104/pp.126.2.485</pub-id><pub-id pub-id-type="pmid">11402179</pub-id></mixed-citation></ref><ref id="B3"><mixed-citation publication-type="journal"><name><surname>Koes</surname><given-names>R</given-names></name><name><surname>Verweij</surname><given-names>W</given-names></name><name><surname>Quattrocchio</surname><given-names>F</given-names></name><article-title>Flavonoids: a colorful model for the regulation and evolution of biochemical pathways</article-title><source>Trends Plant Sci</source><year>2005</year><volume>10</volume><issue>5</issue><fpage>236</fpage><lpage>242</lpage><pub-id pub-id-type="doi">10.1016/j.tplants.2005.03.002</pub-id><pub-id pub-id-type="pmid">15882656</pub-id></mixed-citation></ref><ref id="B4"><mixed-citation publication-type="journal"><name><surname>Peters</surname><given-names>DJ</given-names></name><name><surname>Constabel</surname><given-names>CP</given-names></name><article-title>Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (<italic>Populus tremuloides</italic>)</article-title><source>Plant J</source><year>2002</year><volume>32</volume><issue>5</issue><fpage>701</fpage><lpage>712</lpage><pub-id pub-id-type="doi">10.1046/j.1365-313X.2002.01458.x</pub-id><pub-id pub-id-type="pmid">12472686</pub-id></mixed-citation></ref><ref id="B5"><mixed-citation publication-type="book"><name><surname>Gould</surname><given-names>KS</given-names></name><name><surname>Lister</surname><given-names>C</given-names></name><person-group person-group-type="editor">Andersen M, Markham KR</person-group><article-title>Flavonoid function in plants</article-title><source>Flavonoids: Chemistry, biochemistry and applications</source><year>2006</year><publisher-name>Boca Raton, FL: CRC Press</publisher-name><fpage>397</fpage><lpage>441</lpage></mixed-citation></ref><ref id="B6"><mixed-citation publication-type="journal"><name><surname>Dixon</surname><given-names>RA</given-names></name><name><surname>Paiva</surname><given-names>NL</given-names></name><article-title>Stress-induced phenylpropanoid metabolism</article-title><source>Plant Cell</source><year>1995</year><volume>7</volume><issue>7</issue><fpage>1085</fpage><lpage>1097</lpage><pub-id pub-id-type="doi">10.1105/tpc.7.7.1085</pub-id><pub-id pub-id-type="pmid">12242399</pub-id></mixed-citation></ref><ref id="B7"><mixed-citation publication-type="journal"><name><surname>Chalker-Scott</surname><given-names>L</given-names></name><article-title>Environmental significance of anthocyanins in plant stress responses</article-title><source>Photochem Photobiol</source><year>1999</year><volume>70</volume><issue>1</issue><fpage>1</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1111/j.1751-1097.1999.tb01944.x</pub-id></mixed-citation></ref><ref id="B8"><mixed-citation publication-type="journal"><name><surname>Hernández</surname><given-names>I</given-names></name><name><surname>Alegre</surname><given-names>L</given-names></name><name><surname>Van Breusegem</surname><given-names>F</given-names></name><name><surname>Munné-Bosch</surname><given-names>S</given-names></name><article-title>How relevant are flavonoids as antioxidants in plants?</article-title><source>Trends Plant Sci</source><year>2009</year><volume>14</volume><fpage>125</fpage><lpage>132</lpage><pub-id pub-id-type="doi">10.1016/j.tplants.2008.12.003</pub-id><pub-id pub-id-type="pmid">19230744</pub-id></mixed-citation></ref><ref id="B9"><mixed-citation publication-type="journal"><name><surname>Korkina</surname><given-names>LG</given-names></name><article-title>Phenylpropanoids as naturally occurring antioxidants: from plant defense to human health</article-title><source>Cell Mol Biol</source><year>2007</year><volume>53</volume><fpage>15</fpage><lpage>25</lpage><pub-id pub-id-type="pmid">17519109</pub-id></mixed-citation></ref><ref id="B10"><mixed-citation publication-type="journal"><name><surname>Ververidis</surname><given-names>F</given-names></name><name><surname>Trantas</surname><given-names>E</given-names></name><name><surname>Douglas</surname><given-names>C</given-names></name><name><surname>Vollmer</surname><given-names>G</given-names></name><name><surname>Kretzschmar</surname><given-names>G</given-names></name><name><surname>Panopoulos</surname><given-names>N</given-names></name><article-title>Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part I: chemical diversity, impacts on plant biology and human health</article-title><source>Biotechnol J</source><year>2007</year><volume>2</volume><fpage>1214</fpage><lpage>1234</lpage><pub-id pub-id-type="doi">10.1002/biot.200700084</pub-id><pub-id pub-id-type="pmid">17935117</pub-id></mixed-citation></ref><ref id="B11"><mixed-citation publication-type="journal"><name><surname>Crozier</surname><given-names>A</given-names></name><name><surname>Jaganath</surname><given-names>IB</given-names></name><name><surname>Clifford</surname><given-names>MN</given-names></name><article-title>Dietary phenolics: chemistry, bioavailability and effects on health</article-title><source>Nat Prod Rep</source><year>2009</year><volume>26</volume><issue>8</issue><fpage>1001</fpage><lpage>1043</lpage><pub-id pub-id-type="doi">10.1039/b802662a</pub-id><pub-id pub-id-type="pmid">19636448</pub-id></mixed-citation></ref><ref id="B12"><mixed-citation publication-type="journal"><name><surname>Espley</surname><given-names>RV</given-names></name><name><surname>Hellens</surname><given-names>RP</given-names></name><name><surname>Putterill</surname><given-names>J</given-names></name><name><surname>Stevenson</surname><given-names>DE</given-names></name><name><surname>Kutty-Amma</surname><given-names>S</given-names></name><name><surname>Allan</surname><given-names>AC</given-names></name><article-title>Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10</article-title><source>Plant J</source><year>2007</year><volume>49</volume><issue>3</issue><fpage>414</fpage><lpage>427</lpage><pub-id pub-id-type="doi">10.1111/j.1365-313X.2006.02964.x</pub-id><pub-id pub-id-type="pmid">17181777</pub-id></mixed-citation></ref><ref id="B13"><mixed-citation publication-type="journal"><name><surname>Shirley</surname><given-names>BW</given-names></name><name><surname>Kubasek</surname><given-names>WL</given-names></name><name><surname>Storz</surname><given-names>G</given-names></name><name><surname>Bruggemann</surname><given-names>E</given-names></name><name><surname>Koornneef</surname><given-names>M</given-names></name><name><surname>Ausubel</surname><given-names>FM</given-names></name><name><surname>Goodman</surname><given-names>HM</given-names></name><article-title>Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis</article-title><source>Plant J</source><year>1995</year><volume>8</volume><issue>5</issue><fpage>659</fpage><lpage>671</lpage><pub-id pub-id-type="doi">10.1046/j.1365-313X.1995.08050659.x</pub-id><pub-id pub-id-type="pmid">8528278</pub-id></mixed-citation></ref><ref id="B14"><mixed-citation