SWATH-MS-Based Proteomics Reveals the Regulatory Metabolism of Amaryllidaceae Alkaloids in Three Lycoris Species

Abstract

Alkaloids are a class of nitrogen-containing alkaline organic compounds found in nature, with significant biological activity, and are also important active ingredients in Chinese herbal medicine. Amaryllidaceae plants are rich in alkaloids, among which galanthamine, lycorine, and lycoramine are representative. Since the difficulty and high cost of synthesizing alkaloids have been the major obstacles in industrial production, particularly the molecular mechanism underlying alkaloid biosynthesis is largely unknown. Here, we determined the alkaloid content in Lycoris longituba, Lycoris incarnata, and Lycoris sprengeri, and performed a SWATH-MS (sequential window acquisition of all theoretical mass spectra)-based quantitative approach to detect proteome changes in the three Lycoris. A total of 2193 proteins were quantified, of which 720 proteins showed a difference in abundance between Ll and Ls, and 463 proteins showed a difference in abundance between Li and Ls. KEGG enrichment analysis revealed that differentially expressed proteins are distributed in specific biological processes including amino acid metabolism, starch, and sucrose metabolism, implicating a supportive role for Amaryllidaceae alkaloids metabolism in Lycoris. Furthermore, several key genes collectively known as OMT and NMT were identified, which are probably responsible for galanthamine biosynthesis. Interestingly, RNA processing-related proteins were also abundantly detected in alkaloid-rich Ll, suggesting that posttranscriptional regulation such as alternative splicing may contribute to the biosynthesis of Amaryllidaceae alkaloids. Taken together, our SWATH-MS-based proteomic investigation may reveal the differences in alkaloid contents at the protein levels, providing a comprehensive proteome reference for the regulatory metabolism of Amaryllidaceae alkaloids.

Keywords: Lycoris; SWATH-MS; alkaloids; galanthamine; metabolism.

Figure 1

Phenotype of samples and alkaloid contents of three kinds of Lycoris species. (A) The three kinds of plant materials: Lycoris longituba, Lycoris incarnata, and Lycoris sprengeri. They were collected from the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, China (32.05° N, 118.83° E) in late March when the leaves were growing vigorously (Bars = 3 cm). (B) The total content of three alkaloids in Lycoris longituba was the highest, whereas that in Lycoris sprengeri was the lowest.

Figure 2

The overview and functional classification by KEGG and MAPMAN BIN.of DEPs. (A) The volcano picture shows the distribution of differentially expressed proteins with different fold changes. (B) Venn diagram shows the number of differential proteins compared between the two groups. (C) KEGG pathway classification was performed on the differential proteins of the 2 groups and enriched in the top 10 pathways. Amino acid biosynthesis, starch, and sucrose metabolic pathways are significantly activated. (D) MAPMAN BIN categorization of DEPs; blue and red represent down-regulated and up-regulated proteins, respectively.

Figure 2

The overview and functional classification by KEGG and MAPMAN BIN.of DEPs. (A) The volcano picture shows the distribution of differentially expressed proteins with different fold changes. (B) Venn diagram shows the number of differential proteins compared between the two groups. (C) KEGG pathway classification was performed on the differential proteins of the 2 groups and enriched in the top 10 pathways. Amino acid biosynthesis, starch, and sucrose metabolic pathways are significantly activated. (D) MAPMAN BIN categorization of DEPs; blue and red represent down-regulated and up-regulated proteins, respectively.

Figure 3

Amino acid metabolism is closely related to the biosynthesis of Amaryllidaceae alkaloids in Lycoris. We detected a total of 38 differential proteins in the amino acid metabolism pathway. The highly expressed proteins were mainly concentrated in Ll, which was consistent with the actual detected trend of alkaloid content of the three varieties.

Figure 4

Candidate genes responsible for the biosynthesis of galanthamine in Lycoris. 189251_c7_g1.p1 and 175950_c0_g1.p1 and other genes upregulated in Ll, which provide the raw material for the biosynthesis of galanthamine, consistent with the highest galanthamine content in Ll.

Figure 5

Potenial pathways contribute to the biosynthesis of Amaryllidaceae alkaloids in Lycoris. (A) Some proteins were detected to be enriched in starch and sucrose metabolism pathways suggesting a reciprocal regulation between alkaloids and sugar metabolism. (B) In secondary metabolic pathways, the up-regulated 187456_c3_g1.p1 and 189251_c7_g1.p1 in Ll participate in the biosynthesis of sinapyl alcohol, resulting in an increase in the alkaloid raw material sinapyl alcohol. Eventually, this leads to a rise in alkaloid content.