Drought Stress Inhibits the Accumulation of Rotenoids and the Biosynthesis of Drought-Responsive Phytohormones in Mirabilis himalaica (Edgew.) Heim Calli

Abstract

Background:Mirabilis himalaica, distributed in the high-altitude, arid, and semi-arid regions of Xizang, exhibits great tolerance to drought, which is rich in rotenoids and other secondary metabolites. It is still unknown, though, how drought stress influences rotenoid synthesis in M. himalaica. Methods: In this study, the calli of M. himalaica were subjected to 5% PEG6000 for 0, 20, and 40 h and divided into control group (CK), mild-drought-treated group (M), and high-drought-treated group (H), respectively. We then analyzed the relative content of three main rotenoids in M. himalaica using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). Results: Our findings demonstrated that the content of rotenoids was significantly reduced under drought stress. Transcriptome analysis subsequently revealed 14,525 differentially expressed genes (DEGs) between the different treatments. Furthermore, these DEGs exhibited enrichment in pathways associated with isoflavone biosynthesis and hormone signaling pathways. Key genes with decreased expression patterns during drought stress were also found to be involved in rotenoid accumulation and drought-responsive phytohormone signaling, including abscisic acid (ABA), auxin (IAA), and jasmonic acid (JA). Conclusions: These findings elucidate the molecular processes of drought resistance in M. himalaica and shed light on the relationship between rotenoid production and drought stress in M. himalaica.

Keywords: Mirabilis himalaica; abscisic acid; auxin; drought stress; jasmonate; rotenoids; transcriptome.

Figure 1

Analysis of the relative content of three rotenoids. (A) M. himalaica leaves before and after callus induction; (B) total ion current plots of three rotenoids; (C) the relative content of boeravionon E, boeravionon B, and boeravionon A. Statistical analysis was performed using GraphPad Prism (9.5.1) software. The significance of differences between groups was assessed using one-way ANOVA. Asterisks * indicate statistical significance at p < 0.05.

Figure 2

Gene functional annotation, analysis of the relationship between samples, and KEGG enrichment analysis of DEGs. (A) Venn plot of the genes annotated in the NR, Swiss Prot, KEGG, and KOG databases; (B) PCA between samples; (C) the number of differentially expressed genes (DEGs) in different comparison groups; (D) volcano maps of DEGs of M vs. CK and H vs. CK; (E) M vs. CK DEGs KEGG enrichment bubble map; (F) H vs. CK DEGs KEGG enrichment bubble map; (G) Analysis of the isoflavonoid biosynthesis pathway in M. himalaica associated with the three treatment groups under drought stress. The solid arrow indicates relationships between molecules in the biosynthetic pathway and activation in the signaling pathway, and the dashed arrow indicates indirect connections. Gene expression levels are presented based on the mean RPKM value from three biological replicates, which were log2 transformed and normalized. The color scale from blue to red represents the expression level of DEGs from low to high. CHS: chalcone synthase; CHI: chalcone isomerase; CYP93C: 2-hydroxyisoflavanone synthase; HIDH: 2-hydroxyisoflavanone dehydratase; 7-IOMT: isoflavone-7-O-methyltransferase; CYP81E: isoflavone 2′-hydroxylase.

Figure 3

Analysis of the ABA, auxin, and JA biosynthesis pathway in M. himalaica under drought stress. (A) The relative content of ABA; (B) analysis of ABA biosynthesis pathway in M. himalaica associated with the three treatment groups under drought stress; (C) the relative content of IAA; (D) analysis of auxin biosynthesis pathway in M. himalaica associated with the three treatment groups under drought stress; (E) the relative content of JA; (F) analysis of the JA biosynthesis pathway in M. himalaica associated with the three treatment groups under drought stress. The solid arrow indicates relationships between molecules in the biosynthetic pathway and activation in the signaling pathway, and the dashed arrow indicates indirect connections. Gene expression levels are presented based on the mean RPKM value from three biological replicates, which were log2 transformed and normalized. The color scale from blue to red represents the expression level of DEGs from low to high. crtZ: β-carotene 3-hydroxylase; CYP97A3: β-ring hydroxylase; ZEP: zeaxanthin epoxidase; NSY: neoxanthin synthase; NCED: 9-cis-epoxycarotenoid dioxygenase; ABA2: xanthoxin dehydrogenase; AAO3: abscisic-aldehyde oxidase; YUCCA: indole-3-pyruvate monooxygenase; ipdC: indolepyruvate decarboxylase; ALDH: aldehyde dehydrogenase; CYP79B1_2: tryptophan N-monooxygenase; CYP83B1: aromatic aldoxime N-monooxygenase; SUR1: S-alkyl-thiohydroximate lyase SUR1; UGT74B1: N-hydroxythioamide S-β-glucosyltransferase; ST5A: aromatic desulfoglucosinolate sulfotransferase; PLA2G, SPLA2: secretory phospholipase A2; PLA2G16: HRAS-like suppressor 3; LOX2S: lipoxygenase; AOS: hydroperoxide dehydratase; AOC: allene oxide cyclase; OPR: 12-oxophytodienoic acid reductase; ACX: acyl-CoA oxidase; MFP2: enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; ACCA1: acetyl-CoA acyltransferase. Statistical analysis was performed using one-way ANOVA. Asterisks * and ** indicate statistical significance at p < 0.05 and p < 0.01, respectively.

Figure 4

Analysis of the ABA, auxin, and JA signal transduction pathway in M. himalaica under drought stress. (A) Analysis of the ABA signal transduction pathway in M. himalaica associated with the three treatment groups under drought stress; (B) analysis of the auxin signal transduction pathway in M. himalaica associated with the three treatment groups under drought stress; (C) analysis of the JA signal transduction pathway in M. himalaica associated with the three treatment groups under drought stress. The solid arrow indicates activation in the signaling pathway, and the vertical solid line with a short transverse solid line at the end indicates inhibition. Gene expression levels are presented based on the mean FPKM value from three biological replicates, which were log2 transformed and normalized. The color scale from blue to red represents the expression level of DEGs from low to high. PYR/PYL: pyrabactin resistance 1/pyrabactin-resistance-like; PP2C: protein phosphatase 2C; SNRK2: sucrose nonfermenting related kinases 2; ABF: ABA response element binding factor; ABRE: abscisic acid response element; AUX1: auxin influx carrier; TIR1: transport inhibitor response 1; IAA: auxin-responsive protein IAA; ARF: auxin response factor; CH3: auxin responsive GH3 gene family; SAUR: SAUR family protein; JAR1: jasmonic acid-amino synthetase; COI-1: coronatine-insensitive protein 1; JAZ: jasmonate ZIM domain-containing protein; MYC2: transcription factor MYC2.

Figure 5

Quantitative analysis of genes related to M. himalaica responses to drought stress.

Figure 6

Theoretical molecular mechanism in M. himalaica in response to drought stress. ABA: abscisic acid; ARF: auxin response factor; AuxRE: auxin response element; HIDH: 2-hydroxyisoflavanone dehydratase. The black arrows indicate a drop in content or expression, the blue arrows indicate the flow (next level reaction), and the black broken line arrows indicate transcription and translation.