publication-type="journal"><name><surname>Bogs</surname><given-names>J</given-names></name><name><surname>Ebadi</surname><given-names>A</given-names></name><name><surname>McDavid</surname><given-names>D</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><article-title>Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development</article-title><source>Plant Physiol</source><year>2006</year><volume>140</volume><issue>1</issue><fpage>279</fpage><lpage>291</lpage><pub-id pub-id-type="doi">10.1104/pp.105.073262</pub-id><pub-id pub-id-type="pmid">16377741</pub-id></mixed-citation></ref><ref id="B15"><mixed-citation publication-type="journal"><name><surname>Castellarin</surname><given-names>SD</given-names></name><name><surname>Di Gaspero</surname><given-names>G</given-names></name><name><surname>Marconi</surname><given-names>R</given-names></name><name><surname>Nonis</surname><given-names>A</given-names></name><name><surname>Peterlunger</surname><given-names>E</given-names></name><name><surname>Paillard</surname><given-names>S</given-names></name><name><surname>Adam-Blondon</surname><given-names>AF</given-names></name><name><surname>Testolin</surname><given-names>R</given-names></name><article-title>Colour variation in red grapevines (<italic>Vitis vinifera </italic>L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin</article-title><source>BMC Genomics</source><year>2006</year><volume>7</volume><fpage>12</fpage><pub-id pub-id-type="doi">10.1186/1471-2164-7-12</pub-id><pub-id pub-id-type="pmid">16433923</pub-id></mixed-citation></ref><ref id="B16"><mixed-citation publication-type="journal"><name><surname>Havsteen</surname><given-names>BH</given-names></name><article-title>The biochemistry and medical significance of the flavonoids</article-title><source>Pharmacol Ther</source><year>2002</year><volume>96</volume><issue>2-3</issue><fpage>67</fpage><lpage>202</lpage><pub-id pub-id-type="doi">10.1016/S0163-7258(02)00298-X</pub-id><pub-id pub-id-type="pmid">12453566</pub-id></mixed-citation></ref><ref id="B17"><mixed-citation publication-type="journal"><name><surname>Kennedy</surname><given-names>JA</given-names></name><name><surname>Hayasaka</surname><given-names>Y</given-names></name><name><surname>Vidal</surname><given-names>S</given-names></name><name><surname>Waters</surname><given-names>EJ</given-names></name><name><surname>Jones</surname><given-names>GP</given-names></name><article-title>Composition of grape skin proanthocyanidins at different stages of berry development</article-title><source>J Agric Food Chem</source><year>2001</year><volume>49</volume><issue>11</issue><fpage>5348</fpage><lpage>5355</lpage><pub-id pub-id-type="doi">10.1021/jf010758h</pub-id><pub-id pub-id-type="pmid">11714327</pub-id></mixed-citation></ref><ref id="B18"><mixed-citation publication-type="journal"><name><surname>Adams</surname><given-names>DO</given-names></name><article-title>Phenolics and ripening in grape berries</article-title><source>Am J Enol Vitic</source><year>2006</year><volume>57</volume><issue>3</issue><fpage>249</fpage><lpage>256</lpage></mixed-citation></ref><ref id="B19"><mixed-citation publication-type="journal"><name><surname>Grimplet</surname><given-names>J</given-names></name><name><surname>Deluc</surname><given-names>LG</given-names></name><name><surname>Tillett</surname><given-names>RL</given-names></name><name><surname>Wheatley</surname><given-names>MD</given-names></name><name><surname>Schlauch</surname><given-names>KA</given-names></name><name><surname>Cramer</surname><given-names>GR</given-names></name><name><surname>Cushman</surname><given-names>JC</given-names></name><article-title>Tissue-specific mRNA expression profiling in grape berry tissues</article-title><source>BMC Genomics</source><year>2007</year><volume>8</volume><fpage>187</fpage><pub-id pub-id-type="doi">10.1186/1471-2164-8-187</pub-id><pub-id pub-id-type="pmid">17584945</pub-id></mixed-citation></ref><ref id="B20"><mixed-citation publication-type="journal"><name><surname>Deluc</surname><given-names>L</given-names></name><name><surname>Bogs</surname><given-names>J</given-names></name><name><surname>Walker</surname><given-names>AR</given-names></name><name><surname>Ferrier</surname><given-names>T</given-names></name><name><surname>Decendit</surname><given-names>A</given-names></name><name><surname>Merillon</surname><given-names>JM</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><name><surname>Barrieu</surname><given-names>F</given-names></name><article-title>The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries</article-title><source>Plant Physiol</source><year>2008</year><volume>147</volume><issue>4</issue><fpage>2041</fpage><lpage>2053</lpage><pub-id pub-id-type="doi">10.1104/pp.108.118919</pub-id><pub-id pub-id-type="pmid">18539781</pub-id></mixed-citation></ref><ref id="B21"><mixed-citation publication-type="journal"><name><surname>Kobayashi</surname><given-names>S</given-names></name><name><surname>Goto-Yamamoto</surname><given-names>N</given-names></name><name><surname>Hirochika</surname><given-names>H</given-names></name><article-title>Retrotransposon-induced mutations in grape skin color</article-title><source>Science</source><year>2004</year><volume>304</volume><issue>5673</issue><fpage>982</fpage><pub-id pub-id-type="doi">10.1126/science.1095011</pub-id><pub-id pub-id-type="pmid">15143274</pub-id></mixed-citation></ref><ref id="B22"><mixed-citation publication-type="journal"><name><surname>Walker</surname><given-names>AR</given-names></name><name><surname>Lee</surname><given-names>E</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><article-title>Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus</article-title><source>Plant Mol Biol</source><year>2006</year><volume>62</volume><issue>4-5</issue><fpage>623</fpage><lpage>635</lpage><pub-id pub-id-type="doi">10.1007/s11103-006-9043-9</pub-id><pub-id pub-id-type="pmid">16932847</pub-id></mixed-citation></ref><ref id="B23"><mixed-citation publication-type="journal"><name><surname>Walker</surname><given-names>AR</given-names></name><name><surname>Lee</surname><given-names>E</given-names></name><name><surname>Bogs</surname><given-names>J</given-names></name><name><surname>McDavid</surname><given-names>DA</given-names></name><name><surname>Thomas</surname><given-names>MR</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><article-title>White grapes arose through the mutation of two similar and adjacent regulatory genes</article-title><source>Plant J</source><year>2007</year><volume>49</volume><issue>5</issue><fpage>772</fpage><lpage>785</lpage><pub-id pub-id-type="doi">10.1111/j.1365-313X.2006.02997.x</pub-id><pub-id pub-id-type="pmid">17316172</pub-id></mixed-citation></ref><ref id="B24"><mixed-citation publication-type="journal"><name><surname>This</surname><given-names>P</given-names></name><name><surname>Lacombe</surname><given-names>T</given-names></name><name><surname>Cadle-Davidson</surname><given-names>M</given-names></name><name><surname>Owens</surname><given-names>CL</given-names></name><article-title>Wine grape (<italic>Vitis vinifera </italic>L.) color associates with allelic variation in the domestication gene VvmybA1</article-title><source>Theor