www.nature.com/scientificreports OPEN Molecular mechanisms of tannin accumulation in Rhus galls and genes involved in plant-insect Received: 20 November 2017 Accepted: 11 June 2018 interactions Published: xx xx xxxx Hang Chen 1,2, Juan Liu1, Kai Cui1, Qin Lu1, Chao Wang1,3, Haixia Wu1, Zixiang Yang1, Weifeng Ding 1, Shuxia Shao1, Haiying Wang1, Xiaofei Ling1, Kirst King-Jones4 & Xiaoming Chen1,2 For galling aphids and their hosts, tannins are crucial for plant-insect interactions and for protecting the host plant from herbivory. Due to their peculiar chemical characteristics, tannins from plant galls have been used for medical and chemical purposes for more than 2000 years. In this study, hydrolyzable tannin concentrations in galls increased from gall initiation (38.34% on June 21) to maturation (74.79% on August 8), then decreased gradually thereafter (58.83% on October 12). We identifed a total of 81 genes (named as GTS1-81) with putative roles in gallotannin biosynthesis and 22 genes (TS1-22) in condensed tannin biosynthesis. We determined the expression profles of these genes by real-time PCR over the course of gall development. Multiple genes encoding 1-beta-D-glucosyl transferases were identifed, which may play a vital role in gallotannin accumulation in plant galls. This study is the frst attempt to examine the molecular basis for the regulation of tannin accumulation in insect gallnuts. The diferentially expressed genes we identifed may play important roles in both tannin biosynthesis and plant-insect interactions. Plants and insects have co-existed for more than 350 million years1, and in their interactions both have evolved strategies to overcome each other’s defense systems2,3. Many insect species induce the formation of galls on various plant tissues. Te aphid Schlechtendalia chinensis (Bell) is a tiny insect of the Pemphigidae family (Hemiptera: Aphidoidea). It feeds on the adaxial surface of winged rachides and induces the formation of large, single-chambered galls, called horned galls, on R. chinensis4. Te galls have been used for medical and chemical applications as a source of tannic, gallic, and pyrogallic acids for more than 2,000 years. It has been reported that there are at least 15,000 tons of tannin demands in diferent industrial felds including leatherworking, textile printing and mineral separation each year around the world (Fig. S1). And the whole value of industry related to tannins has added up to 30 billion dollars annually5–8. Tannins are astringent, polyphenolic compounds that bind to and precipitate proteins and various other organic compounds including amino acids and alkaloids. Tannins occur widely in various plant tissues, where they play crucial roles in protecting plants from herbivory, and in regulating plant growth9. Most tannins have molecular weights from 500 to over 3,000 (gallic acid esters) Da, but some can reach up to 20,000 (proanthocya- nidins) (Fig. S2)10,11. Plant tannins are divided into hydrolyzable tannins and favonoid-derived, condensed tan- nins, which are also called proanthocyanidins. Condensed tannins are produced through the shikimate pathway leading to the production of anthocyanins, a pathway that has been investigated intensively at biochemical and genetic levels (Fig. S2)12–14. Te current understanding is that anthocyanins and the other favonoid classes are derived from earlier intermediates in the anthocyanin pathway15–17. Hydrolysable tannins are derivatives of gallic acids. Te simplest hydrolysable tannins, the gallotannins, are simple polygalloyl esters of glucose18. Te prototypical gallotannin is pentagalloyl glucose, and has fve identical 1Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming, China. 2The Key Laboratory of Cultivating and Utilization of Resources Insects, State Forestry Administration, Kunming, China. 3Southwest Forestry University, Kunming, 650224, Yunnan, China. 4Department of Biological Sciences, Biological Sciences Building, University of Alberta, Edmonton, Canada. Correspondence and requests for materials should be addressed to H.C. (email: [email protected]) or X.C. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:9841 | DOI:10.1038/s41598-018-28153-y 1 www.nature.com/scientificreports/ Figure 1. Changes in tannin concentrations in galls and leaves over the course of gallnut growth. (A) Te fundatrix feeds on the winged rachides and initiates gall formation, the arrow pointing the location of early stage of gall on the leaf; (B) hundreds of aphids inside horned gall stimulating the gall growing constantly, the arrow pointing the individual aphid; (C) the change trend of two kinds of tannin contents in gall and in leaves with the growth of gallnut. Te horizontal coordinates are the dry weight percentage of tannins; the vertical coordinates are diferent developmental stage of gallnut from June 21 to October 12. ester linkages that involve aliphatic hydroxyl groups of the core sugar (Fig. S2). Te biosynthetic pathways of hydrolyzable tannins include the biosynthesis of three intermediates, pentagalloylglucoses, gallotannins, and