Transcriptome analysis of buds and leaves using 454 pyrosequencing to discover genes associated with the biosynthesis of active ingredients in Lonicera japonica Thunb

Abstract

Background: Lonicera japonica Thunb. is a plant used in traditional Chinese medicine known for its anti-inflammatory, anti-oxidative, anti-carcinogenic, and antiviral pharmacological properties. The major active secondary metabolites of this plant are chlorogenic acid (CGA) and luteoloside. While the biosynthetic pathways of these metabolites are relatively well known, the genetic information available for this species, especially the biosynthetic pathways of its active ingredients, is limited.

Methodology/principal findings: We obtained one million reads (average length of 400 bp) in a whole sequence run using a Roche/454 GS FLX titanium platform. Altogether, 85.69% of the unigenes covering the entire life cycle of the plant were annotated and 325 unigenes were assigned to secondary metabolic pathways. Moreover, 2039 unigenes were predicted as transcription factors. Nearly all of the possible enzymes involved in the biosynthesis of CGA and luteoloside were discovered in L. japonica. Three hydroxycinnamoyl transferase genes, including two hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes and one hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) gene featuring high similarity to known genes from other species, were cloned. The HCT gene was discovered for the first time in L. japonica. In addition, 188 candidate cytochrome P450 unigenes and 245 glycosyltransferase unigenes were found in the expressed sequence tag (EST) dataset.

Conclusion: This study provides a high quality EST database for L. japonica by 454 pyrosequencing. Based on the EST annotation, a set of putative genes involved in CGA and luteoloside biosynthetic pathways were discovered. The database serves as an important source of public information on genetic markers, gene expression, genomics, and functional genomics in L. japonica.

Figure 1. Distribution of the 454 HQ read and contig lengths from L. japonica.

A. Size distribution of 454 sequencing HQ reads. B. Length distribution of contigs in the EST datasets.

Figure 2. GO and COG classifications of unigenes from L. japonica.

A. GO function classification of transcriptome. B. COG function classification of transcriptome. Purple boxes represent information storage and processing. J: Translation, ribosomal structure, and biogenesis. A: RNA processing and modification. K: Transcription. L: Replication, recombination and repair. B: Chromatin structure and dynamics. Yellow boxes represent cellular processes and signaling. D: Cell cycle control, cell division, and chromosome partitioning. Y: Nuclear structure. V: Defense mechanisms. T: Signal transduction mechanisms. M: Cell wall/membrane/envelope biogenesis. N: Cell motility. Z: Cytoskeleton. W: Extracellular structures. U: Intracellular trafficking, secretion, and vesicular transport. O: Posttranslational modification, protein turnover, and chaperones. Blue boxes represent metabolism. C: Energy production and conversion. G: Carbohydrate transport and metabolism. E: Amino acid transport and metabolism. F: Nucleotide transport and metabolism. H: Coenzyme transport and metabolism. I: Lipid transport and metabolism. P: Inorganic ion transport and metabolism. Q: Secondary metabolite biosynthesis, transport, and catabolism. Gray boxes represent poorly characterized. R: General function prediction only. S: Function unknown.

Figure 3. Percentages of L. japonica unigenes in 11 subcategories of the metabolic pathway category.

Figure 4. Proposed pathways for the biosynthesis of CGA and luteoloside in L. japonica.

The three different routes of CGA biosynthesis are labeled 1, 2, and 3. Enzyme names are shown in the pictures. Each enzyme is annotated with the number of corresponding unigenes shown in parentheses. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl CoA ligase/4-coumarate-CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase; C3H, p-coumarate 3′-hydroxlase; HQT, hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase; UGCT, UDP glucose: cinnamate glucosyl transferase; HCGQT, hydroxycinnamoyl D-glucose: quinate hydroxycinnamoyl transferase. CHS, chalcone synthesis; CHI, chalcone isomerase; FSII, flavonol synthase; F3'H, Flavonoid 3'-monooxygenase.

Figure 5. Protein sequence alignment of putative L. japonica HQTs and HCTs with representative members of the HQT and HCT families.

A. Sequence alignment of the conserved structure motifs in eight L. japonica putative HQTs and HCTs with Solanum lycopersicum (NP001234850) and Nicotiana tabacum (CAE46932). Black boxes indicate the conserved region. B. Neighbor-joining tree of HQTs and HCTs from L. japonica and other plants. The HQTs and HCTs used in phylogenetic analysis were retrieved from NCBI, including Cynara cardunculus var. scolymus (ACJ23164, ADL62854, CAR92145, ACF37072, AFL93687, ABK79689, ABK79690, AFL93686), Coffea canephora (ABO77957, ABO47805, ABO77955), Nicotiana tabacum (CAE46932, Q8GSM7), Solanum lycopersicum (NP001234850), Trifolium pratense (ACI28534), Populus trichocarpa (XP002303858, XP002332068), P. nigra (AEN02914) and C. arabica (ABO40491).