Appl Genet</source><year>2007</year><volume>114</volume><issue>4</issue><fpage>723</fpage><lpage>730</lpage><pub-id pub-id-type="doi">10.1007/s00122-006-0472-2</pub-id><pub-id pub-id-type="pmid">17221259</pub-id></mixed-citation></ref><ref id="B25"><mixed-citation publication-type="journal"><name><surname>Boss</surname><given-names>PK</given-names></name><name><surname>Davies</surname><given-names>C</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><article-title>Anthocyanin composition and anthocyanin pathway gene expression in grapevine sports differing in berry skin colour</article-title><source>Aus J Grape Wine Res</source><year>1996</year><volume>2</volume><issue>3</issue><fpage>163</fpage><lpage>170</lpage><pub-id pub-id-type="doi">10.1111/j.1755-0238.1996.tb00104.x</pub-id></mixed-citation></ref><ref id="B26"><mixed-citation publication-type="journal"><name><surname>Kobayashi</surname><given-names>S</given-names></name><name><surname>Ishimaru</surname><given-names>M</given-names></name><name><surname>Ding</surname><given-names>CK</given-names></name><name><surname>Yakushiji</surname><given-names>H</given-names></name><name><surname>Goto</surname><given-names>N</given-names></name><article-title>Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (<italic>Vitis vinifera</italic>) and their sports with red skin</article-title><source>Plant Sci</source><year>2001</year><volume>160</volume><issue>3</issue><fpage>543</fpage><lpage>550</lpage><pub-id pub-id-type="doi">10.1016/S0168-9452(00)00425-8</pub-id><pub-id pub-id-type="pmid">11166442</pub-id></mixed-citation></ref><ref id="B27"><mixed-citation publication-type="journal"><name><surname>Boss</surname><given-names>PK</given-names></name><name><surname>Davies</surname><given-names>C</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><article-title>Expression of anthocyanin biosynthesis pathway genes in red and white grapes</article-title><source>Plant Mol Biol</source><year>1996</year><volume>32</volume><issue>3</issue><fpage>565</fpage><lpage>569</lpage><pub-id pub-id-type="doi">10.1007/BF00019111</pub-id><pub-id pub-id-type="pmid">8980508</pub-id></mixed-citation></ref><ref id="B28"><mixed-citation publication-type="journal"><name><surname>Verries</surname><given-names>C</given-names></name><name><surname>Guiraud</surname><given-names>JL</given-names></name><name><surname>Souquet</surname><given-names>JM</given-names></name><name><surname>Vialet</surname><given-names>S</given-names></name><name><surname>Terrier</surname><given-names>N</given-names></name><name><surname>Olle</surname><given-names>D</given-names></name><article-title>Validation of an extraction method on whole pericarp of grape berry (<italic>Vitis vinifera </italic>L. cv. Shiraz) to study biochemical and molecular aspects of flavan-3-ol synthesis during berry development</article-title><source>J Agric Food Chem</source><year>2008</year><volume>56</volume><issue>14</issue><fpage>5896</fpage><lpage>5904</lpage><pub-id pub-id-type="doi">10.1021/jf800028k</pub-id><pub-id pub-id-type="pmid">18582087</pub-id></mixed-citation></ref><ref id="B29"><mixed-citation publication-type="journal"><name><surname>Liakopoulos</surname><given-names>G</given-names></name><name><surname>Nikolopoulos</surname><given-names>D</given-names></name><name><surname>Klouvatou</surname><given-names>A</given-names></name><name><surname>Vekkos</surname><given-names>KA</given-names></name><name><surname>Manetas</surname><given-names>Y</given-names></name><name><surname>Karabourniotis</surname><given-names>G</given-names></name><article-title>The photoprotective role of epidermal anthocyanins and surface pubescence in young leaves of grapevine (<italic>Vitis vinifera</italic>)</article-title><source>Ann Bot</source><year>2006</year><volume>98</volume><issue>1</issue><fpage>257</fpage><lpage>265</lpage><pub-id pub-id-type="doi">10.1093/aob/mcl097</pub-id><pub-id pub-id-type="pmid">16704996</pub-id></mixed-citation></ref><ref id="B30"><mixed-citation publication-type="other"><name><surname>Rayapati</surname><given-names>AN</given-names></name><name><surname>O'Neil</surname><given-names>S</given-names></name><name><surname>Walsh</surname><given-names>D</given-names></name><article-title>Grapevine leafroll disease. WSU Extension Bulletin</article-title><year>2008</year><fpage>20</fpage><ext-link ext-link-type="uri" xlink:href="http://cru.cahe.wsu.edu/CEPublications/eb2027e/eb2027e.pdf">http://cru.cahe.wsu.edu/CEPublications/eb2027e/eb2027e.pdf</ext-link><comment>EB2027E</comment></mixed-citation></ref><ref id="B31"><mixed-citation publication-type="book"><name><surname>Martelli</surname><given-names>GP</given-names></name><article-title>Grapevine virology highlights 2006-09</article-title><source>Proceedings of the 16th Meeting of International Council for the study of Virus and Virus-like Diseases of Grapevine: 31 Aug - 4 Sep 2009</source><year>2009</year><publisher-name>Dijon, France</publisher-name><fpage>15</fpage><lpage>23</lpage></mixed-citation></ref><ref id="B32"><mixed-citation publication-type="journal"><name><surname>Jarugula</surname><given-names>S</given-names></name><name><surname>Gowda</surname><given-names>S</given-names></name><name><surname>Dawson</surname><given-names>WO</given-names></name><name><surname>Naidu</surname><given-names>RA</given-names></name><article-title>3'-coterminal subgenomic RNAs and putative cis-acting elements of <italic>Grapevine leafroll-associated virus 3 </italic>reveals 'unique' features of gene expression strategy in the genus <italic>Ampelovirus</italic></article-title><source>Virology J</source><year>2010</year><volume>7</volume><fpage>180</fpage><pub-id pub-id-type="doi">10.1186/1743-422X-7-180</pub-id></mixed-citation></ref><ref id="B33"><mixed-citation publication-type="journal"><name><surname>Cabaleiro</surname><given-names>C</given-names></name><name><surname>Segura</surname><given-names>A</given-names></name><name><surname>Garcia-berrios</surname><given-names>JJ</given-names></name><article-title>Effects of grapevine leafroll-associated virus 3 on the physiology and must of <italic>Vitis vinifera </italic>L. cv. Albarifio following contamination in the field</article-title><source>Am J Enol Vitic</source><year>1999</year><volume>50</volume><issue>1</issue><fpage>40</fpage><lpage>44</lpage></mixed-citation></ref><ref id="B34"><mixed-citation publication-type="book"><name><surname>Golino</surname><given-names>DA</given-names></name><name><surname>Wolpert</surname><given-names>J</given-names></name><name><surname>Sim</surname><given-names>ST</given-names></name><name><surname>Benz</surname><given-names>J</given-names></name><name><surname>Anderson</surname><given-names>M</given-names></name><name><surname>Rowhani</surname><given-names>A</given-names></name><article-title>Virus effects on vine growth and fruit components of Cabernet Sauvignon on six rootstocks</article-title><source>Proceedings of the 16th Meeting of International Council for the study of Virus and Virus-like Diseases of Grapevine: 31 Aug - 4 Sep 