ellagitannins19. Schlechtendalia chinensis is an insect with a complex life cycle that includes the development of gall-dwelling colonies on its host R. chinensis. In early spring in China, sexuparae migrate from the moss to the trunk of R. chinensis tree to produce sexually reproductive females and males that subsequently mate to produce fundatrices. A fundatrix feeds on the winged rachides of R. chinensis and then initiates gall formation (Fig. 1A,B)20,21. Afer a horned gall matures and dehisces in autumn, the alate fundatrigeniae migrate from the gall to the moss, its over-wintering host22. Although the biological characteristics of R. chinensis and its herbivore S. chinensis have been examined by dif- ferent labs over the years23,24, at present, there are no genomic resources available for R. chinensis. A better under- standing of the molecular underpinnings for tannin biosynthesis would potentially beneft the tannin industry, which is currently hampered by the lack of information available on transcriptional, proteomic, and genetic anal- yses of R. chinensis. Next generation sequencing technologies have proven to be a rapid and cost-efective means for analyzing the genome and transcriptome of non-model species25–27. Our long-term goal is to identify genes with functions in the biosynthesis of tannins and to modify these genes genetically to increase the efciency of tannin production. Te specifc objective of this study was to generate a transcriptome that provides a foundation for future studies leading to a better understanding ofwhy tannin accumulation occurs so rapidly in galls growing on R. chinensis and the co-evolution trends between insects and their host plants. Results Accumulation of tannins in horned galls and leaves. To determine the best time for sample collection for transcriptomic analyses, we measured gallotannin concentrations in galls and leaves throughout an entire season (June through October). Gallotannin concentrations in galls were nearly doubled in the frst six weeks, from 38.34% on June 21 to 74.79% of dry gallnut weight on August 8. Afer that, gallotannin concentrations grad- ually decreased to 58.83% of dry gallnut weight over the next ten weeks (Fig. 1C, Table S1). Te concentrations of gallotannin in leaf samples were considerably lower compared to those in gallnuts. Still, there was a signifcant increase in gallotanninin leaves during the process of gall initiation to maturation. On June 21, at the beginning of gallnut formation, the gallotannin abundance in leaves was only 2.79% of dry weight (Table S2), which was then nearly doubled to 4.90% by August 8. Afer the peak, a gradual decrease was observed during the September- October period, reaching 3.13% of dry weight on October 12 (Fig. 1C). Te abundance of condensed tannins exhibited a fuctuating pattern. Te initial level of condensed tannins in galls was 0.70%, which then decreased to 0.32% by August 8. Afer that, a gradual increase to 0.39% was observed on August 28, followed by a drop to 0.32% again on September 24. On October 12, the abundance increased to 1.92% (Fig. 1C, Table S3). Diferent from gallotannins, the abundance of condensed tannins was much higher in leaf samples than in gallnuts. Te accumulation of condensed tannins was fast in the frst two months, from 5.78% of dry leaf weight on June 21 to 12.50% on August 8. Afer that condensed tannin abundance gradually tapered of to 6.47% on August 28, followed by an increase to 11.34% towards the end of the season (Fig. 1C, Table S4). SCIENTIFIC REPORTS | (2018) 8:9841 | DOI:10.1038/s41598-018-28153-y 2 www.nature.com/scientificreports/ Database Numbers of Unigene Percent (%) Nr 36,153 60.74% Nt 31,128 52.30% Swiss-Prot 24,856 41.76% KEGG 21,880 36.76% COG 14,409 24.21% GO 28,491 47.87% Table 1. Annotation result statistics in diferent databases. De novo transcriptome assembly and annotation. Te Illumina sequencing data of R. chinensis gen- erated 86,156,236 raw reads covering a total of 7,203,025,980 nucleotides (nt) from 15 libraries (fve time points and three diferent types of tissues). Afer removing adapter sequences, ambiguous nucleotides and low-quality sequences, 80,033,622 clean reads were retained. Te average length of reads was 486 nt. Te clean reads were assembled into 59,522 unigenes with average length 889 and N50 1675 nt. Te size statistics of assembled uni- genes were shown in Supplementary Information: Tables S5, S6, and Fig. S3. We annotated gene function of all unigenes based on common databases, including the NCBI database Non-redundant protein sequences (Nr), NCBI nucleotide sequences (Nt), the manually annotated and curated protein sequence database (Swiss-Prot), Kyoto Encyclopedia of Genes and Genomes Ortholog database (KEGG), Clusters of Orthologous Groups of proteins (COG) and Gene Ontology (GO) databases. Annotation results of unigenes are shown in Table 1. A total of 36,153 unigenes (60.74% of all unigenes) returned above-cutof BLAST (E value < 10−5) results when searched against the Nr nucleotide database.