Transcriptome analysis of Asparagus officinalis reveals genes involved in the biosynthesis of rutin and protodioscin

Abstract

Garden asparagus (Asparagus officinalis L.) is a popular vegetable cultivated worldwide. The secondary metabolites in its shoot are helpful for human health. We analyzed A. officinalis transcriptomes and identified differentially expressed genes (DEGs) involved in the biosynthesis of rutin and protodioscin, which are health-promoting functional compounds, and determined their association with stem color. We sequenced the complete mRNA transcriptome using the Illumina high-throughput sequencing platform in one white, three green, and one purple asparagus cultivars. A gene set was generated by de novo assembly of the transcriptome sequences and annotated using a BLASTx search. To investigate the relationship between the contents of rutin and protodioscin and their gene expression levels, rutin and protodioscin were analyzed using high-performance liquid chromatography. A secondary metabolite analysis using high-performance liquid chromatography showed that the rutin content was higher in green asparagus, while the protodioscin content was higher in white asparagus. We studied the genes associated with the biosynthesis of the rutin and protodioscin. The transcriptomes of the five cultivars generated 336 599 498 high-quality clean reads, which were assembled into 239 873 contigs with an average length of 694 bp, using the Trinity v2.4.0 program. The green and white asparagus cultivars showed 58 932 DEGs. A comparison of rutin and protodioscin biosynthesis genes revealed that 12 of the 57 genes associated with rutin and two of the 50 genes associated with protodioscin showed more than four-fold differences in expression. These DEGs might have caused a variation in the contents of these two metabolites between green and white asparagus. The present study is possibly the first to report transcriptomic gene sets in asparagus. The DEGs putatively involved in rutin and protodioscin biosynthesis might be useful for molecular engineering in asparagus.

Fig 1. GO analysis of asparagus.

(A: biological process, B: cellular component, C: molecular function).

Fig 2. Distribution of expression levels between Atlas_G of green shoot asparagus and Atlas_W of white shoot asparagus.

Fig 3. Biosynthetic pathways and compartmentalization of flavonoid synthesis in plants.

4CL, 4-coumarate:CoA ligase; C4H, cinnamate-4-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; F3Rha, flavonol-3-O-glucoside l-rhamnosyltransferase; FLS, flavonol synthase; FNS, flavone synthase; PAL, phenylalanine ammonia-lyase. The numbering of the 3′, 4′, and 5′ carbon positions is shown in the structure. Enzyme names are in italics.

Fig 4. Biosynthetic pathways and compartmentalization of terpenoid synthesis in plants.

AACT, acetoacetyl-CoA thiolase; AcAc-CoA, acetoacetyl-CoA; CDP-ME, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol; CDP-ME2P, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol phosphate; CMK, CDP-ME kinase; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-d-xylulose 5-phosphate; DXR, DXP reductoisomerase; DXS, DXP synthase; FDS, farnesyl diphosphate synthase; FPP, farnesyl diphosphate; G3P, glyceraldehyde-3-phosphate; GDS, geranyl diphosphate synthase; GPP, geranyl diphosphate; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl diphosphate; ISPS, isoprene synthase; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; MDS, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; ME-2,4cPP, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate; MEP, 2-C-methyl-d-erythritol 4-phosphate; MVD, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; TPS, terpene synthase. Enzyme names are in italics.

Fig 5. Heat map of expression differences in 57 flavonoid and 50 terpenoid biosynthesis genes of asparagus based on FPKM values (names in yellow represent flavonoid-related genes and names in pink represent terpenoid-related genes).

Fig 6. Comparison of the expression patterns of genes involved in flavonoid and terpenoid biosynthesis in Atlas_W of white shoot asparagus and Atlas_G of green shoot asparagus between the RNA-seq FPKM and qRT-PCR data.

(A: RNA-seq FPKM data, B: qRT-PCR data). Values in a row with different letters are significantly different (p < 0.05), mean ± SD (n = 3).

Fig 7. Rutin and protodioscin contents in Atlas_W of white shoot asparagus and Atlas_G of green shoot asparagus.

Values in a row with different letters are significantly different (p < 0.05), mean ± SD (n = 3).