2009</source><year>2009</year><publisher-name>Dijon, France</publisher-name><fpage>245</fpage><lpage>246</lpage></mixed-citation></ref><ref id="B35"><mixed-citation publication-type="journal"><name><surname>Komar</surname><given-names>V</given-names></name><name><surname>Vigne</surname><given-names>E</given-names></name><name><surname>Demangeat</surname><given-names>G</given-names></name><name><surname>Lemaire</surname><given-names>O</given-names></name><name><surname>Fuchs</surname><given-names>M</given-names></name><article-title>Comparative performance analysis of virus-infected <italic>Vitis vinifera </italic>cv. Savagnin rose grafted onto three rootstocks</article-title><source>Am J Enol Vitic</source><year>2010</year><volume>61</volume><issue>1</issue><fpage>68</fpage><lpage>73</lpage></mixed-citation></ref><ref id="B36"><mixed-citation publication-type="journal"><name><surname>Huggett</surname><given-names>J</given-names></name><name><surname>Dheda</surname><given-names>K</given-names></name><name><surname>Bustin</surname><given-names>S</given-names></name><name><surname>Zumla</surname><given-names>A</given-names></name><article-title>Real-time RT-PCR normalisation; strategies and considerations</article-title><source>Genes Immun</source><year>2005</year><volume>6</volume><issue>4</issue><fpage>279</fpage><lpage>284</lpage><pub-id pub-id-type="doi">10.1038/sj.gene.6364190</pub-id><pub-id pub-id-type="pmid">15815687</pub-id></mixed-citation></ref><ref id="B37"><mixed-citation publication-type="journal"><name><surname>Nicot</surname><given-names>N</given-names></name><name><surname>Hausman</surname><given-names>JF</given-names></name><name><surname>Hoffmann</surname><given-names>L</given-names></name><name><surname>Evers</surname><given-names>D</given-names></name><article-title>Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress</article-title><source>J Exp Bot</source><year>2005</year><volume>56</volume><issue>421</issue><fpage>2907</fpage><lpage>2914</lpage><pub-id pub-id-type="doi">10.1093/jxb/eri285</pub-id><pub-id pub-id-type="pmid">16188960</pub-id></mixed-citation></ref><ref id="B38"><mixed-citation publication-type="journal"><name><surname>Mackay</surname><given-names>IM</given-names></name><name><surname>Arden</surname><given-names>KE</given-names></name><name><surname>Nitsche</surname><given-names>A</given-names></name><article-title>Real-time PCR in virology</article-title><source>Nucl Acids Res</source><year>2002</year><volume>30</volume><issue>6</issue><fpage>1292</fpage><lpage>1305</lpage><pub-id pub-id-type="doi">10.1093/nar/30.6.1292</pub-id><pub-id pub-id-type="pmid">11884626</pub-id></mixed-citation></ref><ref id="B39"><mixed-citation publication-type="journal"><name><surname>Bustin</surname><given-names>S</given-names></name><article-title>Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems</article-title><source>J Mol Endocrinol</source><year>2002</year><volume>29</volume><issue>1</issue><fpage>23</fpage><lpage>39</lpage><pub-id pub-id-type="doi">10.1677/jme.0.0290023</pub-id><pub-id pub-id-type="pmid">12200227</pub-id></mixed-citation></ref><ref id="B40"><mixed-citation publication-type="journal"><name><surname>Czechowski</surname><given-names>T</given-names></name><name><surname>Bari</surname><given-names>RP</given-names></name><name><surname>Stitt</surname><given-names>M</given-names></name><name><surname>Scheible</surname><given-names>WR</given-names></name><name><surname>Udvardi</surname><given-names>MK</given-names></name><article-title>Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes</article-title><source>Plant J</source><year>2004</year><volume>38</volume><issue>2</issue><fpage>366</fpage><lpage>379</lpage><pub-id pub-id-type="doi">10.1111/j.1365-313X.2004.02051.x</pub-id><pub-id pub-id-type="pmid">15078338</pub-id></mixed-citation></ref><ref id="B41"><mixed-citation publication-type="journal"><name><surname>Gachon</surname><given-names>C</given-names></name><name><surname>Mingam</surname><given-names>A</given-names></name><name><surname>Charrier</surname><given-names>B</given-names></name><article-title>Real-time PCR: what relevance to plant studies?</article-title><source>J Exp Bot</source><year>2004</year><volume>55</volume><issue>402</issue><fpage>1445</fpage><lpage>1454</lpage><pub-id pub-id-type="doi">10.1093/jxb/erh181</pub-id><pub-id pub-id-type="pmid">15208338</pub-id></mixed-citation></ref><ref id="B42"><mixed-citation publication-type="journal"><name><surname>Bustin</surname><given-names>SA</given-names></name><name><surname>Benes</surname><given-names>V</given-names></name><name><surname>Nolan</surname><given-names>T</given-names></name><name><surname>Pfaffl</surname><given-names>MW</given-names></name><article-title>Quantitative real-time RT-PCR--a perspective</article-title><source>J Mol Endocrinol</source><year>2005</year><volume>34</volume><issue>3</issue><fpage>597</fpage><lpage>601</lpage><pub-id pub-id-type="doi">10.1677/jme.1.01755</pub-id><pub-id pub-id-type="pmid">15956331</pub-id></mixed-citation></ref><ref id="B43"><mixed-citation publication-type="journal"><name><surname>Vandesompele</surname><given-names>J</given-names></name><name><surname>De Preter</surname><given-names>K</given-names></name><name><surname>Pattyn</surname><given-names>F</given-names></name><name><surname>Poppe</surname><given-names>B</given-names></name><name><surname>Van Roy</surname><given-names>N</given-names></name><name><surname>De Paepe</surname><given-names>A</given-names></name><name><surname>Speleman</surname><given-names>F</given-names></name><article-title>Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes</article-title><source>Genome Biol</source><year>2002</year><volume>3</volume><issue>7</issue><fpage>research0034.0031</fpage><lpage>research0034.0011</lpage><pub-id pub-id-type="doi">10.1186/gb-2002-3-7-research0034</pub-id></mixed-citation></ref><ref id="B44"><mixed-citation publication-type="journal"><name><surname>Rieu</surname><given-names>I</given-names></name><name><surname>Powers</surname><given-names>SJ</given-names></name><article-title>Real-time quantitative RT-PCR: Design, calculations, and statistics</article-title><source>Plant Cell</source><year>2009</year><volume>21</volume><fpage>1031</fpage><lpage>1033</lpage><pub-id pub-id-type="doi">10.1105/tpc.109.066001</pub-id><pub-id pub-id-type="pmid">19395682</pub-id></mixed-citation></ref><ref id="B45"><mixed-citation publication-type="journal"><name><surname>Radonic</surname><given-names>A</given-names></name><name><surname>Thulke</surname><given-names>S</given-names></name><name><surname>Mackay</surname><given-names>IM</given-names></name><name><surname>Landt</surname><given-names>O</given-names></name><name><surname>Siegert</surname><given-names>W</given-names></name><name><surname>Nitsche</surname><given-names>A</given-names></name><article-title>Guideline to reference gene selection for quantitative real-time PCR</article-title><source>Biochem Biophys Res Commun</source><year>2004</year><volume>313</volume><issue>4</issue><fpage>856</fpage><lpage>862</lpage><pub-id