RNA-sequencing-based transcriptome and biochemical analyses of steroidal saponin pathway in a complete set of Allium fistulosum-A. cepa monosomic addition lines

Abstract

The genus Allium is a rich source of steroidal saponins, and its medicinal properties have been attributed to these bioactive compounds. The saponin compounds with diverse structures play a pivotal role in Allium's defense mechanism. Despite numerous studies on the occurrence and chemical structure of steroidal saponins, their biosynthetic pathway in Allium species is poorly understood. The monosomic addition lines (MALs) of the Japanese bunching onion (A. fistulosum, FF) with an extra chromosome from the shallot (A. cepa Aggregatum group, AA) are powerful genetic resources that enable us to understand many physiological traits of Allium. In the present study, we were able to isolate and identify Alliospiroside A saponin compound in A. fistulosum with extra chromosome 2A from shallot (FF2A) and its role in the defense mechanism against Fusarium pathogens. Furthermore, to gain molecular insight into the Allium saponin biosynthesis pathway, high-throughput RNA-Seq of the root, bulb, and leaf of AA, MALs, and FF was carried out using Illumina's HiSeq 2500 platform. An open access Allium Transcript Database (Allium TDB, http://alliumtdb.kazusa.or.jp) was generated based on RNA-Seq data. The resulting assembled transcripts were functionally annotated, revealing 50 unigenes involved in saponin biosynthesis. Differential gene expression (DGE) analyses of AA and MALs as compared with FF (as a control) revealed a strong up-regulation of the saponin downstream pathway, including cytochrome P450, glycosyltransferase, and beta-glucosidase in chromosome 2A. An understanding of the saponin compounds and biosynthesis-related genes would facilitate the development of plants with unique saponin content and, subsequently, improved disease resistance.

Fig 1. Allium cepa Aggregatum group (AA), monosomic addition lines (MALs = FF1A, FF2A, FF3A, FF4A, FF5A, FF6A, FF7A, and FF8A) and A. fistulosum (FF) saponin TLC profile.

(A) Total saponin profile visualized by p-anisaldehyde reagent; arrow indicates the Alliospiroside A accumulation in AA and FF2A. (B) Furostanol saponin profile visualized by Ehrlich's reagent; arrow indicates the furostanol saponin accumulation in AA, FF1A, and FF2A.

Fig 2. Box plot diagram showing the changes in (A) root, (B) bulb (C) and leaf saponin contents (mg g−1 DW) in Allium cepa Aggregatum group (AA), monosomic addition lines (MALs = FF1A, FF2A, FF3A, FF4A, FF5A, FF6A, FF7A, and FF8A) and A. fistulosum (FF).

Values represent the maximum, third quartile, median, first quartile and minimum of three independent replicates (n = 3). Different letters indicate statistically significant difference at P < 0.05 according to Tukey’s honest significant difference (HSD) post-hoc test.

Fig 3. The biological assays of Allium cepa Aggregatum group (AA), monosomic addition lines (MALs = FF1A, FF2A, FF3A, FF4A, FF5A, FF6A, FF7A, and FF8A) and A. fistulosum (FF) crude saponin extracts and Alliospiroside A against Fusarium oxysporum f.sp. cepa.

(A) AA-, MALs- and FF-root saponin extracts antifungal activity against F. oxysporum f.sp. cepa strain TA and AF22. (B) Potato dextrose agar (PDA) plates of Alliospiroside A antifungal activity against F. oxysporum f.sp. cepa strain TA and AF22. (C) Alliospiroside A and furostanol saponin antifungal activity against F. oxysporum f.sp. cepa strain TA and AF22. (D) Alliospiroside A chemical structure. Antifungal activity values are means of three independent replication (n = 3) ± standard errors (SEs). Different letters indicate statistically significant differences according to Tukey’s honestly significant difference (HSD) post-hoc test.

Fig 4. Transcriptomic profiling of steroidal saponin pathway in the root, bulb and leaf of Allium cepa Aggregatum group (AA) and monosomic addition lines (MALs = FF1A, FF2A, FF3A, FF4A, FF5A, FF6A, FF7A and FF8A) as compared with A. fistulosum (FF) as control.

(A) Schematic representation of the steroidal saponin biosynthesis pathway and heatmap clustering of the 50 unigene detected in this study which functionally involved in the upstream (B) and downstream (C) saponin biosynthesis pathway. Heatmap constructed using FPKM log2 fold change.

Fig 5. Representative model of Alliospiroside A biosynthesis and defense mechanism in resistance and susceptible Allium genotypes against Fusarium pathogens.