Comparative transcriptome and metabolite profiling of four tissues from Alisma orientale (Sam.) Juzep reveals its inflorescence developmental and medicinal characteristics

Abstract

Alisma orientale (Sam.) Juzep (A. orientale) is an important medicinal plant in traditional Chinese medicine. In this study, de novo RNA-seq of A. orientale was performed based on the cDNA libraries from four different tissues, roots, leaves, scapes and inflorescences. A total of 41,685 unigenes were assembled, 25,024 unigene functional annotations were obtained by searching against the five public sequence databases, and 3,411 simple sequence repeats in A. orientale were reported for the first time. 15,402 differentially expressed genes were analysed. The morphological characteristics showed that compared to the other tissues, the leaves had more chlorophyll, the scapes had more vascular bundles, and the inflorescences contained more starch granules and protein. In addition, the metabolic profiles of eight kinds of alisols metabolite profiling, which were measured by ultra-Performance liquid chromatography-triple quadrupole-mass spectrometry showed that alisol B 23-acetate and alisol B were the major components of the four tissues at amounts of 0.068~0.350 mg/g and 0.046~0.587 mg/g, respectively. In addition, qRT-PCR validated that farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-CoA reductase should be considered the critical candidate genes involved in alisol biosynthesis. These transcriptome and metabolic profiles of A. orientale may help clarify the molecular mechanisms underlying the medicinal characteristics of A. orientale.

Figure 1

Microstructure structure of the roots, leaves, scapes and inflorescences of A. orientale under I-KI and fast green exposure. (A–D) Transverse section of the root (20x), leaf (100x), scape (100x) and inflorescence (50x); (E–H) vertical section of the root (50x), leaf (50x), scape (100x) and inflorescence (200x). When stained with I-KI, the starch granules are blue or blue-black, and the protein is yellow. When stained with fast green, the cell wall is green.

Figure 2

Venn diagram of annotated unigenes of A. orientale.

Figure 3

KOG function classifications of the A. orientale unigenes.

Figure 4

Level2 GO terms of the A. orientale unigenes.

Figure 5

Frequencies of the SSRs identified in A. orientale unigenes.

Figure 6

Annotated DEG numbers among various tissues of A. orientale.

Figure 7

qRT-PCR validation of the DEGs involved in the biosynthesis of alisol and its derivatives. PIP: aquaporin PIP1-3, TIP: aquaporin TIP2-1, NIP: aquaporin NIP5-1, SIP: aquaporin SIP1-1, DHN: dehydrin, C4H: cinnamate 4-hydroxylase, PAL: phenylalanine ammonia-lyase, TUB: beta-tubulin 4, CYP: cytochrome P450 CYP73A100-like, UGD: UDP-glucose 6-dehydrogenase, SQS: farnesyl-diphosphate farnesyltransferase, FPS: farnesyl pyrophosphate synthase, HMR: 3-hydroxy-3-methylglutaryl-CoA reductase, and MVD: diphosphomevalonate decarboxylase.

Figure 8

Contents of the eight triterpenoids in A. orientale. Inflorescence compared to root, ^ΔΔp < 0.01, inflorescence compared to leaf, ^*p < 0.05, ^**p < 0.01.