pub-id-type="doi">10.1016/j.bbrc.2003.11.177</pub-id><pub-id pub-id-type="pmid">14706621</pub-id></mixed-citation></ref><ref id="B46"><mixed-citation publication-type="journal"><name><surname>Remans</surname><given-names>T</given-names></name><name><surname>Smeets</surname><given-names>K</given-names></name><name><surname>Opdenakker</surname><given-names>K</given-names></name><name><surname>Mathijsen</surname><given-names>D</given-names></name><name><surname>Vangronsveld</surname><given-names>J</given-names></name><name><surname>Cuypers</surname><given-names>A</given-names></name><article-title>Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations</article-title><source>Planta</source><year>2008</year><volume>227</volume><issue>6</issue><fpage>1343</fpage><lpage>1349</lpage><pub-id pub-id-type="doi">10.1007/s00425-008-0706-4</pub-id><pub-id pub-id-type="pmid">18273637</pub-id></mixed-citation></ref><ref id="B47"><mixed-citation publication-type="journal"><name><surname>Gutierrez</surname><given-names>L</given-names></name><name><surname>Mauriat</surname><given-names>M</given-names></name><name><surname>Guenin</surname><given-names>S</given-names></name><name><surname>Pelloux</surname><given-names>J</given-names></name><name><surname>Lefebvre</surname><given-names>JF</given-names></name><name><surname>Louvet</surname><given-names>R</given-names></name><name><surname>Rusterucci</surname><given-names>C</given-names></name><name><surname>Moritz</surname><given-names>T</given-names></name><name><surname>Guerineau</surname><given-names>F</given-names></name><name><surname>Bellini</surname><given-names>C</given-names></name><name><surname>Van Wuytswinkel</surname></name><article-title>The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants</article-title><source>Plant Biotechnol J</source><year>2008</year><volume>6</volume><issue>6</issue><fpage>609</fpage><lpage>618</lpage><pub-id pub-id-type="doi">10.1111/j.1467-7652.2008.00346.x</pub-id><pub-id pub-id-type="pmid">18433420</pub-id></mixed-citation></ref><ref id="B48"><mixed-citation publication-type="journal"><name><surname>Gutierrez</surname><given-names>L</given-names></name><name><surname>Mauriat</surname><given-names>M</given-names></name><name><surname>Pelloux</surname><given-names>J</given-names></name><name><surname>Bellini</surname><given-names>C</given-names></name><name><surname>Van Wuytswinkel</surname><given-names>O</given-names></name><article-title>Towards a systematic validation of references in real-time RT-PCR</article-title><source>Plant Cell</source><year>2008</year><volume>20</volume><issue>7</issue><fpage>1734</fpage><lpage>1735</lpage><pub-id pub-id-type="doi">10.1105/tpc.108.059774</pub-id><pub-id pub-id-type="pmid">18664615</pub-id></mixed-citation></ref><ref id="B49"><mixed-citation publication-type="journal"><name><surname>Bustin</surname><given-names>SA</given-names></name><name><surname>Benes</surname><given-names>V</given-names></name><name><surname>Garson</surname><given-names>JA</given-names></name><name><surname>Hellemans</surname><given-names>J</given-names></name><name><surname>Huggett</surname><given-names>J</given-names></name><name><surname>Kubista</surname><given-names>M</given-names></name><name><surname>Mueller</surname><given-names>R</given-names></name><name><surname>Nolan</surname><given-names>T</given-names></name><name><surname>Pfaffl</surname><given-names>MW</given-names></name><name><surname>Shipley</surname><given-names>GL</given-names></name><name><surname>Vandesompele</surname><given-names>J</given-names></name><name><surname>Wittwer</surname><given-names>CT</given-names></name><article-title>The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments</article-title><source>Clin Chem</source><year>2009</year><volume>55</volume><issue>4</issue><fpage>611</fpage><lpage>622</lpage><pub-id pub-id-type="doi">10.1373/clinchem.2008.112797</pub-id><pub-id pub-id-type="pmid">19246619</pub-id></mixed-citation></ref><ref id="B50"><mixed-citation publication-type="journal"><name><surname>Hu</surname><given-names>R</given-names></name><name><surname>Fan</surname><given-names>C</given-names></name><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Fu</surname><given-names>YF</given-names></name><article-title>Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR</article-title><source>BMC Mol Biol</source><year>2009</year><volume>10</volume><issue>1</issue><fpage>93</fpage><pub-id pub-id-type="doi">10.1186/1471-2199-10-93</pub-id><pub-id pub-id-type="pmid">19785741</pub-id></mixed-citation></ref><ref id="B51"><mixed-citation publication-type="journal"><name><surname>Maule</surname><given-names>A</given-names></name><name><surname>Leh</surname><given-names>V</given-names></name><name><surname>Lederer</surname><given-names>C</given-names></name><article-title>The dialogue between viruses and hosts in compatible interactions</article-title><source>Curr Opin Plant Biol</source><year>2002</year><volume>5</volume><issue>4</issue><fpage>279</fpage><lpage>284</lpage><pub-id pub-id-type="doi">10.1016/S1369-5266(02)00272-8</pub-id><pub-id pub-id-type="pmid">12179959</pub-id></mixed-citation></ref><ref id="B52"><mixed-citation publication-type="journal"><name><surname>Culver</surname><given-names>JN</given-names></name><name><surname>Padmanabhan</surname><given-names>MS</given-names></name><article-title>Virus-induced disease: altering host physiology one interaction at a time</article-title><source>Annu Rev Phytopathol</source><year>2007</year><volume>45</volume><fpage>221</fpage><lpage>243</lpage><pub-id pub-id-type="doi">10.1146/annurev.phyto.45.062806.094422</pub-id><pub-id pub-id-type="pmid">17417941</pub-id></mixed-citation></ref><ref id="B53"><mixed-citation publication-type="journal"><name><surname>Dunoyer</surname><given-names>P</given-names></name><name><surname>Voinnet</surname><given-names>O</given-names></name><article-title>The complex interplay between plant viruses and host RNA-silencing pathways</article-title><source>Curr Opin Plant Biol</source><year>2005</year><volume>8</volume><issue>4</issue><fpage>415</fpage><lpage>423</lpage><pub-id pub-id-type="doi">10.1016/j.pbi.2005.05.012</pub-id><pub-id pub-id-type="pmid">15939663</pub-id></mixed-citation></ref><ref id="B54"><mixed-citation publication-type="journal"><name><surname>Whitham</surname><given-names>SA</given-names></name><name><surname>Yang</surname><given-names>C</given-names></name><name><surname>Goodin</surname><given-names>MM</given-names></name><article-title>Global impact: elucidating plant responses to viral infection</article-title><source>Mol Plant Microbe Interact</source><year>2006</year><volume>19</volume><issue>11</issue><fpage>1207</fpage><lpage>1215</lpage><pub-id pub-id-type="doi">10.1094/MPMI-19-1207</pub-id><pub-id pub-id-type="pmid">17073303</pub-id></mixed-citation></ref><ref id="B55"><mixed-citation publication-type="journal"><name><surname>Lovdal</surname><given-names>T</given-names></name><name><surname>Lillo</surname><given-names>C</given-names></name><article-title>Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress</article-title><source>Anal