Identification and developmental expression profiling of putative alkaloid biosynthetic genes in Corydalis yanhusuo bulbs

Abstract

Alkaloids in bulbs of Corydalis (C.) yanhusuo are the major pharmacologically active compounds in treatment of blood vessel diseases, tumors and various pains. However, due to the absence of gene sequences in C. yanhusuo, the genes involved in alkaloid biosynthesis and their expression during bulb development remain unknown. We therefore established the first transcriptome database of C. yanhusuo via Illumina mRNA-Sequencing of a RNA composite sample collected at Bulb initiation (Day 0), early enlargement (Day 10) and maturation (Day 30). 25,013,630 clean 90 bp paired-end reads were de novo assembled into 47,081 unigenes with an average length of 489 bp, among which 30,868 unigenes (65.56%) were annotated in four protein databases. Of 526 putative unigenes involved in biosynthesis o f various alkaloids, 187 were identified as the candidate genes involved in the biosynthesis of benzylisoquinoline alkaloids (BIAs), the only alkaloid type reported in C. yanhusuo untill now. BIAs biosynthetic genes were highly upregulated in the overall pathway during bulb development. Identification of alkaloid biosynthetic genes in C. yanhusuo provide insights on pathways and molecular regulation of alkaloid biosynthesis, to initiate metabolic engineering in order to improve the yield of interesting alkaloids and to identify potentially new alkaloids predicted from the transcriptomic information.

Figure 1

Figure 2. Bulb initiation and development in C. yanhusuo.

Day 0: Several rhizomes emerged from the mother bulb and no new bulblets were visible; Day 5: new bulblets indicated by a arrow were visible at nodes of rhizomes; Day 10–25: bulbs in enlargement.; 2-3 bulblets were formed on each rhizome. Formation of a new bulb to renew mother bulb in situ was simultaneously observed at Day 10; Day 30–40: Bulbs at maturation. Numbers on the images indicated days after the formation of new bulblet initiation, counted from the first sampling.

Figure 3. Expression profiling of C. yanhusuo Unigenes during the bulb development.

(a) Number of Unigenes expressed at defined expression levels Numbers on the Y-axis were in units of 1,000. (b) Numbers under the stage name are total numbers of DGEs detected by uniquely-mapped reads at the corresponding stage; Numbers in black on Ven diagram meant number of shared and unique genes among the three developmental stages; Numbers in red on the Ven diagram are numbers of shared and unique DEGs among the three developmental stages. a total of 13,354 DEGs were identified in at least one pairwise comparison of developmental stages. (c,d) Numbers in () showed are total numbers of DEGs up- or down-regulated between two compared stages. Numbers on Ven diagram are numbers of DEGs shared or unique among different comparisons.

Figure 4. Expression patterns of BIAs biosynthetic unigenes during C. yanhusuo bulb development.

(a) Biosynthetic pathways leading to major benzylisoquinoline alkaloids (BIAs) are based on Beaudoin and Facchini. Pathways: black arrows 1-benzylisoquinoline; dark blue arrows, papaverine; green arrows, morphinan; purple arrows, phthalideisoquinoline; light blue arrows, protoberberine; yellow arrows, bisbenzylisoquinoline; grey arows, aporphine; pink arrows, benzo[c]phenanthridine; Enzymes for which corresponding genes have been isolated from other plants were shown in green. Enzymes for which corresponding genes have not been isolated from any plant are shown in black. The number of identified C. yanhusuo unigenes is shown in () following the abbreviated nameof the corresponding enzyme whose full name can be found in Table S1. Intermediates or alkaloid compounds that have been identified in C. yanhusuo are shaded in gray. Although coptisine, tetrahydrojatrorrhizine, jatrorrhizine, allocryptopine, N-methyltetrahydrocolumbamine, N-methyltetrahydrocoptisine and columbamine are also present in C. yanhusuo bulbs, their final biosynthetic steps mainly involved STOX and are not displayed here since STOX catalyzes the formation of many such metabolites. Two unigenes identified as Pavine N-methyltransferase are not listed here. Abbreviations unlisted in Table S1: 4HPP, 4- Hydroxyphenylpyruvate; 4HPAA, 4- Hydroxy- phenylacetaldehyde; 3OHase tyrosine/tyramine 3-hydroxylase; 4HPPDC 4-hydroxyphenylpuruvate decarboxylase; 3′OHase uncharacterized 3′-hydroxylase; 3′OMT uncharacterized 3′-O-methyltransferase. (b) Expression patterns of representative DEGs. Only one of the differentially expressed BIA unigenes with similar expression patterns is shown here to represent different expression patterns of the gene family. The grids with 10 different colors from dark green to red show the relative expression levels to maximum RPKM values: 0–10, 10–20, 20–30, 30–40, 40–50, 50–60, 60–70, 70–80, 80–90 and 90–100%, respectively. The detailed value for each gene is given in Table S7. Unigene IDs are shown on the left. On the right of the grids, the numbers of differentially expressed BIA unigenes and DEGs with similar expression patterns are shown in (), following the abbreviated names of the corresponding enzymes. Expression of the enzymes functioning in multiple branch pathways is displayed just once.

Figure 5. Expression patterns of Monoterpenoid Indole Alkaloids (MIAs) biosynthetic unigenes during bulb development.

(a) Biosynthetic pathways of MIAs are based on PlantCYC (9.5), Gidding et al., De Luca et al. and Verma et al.. Enzymes for which corresponding genes have been isolated from other plants are shown in green next to the arrows. Two or more known steps between two intermediates, whose cDNA sequences were not isolated, are omitted here but indicated by two continuous arrows. Black arrows, secologanin and strictosidine biosynthesis; blue arrows, ajmaline and raucaffricine biosynthesis after the branch-point intermediate aglycone; pink arrows, tabersonine-derived alkaloid biosynthesis after the branch-point intermediate aglycone, represented by vindoline and vinblastine biosynthesis. Full names of enzymes represented by their abbreviated names can be found in Table S1. (b) Expression patterns of representative DEGs. Illustrations are provided in Fig. 4b.

Figure 6. Expression patterns of Tropane and Pyridine alkaloids biosynthetic unigenes over C. yanhusuo bulb development.

(a) Biosynthetic pathways are based on Dewey and Xie, Kushwaha et al. and Bedewitz et al.. Black arrows, N-methyl-Δ¹-pyrrolinium cation biosynthesis; blue arrows, various tropane alkaloids biosynthesis; pink arrows, pyridine alkaloid biosynthesis, represented by nicotine biosynthesis. Enzymes for which corresponding genes have been isolated from other plants are shown in green next to the arrows. Two or more known steps between two intermediates, whose cDNA sequences haven’t been isolated, are omitted here but indicated by continuous arrows. Full names of enzymes by abbreviations are provided in Table S1. NAMN, nicotinic acid mononucleotide. (b) Expression patterns of representative DEGs. Illustrations are provided in Fig. 4b.

Figure 7. Expression patterns of betalain alkaloids biosynthetic unigenes duringbulb development.

(a) The betalain biosynthetic pathway is modified from Suzuki et al.. Full names of abbreviated enzymes can be found in Table S1. (b) Expression patterns of representative DEGs. Illustrations are provided in Fig. 4b.

Figure 8. Chromatograms of BIA standards at 280 nm

(a) and C. yanhusuo bulb extract monitored at 280 (b), 470 (c) and 535 nm (d). Numbers in (a) and (b) refer to magnoflorine (1), corytuberine (2), tetrahydropalmatine (3) sanguinarine (4) and dihydrosanguinarine (5). Numbers in (c) refer to unknown components detected at 470 nm.