Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa Lijun Wang1,2., Yuanzhong Jiang1., Li Yuan1., Wanxiang Lu2,3, Li Yang1, Abdul Karim1, Keming Luo1,2,3* 1 Key Laboratory of Eco-environments of Three Gorges Reservoir Region, Ministry of Education, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing, China, 2 College of Horticulture and Landscape Architecture, Southwest University, Chongqing, China, 3 Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China Abstract Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (2)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P,0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus. Citation: Wang L, Jiang Y, Yuan L, Lu W, Yang L, et al. (2013) Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa. PLoS ONE 8(5): e64664. doi:10.1371/journal.pone.0064664 Editor: Deyu Fang, Northwestern University Feinberg School of Medicine, United States of America Received February 6, 2013; Accepted April 17, 2013; Published May 31, 2013 Copyright: ß 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (31171620), the program for New Century Excellent Talents in University (NCET-11-0700) and The Research Fund for the Doctoral Program of Higher Education (20110182110004). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction has been characterized genetically and biochemically in a number of plant species including Arabidopsis thaliana and Medicago truncatula Flavonoids are a large group of plant secondary metabolites that [12]. At the beginning of the flavonoid biosynthetic pathway, the comprise several classes of compounds (e.g. anthocyanins, enzyme chalcone synthase (CHS) carries out the condensation of flavonols, isoflavones and flavan-3-ols) and accumulate in a wide one molecule of 4-coumaroyl-CoA and three molecules of variety of plant tissues [1]. Proanthocyanidins (PAs), also called as malonyl-CoA to yield naringenin chalcone. Chalcone is isomerised condensed tannins, are oligomers or polymers of flavan-3-ols and to a flavanone via the enzyme chalcone flavanone isomerase are among the major flavonoid compounds found in higher plants (CHI). Sharing these central intermediates, the pathway divided [2,3]. As a final product of the flavonoid pathway, PAs play an into several side branches, each producing a different class of important role in the protection of plants against herbivores and flavonoids. The stereospecific 3ß-hydroxylation of (2S)-flavanones pathogens [1,4,5]. PAs in forage crops can protect ruminants are catalyzed to dihydroflavonols by flavanone 3-hydroxylase against pasture bloat [6,7,8]. PAs also act as antioxidants with (F39H). For the biosynthesis of anthocyanins, dihydroflavonols are beneficial effects for human health by protection against free catalyzed to flavan-3,4-diols (leucoanthocyanins) by dihydroflavo- radical-mediated injury and cardiovascular disease [9,10]. Addi- nol reductase (DFR), then leucoanthocyanins are converted to tionally, PAs also contribute to the astringency and taste of many anthocyanidins by anthocyanidin synthase (ANS). The formation fruits and the quality of other plant products, such as wine, tea of glucosides is catalyzed by UDP glucose-flavonoid 3-o-glucosyl (Camellia sinensis), and cocoa [11]. Therefore, an understanding of transferase (UFGT), which stabilizes the anthocyanidins by 3-O- the mechanisms leading to the formation of PA polymers and its glucosylation [13,14]. regulation is important for regulation of PA biosynthesis in plants. The biosynthesis of PAs and anthocyanins begins with the same Flavonoids are synthesized through the phenylpropanoid upstream avonoid pathway, leading to the synthesis of avan-3-ol pathway. PA biosynthesis shares the common avonoid biosynthetic units such as catechin and epicatechin [15,16]. Catechin is pathway with anthocyanins and avonols. The common pathway PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e64664 Characterization of ANR1 and LAR1 from Populus traditionally derived from leucocyanidin by the catalyzation of Furthermore, these genes encoding ANR and LAR isoforms from leucoanthocyanidin reductase (LAR) [15], while epicatechins are P. trichocarpa were cloned and characterized. Expression analysis of synthesized from cyanidin by anthocyanidin reductase (ANR), these genes in the empty-vector control and transgenic lines which is encoded by the BANYULS (BAN) gene and has been showing different accumulation of PAs has developed our initially characterized in Arabidopsis [17]. Ectopic expression of understanding of the genetic control of PA biosynthesis in poplar. BAN in tobacco flower petals and Arabidopsis leaves results in loss of Our findings established the roles of PtrANR and PtrLAR in the anthocyanins and accumulation of PAs, suggesting that there is an PA biosynthesis pathway in poplar and may be of importance in interaction between anthocyanidin and PA pathways [17]. VvANR, understanding the genetic mechanisms controlling PA accumula- encoded by a single gene, has been also isolated and characterized tion in poplar as well as other plants. in grapevines (Vitis vinifera) [18]. More recently, three ANR genes were reported in apple (Malus6domestica Borkh.), and introduction Materials and Methods of these genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin [19]. This Plant Growth Conditions and Stress Treatments finding suggests that the ANR gene may be capable of generating Populus tomentosa Carr. (clone 73) is native in China. In this study, catechin via an alternative route. we collected poplar leaves from the vicinity of Yangling. No LAR activity has been found in several plants and its activity specific permission was required for any locations/activities. It was correlated with PA accumulation [2,15,20,21]. The functionality sure that the field studies did not involve endangered or protected of LAR has been determined in Desmodium uncinatum, in which the species. Poplar plants were grown in the greenhouse at 25uC under recombinant LAR protein catalyzed the conversion of leucocya- a 14-/10-h light/dark cycle with supplemental light (4500 lux). nidin, leucodelphinidin, or leucopelargonidin to the corresponding Three-month-old poplar seedlings grown in the greenhouse were 2,3-trans-avan-3-ol (catechin) [5]. This finding clearly established subjected to stress experiments analysis of transgenics. Upon the role of DuLARs in PA biosynthesis. Two grapevine LAR harvest of leaves, midveins and necrotic tissue were removed and orthologs were also isolated and had different patterns of tissues were frozen in liquid nitrogen and stored at 280uC until expression in skin and seeds [18]. In M. truncatula, a single LAR analyzed. Leaves within the range of Leaf Plastochron Index (LPI)- gene has been cloned and characterized and transgenic tobacco 8to212 [27], which correspond to the youngest fully expand plants constitutively overexpressing MtLAR showed reduced leaves, were used for all stress experiments. anthocyanin content, but no catechin or increased levels of PAs For pathogen inoculations, leaf discs were inoculated with were detected either in leaves or in flowers, suggesting the poor Melampsora brunnea f.sp. multigermtubi (Mb) as previously described correlation between LAR expression and PA biosynthesis in [26]. Briefly, leaf discs were inoculated with Mbm urediniospores Medicago [22].