Biochem</source><year>2009</year><volume>387</volume><issue>2</issue><fpage>238</fpage><lpage>242</lpage><pub-id pub-id-type="doi">10.1016/j.ab.2009.01.024</pub-id><pub-id pub-id-type="pmid">19454243</pub-id></mixed-citation></ref><ref id="B56"><mixed-citation publication-type="journal"><name><surname>Reid</surname><given-names>KE</given-names></name><name><surname>Olsson</surname><given-names>N</given-names></name><name><surname>Schlosser</surname><given-names>J</given-names></name><name><surname>Peng</surname><given-names>F</given-names></name><name><surname>Lund</surname><given-names>ST</given-names></name><article-title>An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development</article-title><source>BMC Plant Biol</source><year>2006</year><volume>6</volume><fpage>27</fpage><pub-id pub-id-type="doi">10.1186/1471-2229-6-27</pub-id><pub-id pub-id-type="pmid">17105665</pub-id></mixed-citation></ref><ref id="B57"><mixed-citation publication-type="journal"><name><surname>Kobayashi</surname><given-names>H</given-names></name><name><surname>Suzuki</surname><given-names>S</given-names></name><name><surname>Tanzawa</surname><given-names>F</given-names></name><name><surname>Takayanagi</surname><given-names>T</given-names></name><article-title>Low expression of flavonoid 3',5'-hydroxylase (F3',5'H) associated with cyanidin-based anthocyanins in grape leaf</article-title><source>Am J Enol Vitic</source><year>2009</year><volume>60</volume><issue>3</issue><fpage>362</fpage><lpage>367</lpage></mixed-citation></ref><ref id="B58"><mixed-citation publication-type="journal"><name><surname>Bogs</surname><given-names>J</given-names></name><name><surname>Downey</surname><given-names>MO</given-names></name><name><surname>Harvey</surname><given-names>JS</given-names></name><name><surname>Ashton</surname><given-names>AR</given-names></name><name><surname>Tanner</surname><given-names>GJ</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><article-title>Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves</article-title><source>Plant Physiol</source><year>2005</year><volume>139</volume><issue>2</issue><fpage>652</fpage><lpage>663</lpage><pub-id pub-id-type="doi">10.1104/pp.105.064238</pub-id><pub-id pub-id-type="pmid">16169968</pub-id></mixed-citation></ref><ref id="B59"><mixed-citation publication-type="journal"><name><surname>Hummer</surname><given-names>W</given-names></name><name><surname>Schreier</surname><given-names>P</given-names></name><article-title>Analysis of proanthocyanidins</article-title><source>Mol Nutr Food Res</source><year>2008</year><volume>52</volume><issue>12</issue><fpage>1381</fpage><lpage>1398</lpage><pub-id pub-id-type="doi">10.1002/mnfr.200700463</pub-id><pub-id pub-id-type="pmid">19065593</pub-id></mixed-citation></ref><ref id="B60"><mixed-citation publication-type="journal"><name><surname>Treutter</surname><given-names>D</given-names></name><article-title>Significance of flavonoids in plant resistance: a review</article-title><source>Environ Chem Lett</source><year>2006</year><volume>4</volume><issue>3</issue><fpage>147</fpage><lpage>157</lpage><pub-id pub-id-type="doi">10.1007/s10311-006-0068-8</pub-id></mixed-citation></ref><ref id="B61"><mixed-citation publication-type="journal"><name><surname>Goto-Yamamoto</surname><given-names>N</given-names></name><name><surname>Wan</surname><given-names>GH</given-names></name><name><surname>Masaki</surname><given-names>K</given-names></name><name><surname>Kobayashi</surname><given-names>S</given-names></name><article-title>Structure and transcription of three chalcone synthase genes of grapevine (<italic>Vitis vinifera</italic>)</article-title><source>Plant Sci</source><year>2002</year><volume>162</volume><fpage>867</fpage><lpage>872</lpage><pub-id pub-id-type="doi">10.1016/S0168-9452(02)00042-0</pub-id></mixed-citation></ref><ref id="B62"><mixed-citation publication-type="journal"><name><surname>Ageorges</surname><given-names>A</given-names></name><name><surname>Fernandez</surname><given-names>L</given-names></name><name><surname>Vialet</surname><given-names>S</given-names></name><name><surname>Merdinoglu</surname><given-names>D</given-names></name><name><surname>Terrier</surname><given-names>N</given-names></name><name><surname>Romieu</surname><given-names>C</given-names></name><article-title>Four specific isogenes of the anthocyanin metabolic pathway are systematically co-expressed with the red colour of grape berries</article-title><source>Plant Sci</source><year>2006</year><volume>170</volume><issue>2</issue><fpage>372</fpage><lpage>383</lpage><pub-id pub-id-type="doi">10.1016/j.plantsci.2005.09.007</pub-id></mixed-citation></ref><ref id="B63"><mixed-citation publication-type="journal"><name><surname>Griesbach</surname><given-names>RJ</given-names></name><name><surname>Beck</surname><given-names>RM</given-names></name><name><surname>Hammond</surname><given-names>J</given-names></name><name><surname>Stommel</surname><given-names>JR</given-names></name><article-title>Gene expression in the star mutation of <italic>Petunia × hybrida </italic>Vilm</article-title><source>J Amer Soc Hort Sci</source><year>2007</year><volume>132</volume><issue>5</issue><fpage>680</fpage><lpage>690</lpage></mixed-citation></ref><ref id="B64"><mixed-citation publication-type="journal"><name><surname>Teycheney</surname><given-names>PY</given-names></name><name><surname>Tepfer</surname><given-names>M</given-names></name><article-title>Virus-specific spatial differences in the interference with silencing of the chs-A gene in non-transgenic petunia</article-title><source>J Gen Virol</source><year>2001</year><volume>82</volume><issue>Pt 5</issue><fpage>1239</fpage><lpage>1243</lpage><pub-id pub-id-type="pmid">11297699</pub-id></mixed-citation></ref><ref id="B65"><mixed-citation publication-type="journal"><name><surname>Senda</surname><given-names>M</given-names></name><name><surname>Masuta</surname><given-names>C</given-names></name><name><surname>Ohnishi</surname><given-names>S</given-names></name><name><surname>Goto</surname><given-names>K</given-names></name><name><surname>Kasai</surname><given-names>A</given-names></name><name><surname>Sano</surname><given-names>T</given-names></name><name><surname>Hong</surname><given-names>JS</given-names></name><name><surname>MacFarlane</surname><given-names>S</given-names></name><article-title>Patterning of virus-infected <italic>Glycine max </italic>seed coat is associated with suppression of endogenous silencing of chalcone synthase genes</article-title><source>Plant Cell</source><year>2004</year><volume>16</volume><issue>4</issue><fpage>807</fpage><lpage>818</lpage><pub-id pub-id-type="doi">10.1105/tpc.019885</pub-id><pub-id pub-id-type="pmid">15037735</pub-id></mixed-citation></ref><ref id="B66"><mixed-citation publication-type="journal"><name><surname>Koseki</surname><given-names>M</given-names></name><name><surname>Goto</surname><given-names>K</given-names></name><name><surname>Masuta</surname><given-names>C</given-names></name><name><surname>Kanazawa</surname><given-names>A</given-names></name><article-title>The star-type color pattern in <italic>Petunia hybrida </italic>'red Star' flowers is induced by sequence-specific degradation of chalcone synthase RNA</article-title><source>Plant Cell Physiol</source><year>2005</year><volume>46</volume><issue>11</issue><fpage>1879</fpage><lpage>1883</lpage><pub-id pub-id-type="doi">10.1093/pcp/pci192</pub-id><pub-id pub-id-type="pmid">16143597</pub-id></mixed-citation></ref><ref id="B67"><mixed-citation publication-type="journal"><name><surname>Kortekamp</surname><given-names>A</given-names></name><article-title>Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen</article-title><source>Plant Physiol Biochem</source><year>2006</year><volume>44</volume><issue>1</issue><fpage>58</fpage><lpage>67</lpage><pub-id pub-id-type="doi">10.1016/j.plaphy.2006.01.008</pub-id><pub-id pub-id-type="pmid">16531058</pub-id></mixed-citation></ref><ref id="B68"><mixed-citation publication-type="journal"><name><surname>Hren</surname><given-names>M</given-names></name><name><surname>Nikolic</surname><given-names>P</given-names></name><name><surname>Rotter</surname><given-names>A</given-names></name><name><surname>Blejec</surname><given-names>A</given-names></name><name><surname>Terrier</surname><given-names>N</given-names></name><name><surname>Ravnikar</surname><given-names>M</given-names></name><name><surname>Dermastia</surname><given-names>M</given-names></name><name><surname>Gruden</surname><given-names>K</given-names></name><article-title>'Bois noir' phytoplasma induces significant reprogramming of the leaf transcriptome in the field grown grapevine</article-title><source>BMC Genomics</source><year>2009</year><volume>10</volume><fpage>460</fpage><pub-id pub-id-type="doi">10.1186/1471-2164-10-460</pub-id><pub-id pub-id-type="pmid">19799775</pub-id></mixed-citation></ref><ref id="B69"><mixed-citation publication-type="journal"><name><surname>Rotter</surname><given-names>A</given-names></name><name><surname>Camps</surname><given-names>C</given-names></name><name><surname>Lohse</surname><given-names>M</given-names></name><name><surname>Kappel</surname><given-names>C</given-names></name><name><surname>Pilati</surname><given-names>S</given-names></name><name><surname>Hren</surname><given-names>M</given-names></name><name><surname>Stitt</surname><given-names>M</given-names></name><name><surname>Coutos-Thevenot</surname><given-names>P</given-names></name><name><surname>Moser</surname><given-names>C</given-names></name><name><surname>Usadel</surname><given-names>B</given-names></name><name><surname>Delrot</surname><given-names>S</given-names></name><name><surname>Gruden</surname><given-names>K</given-names></name><article-title>Gene expression profiling in susceptible interaction of grapevine with its fungal pathogen Eutypa lata: extending MapMan ontology for grapevine</article-title><source>BMC Plant Biol</source><year>2009</year><volume>9</volume><fpage>104</fpage><pub-id pub-id-type="doi">10.1186/1471-2229-9-104</pub-id><pub-id pub-id-type="pmid">19656401</pub-id></mixed-citation></ref><ref id="B70"><mixed-citation publication-type="journal"><name><surname>Miranda</surname><given-names>M</given-names></name><name><surname>Ralph</surname><given-names>SG</given-names></name><name><surname>Mellway</surname><given-names>R</given-names></name><name><surname>White</surname><given-names>R</given-names></name><name><surname>Heath</surname><given-names>MC</given-names></name><name><surname>Bohlmann</surname><given-names>J</given-names></name><name><surname>Constabel</surname><given-names>CP</given-names></name><article-title>The transcriptional response of hybrid poplar (<italic>Populus trichocarpa </italic>× <italic>P. deltoides</italic>) to infection by <italic>Melampsora medusae </italic>leaf rust involves induction of flavonoid pathway genes leading to the accumulation of proanthocyanidins</article-title><source>Mol Plant-Microbe Interact</source><year>2007</year><volume>20</volume><issue>7</issue><fpage>816</fpage><lpage>831</lpage><pub-id pub-id-type="doi">10.1094/MPMI-20-7-0816</pub-id><pub-id pub-id-type="pmid">17601169</pub-id></mixed-citation></ref><ref id="B71"><mixed-citation publication-type="journal"><name><surname>Kobayashi</surname><given-names>S</given-names></name><name><surname>Ishimaru</surname><given-names>M</given-names></name><name><surname>Hiraoka</surname><given-names>K</given-names></name><name><surname>Honda</surname><given-names>C</given-names></name><article-title>Myb-related genes of the Kyoho grape (<italic>Vitis labruscana</italic>) regulate anthocyanin biosynthesis</article-title><source>Planta</source><year>2002</year><volume>215</volume><issue>6</issue><fpage>924</fpage><lpage>933</lpage><pub-id pub-id-type="doi">10.1007/s00425-002-0830-5</pub-id><pub-id pub-id-type="pmid">12355152</pub-id></mixed-citation></ref><ref id="B72"><mixed-citation publication-type="journal"><name><surname>Ramsay</surname><given-names>NA</given-names></name><name><surname>Glover</surname><given-names>BJ</given-names></name><article-title>MYB-bHLH-WD40 protein complex and the evolution of cellular diversity</article-title><source>Trends Plant Sci</source><year>2005</year><volume>10</volume><issue>2</issue><fpage>63</fpage><lpage>70</lpage><pub-id pub-id-type="doi">10.1016/j.tplants.2004.12.011</pub-id><pub-id pub-id-type="pmid">15708343</pub-id></mixed-citation></ref><ref id="B73"><mixed-citation publication-type="journal"><name><surname>Lovdal</surname><given-names>T</given-names></name><name><surname>Olsen</surname><given-names>KM</given-names></name><name><surname>Slimestad</surname><given-names>R</given-names></name><name><surname>Verheul</surname><given-names>M</given-names></name><name><surname>Lillo</surname><given-names>C</given-names></name><article-title>Synergetic effects of nitrogen depletion, temperature, and light on the content of phenolic compounds and gene expression in leaves of tomato</article-title><source>Phytochemistry</source><year>2010</year><volume>71</volume><issue>5-6</issue><fpage>605</fpage><lpage>613</lpage><pub-id pub-id-type="doi">10.1016/j.phytochem.2009.12.014</pub-id><pub-id pub-id-type="pmid">20096428</pub-id></mixed-citation></ref><ref id="B74"><mixed-citation publication-type="journal"><name><surname>Field</surname><given-names>TS</given-names></name><name><surname>Lee</surname><given-names>DW</given-names></name><name><surname>Holbrook</surname><given-names>NM</given-names></name><article-title>Why leaves turn red in autumn. The role of anthocyanins in senescing leaves of red-osier dogwood</article-title><source>Plant Physiol</source><year>2001</year><volume>127</volume><fpage>566</fpage><lpage>574</lpage><pub-id pub-id-type="doi">10.1104/pp.010063</pub-id><pub-id pub-id-type="pmid">11598230</pub-id></mixed-citation></ref><ref id="B75"><mixed-citation publication-type="journal"><name><surname>Mekuria</surname><given-names>TA</given-names></name><name><surname>Soule</surname><given-names>MJ</given-names></name><name><surname>Jarugula</surname><given-names>S</given-names></name><name><surname>Naidu</surname><given-names>RA</given-names></name><article-title>Current status of grapevine viruses in Washington State vineyards [abstract]</article-title><source>Phytopathology</source><year>2009</year><volume>99</volume><fpage>S83</fpage><pub-id pub-id-type="doi">10.1094/PHYTO-99-12-1394</pub-id></mixed-citation></ref><ref id="B76"><mixed-citation publication-type="journal"><name><surname>Porra</surname><given-names>RJ</given-names></name><article-title>The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b</article-title><source>Photosynth Res</source><year>2002</year><volume>73</volume><issue>1-3</issue><fpage>149</fpage><lpage>156</lpage><pub-id pub-id-type="doi">10.1023/A:1020470224740</pub-id><pub-id pub-id-type="pmid">16245116</pub-id></mixed-citation></ref><ref id="B77"><mixed-citation publication-type="journal"><name><surname>Lichtenthaler</surname><given-names>HK</given-names></name><name><surname>Welburn</surname></name><article-title>Determination of total carotenoids and chlorophylls a and b of leaf extracts in different solvents</article-title><source>Biochem Soc Trans</source><year>1983</year><volume>11</volume><fpage>591</fpage><lpage>592</lpage></mixed-citation></ref><ref id="B78"><mixed-citation publication-type="journal"><name><surname>Lefever</surname><given-names>S</given-names></name><name><surname>Hellemans</surname><given-names>J</given-names></name><name><surname>Pattyn</surname><given-names>F</given-names></name><name><surname>Przybylski</surname><given-names>DR</given-names></name><name><surname>Taylor</surname><given-names>C</given-names></name><name><surname>Geurts</surname><given-names>R</given-names></name><name><surname>Untergasser</surname><given-names>A</given-names></name><name><surname>Vandesompele</surname><given-names>J</given-names></name><article-title>RDML: structured language and reporting guidelines for real-time quantitative PCR data</article-title><source>Nucleic Acids Res</source><year>2009</year><volume>37</volume><issue>7</issue><fpage>2065</fpage><lpage>2069</lpage><pub-id pub-id-type="doi">10.1093/nar/gkp056</pub-id><pub-id pub-id-type="pmid">19223324</pub-id></mixed-citation></ref><ref id="B79"><mixed-citation publication-type="journal"><name><surname>Downey</surname><given-names>MO</given-names></name><name><surname>Rochfort</surname><given-names>S</given-names></name><article-title>Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectral identification of anthocyanins and flavonols in Shiraz grape skin</article-title><source>J Chromat A</source><year>2008</year><volume>1201</volume><fpage>43</fpage><lpage>47</lpage><pub-id pub-id-type="doi">10.1016/j.chroma.2008.06.002</pub-id></mixed-citation></ref><ref id="B80"><mixed-citation publication-type="journal"><name><surname>Harbertson</surname><given-names>JF</given-names></name><name><surname>Kennedy</surname><given-names>JA</given-names></name><name><surname>Adams</surname><given-names>DO</given-names></name><article-title>Tannin in skins and seeds of cabernet sauvignon, syrah, and pinot noir berries during ripening</article-title><source>Am J Enol Vitic</source><year>2002</year><volume>53</volume><issue>1</issue><fpage>54</fpage><lpage>59</lpage></mixed-citation></ref><ref id="B81"><mixed-citation publication-type="journal"><name><surname>Boss</surname><given-names>PK</given-names></name><name><surname>Davies</surname><given-names>C</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><article-title>Analysis of the expression of anthocyanin pathway genes in developing <italic>Vitis vinifera </italic>L. cv Shiraz grape berries and the implications for pathway regulation</article-title><source>Plant Physiol</source><year>1996</year><volume>111</volume><issue>4</issue><fpage>1059</fpage><lpage>1066</lpage><pub-id pub-id-type="pmid">12226348</pub-id></mixed-citation></ref><ref id="B82"><mixed-citation publication-type="journal"><name><surname>Fujita</surname><given-names>A</given-names></name><name><surname>Soma</surname><given-names>N</given-names></name><name><surname>Goto-Yamamoto</surname><given-names>N</given-names></name><name><surname>Mizuno</surname><given-names>A</given-names></name><name><surname>Kiso</surname><given-names>K</given-names></name><name><surname>Hashizume</surname><given-names>K</given-names></name><article-title>Effect of shading on proanthocyanidin biosynthesis in the grape berry</article-title><source>J Japan Soc Hort Sci</source><year>2007</year><volume>76</volume><issue>2</issue><fpage>112</fpage><lpage>119</lpage><pub-id pub-id-type="doi">10.2503/jjshs.76.112</pub-id></mixed-citation></ref><ref id="B83"><mixed-citation publication-type="journal"><name><surname>Menzel</surname><given-names>W</given-names></name><name><surname>Jelkmann</surname><given-names>W</given-names></name><name><surname>Maiss</surname><given-names>E</given-names></name><article-title>Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control</article-title><source>J Virol Methods</source><year>2002</year><volume>99</volume><issue>1-2</issue><fpage>81</fpage><lpage>92</lpage><pub-id pub-id-type="doi">10.1016/S0166-0934(01)00381-0</pub-id><pub-id pub-id-type="pmid">11684306</pub-id></mixed-citation></ref><ref id="B84"><mixed-citation publication-type="journal"><name><surname>Jeong</surname><given-names>ST</given-names></name><name><surname>Goto-Yamamoto</surname><given-names>N</given-names></name><name><surname>Kobayashi</surname><given-names>S</given-names></name><name><surname>Esaka</surname><given-names>M</given-names></name><article-title>Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins</article-title><source>Plant Sci</source><year>2004</year><volume>167</volume><issue>2</issue><fpage>247</fpage><lpage>252</lpage><pub-id pub-id-type="doi">10.1016/j.plantsci.2004.03.021</pub-id></mixed-citation></ref><ref id="B85"><mixed-citation publication-type="journal"><name><surname>Downey</surname><given-names>MO</given-names></name><name><surname>Harvey</surname><given-names>JS</given-names></name><name><surname>Robinson</surname><given-names>SP</given-names></name><article-title>Synthesis of flavonols and expression of flavonol synthase genes in the developing grape berries of Shiraz and Chardonnay (<italic>Vitis vinifera </italic>L.)</article-title><source>Aus J Grape Wine Res</source><year>2003</year><volume>9</volume><issue>2</issue><fpage>110</fpage><lpage>121</lpage><pub-id pub-id-type="doi">10.1111/j.1755-0238.2003.tb00261.x</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Bois/explor/MelampsoraV2/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000587 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000587 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Bois
|area= MelampsoraV2
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:2956537
|texte= Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:20731850" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a MelampsoraV2
| This area was generated with Dilib version V0.6.38. |  |