ABA and SA Participate in the Regulation of Terpenoid Metabolic Flux Induced by Low-Temperature within Conyza blinii

Abstract

Under dry-hot valley climates, Conyza blinii (also known as Jin Long Dan Cao), suffers from nocturnal low-temperature stress (LTS) during winter. Here, to investigate the biological significance of terpenoid metabolism during LTS adaptation, the growth state and terpenoid content of C. blinii under different LTS were detected, and analyzed with the changes in phytohormone. When subjected to LTS, the results demonstrated that the growth activity of C. blinii was severely suppressed, while the metabolism activity was smoothly stimulated. Meanwhile, the fluctuation in phytohormone content exhibited three different physiological stages, which are considered the stress response, signal amplification, and stress adaptation. Furthermore, drastic changes occurred in the distribution and accumulation of terpenoids, such as blinin (diterpenoids from MEP) accumulating specifically in leaves and oleanolic acid (triterpenoids from MVA) accumulating evenly and globally. The gene expression of MEP and MVA signal transduction pathways also changes under LTS. In addition, a pharmacological study showed that it may be the ABA-SA crosstalk driven by the LTS signal, that balances the metabolic flux in the MVA and MEP pathways in an individual manner. In summary, this study reveals the different standpoints of ABA and SA, and provides a research foundation for the optimization of the regulation of terpenoid metabolic flux within C. blinii.

Keywords: Conyza blinii (Jin Long Dan Cao); low-temperature stress; phytohormone fluctuation; terpenoid metabolic flux.

Figure 1

The morphology and characteristics of C. blinii during nocturnal LTS. C. blinii was subjected to nocturnal LTS for 9 weeks, and then the morphology and defoliation (A), the number of active GTs (B), the pectin and cellulose content in each tissue (C), and the photosynthetic pigment content (D) were detected. The CK group did not receive nocturnal LTS treatment.

Figure 2

The content and distribution of blinin and oleanolic acid within C. blinii tissues during nocturnal LTS. After being subjected to nocturnal LTS for 1 week, an obvious accumulation occurred regarding blinin and oleanolic acid within leaves (A). During monitoring of the 9 weeks of nocturnal LTS, blinin displayed a specific distribution within leaves (B), while oleanolic acid displayed an even distribution (C). In order to observe the balance between MVA and MEP metabolic pathways, we calculated the percentage of blinin and oleanolic acid in leaves (D), stems (E) and roots (F) respectively. Three independent strains were selected. Different letters indicate significant differences between CK and experimental groups at the same time (a, b, c, p < 0.05).

Figure 3

The content and distribution of phytohormones within C. blinii tissues during nocturnal LTS. We monitored and analyzed the trend of phytohormone content in roots (A), stems (C) and leaves (E) from 1 to 9 weeks of nocturnal LTS, and calculated the percentage of phytohormones (B,D,F). Unexpectedly, the contents of ZT and BR in this study were one order of magnitude lower than that of other plant hormones, so we independently displayed the trend (F,G). Three independent strains were selected. Different letters indicate significant differences between CK and experimental groups at the same time (a, b, c, p < 0.05).

Figure 4

The functional verification of ABA and SA effects on terpenoid metabolism during nocturnal LTS in leaves. The correlation network among phytohormone, blinin and oleanolic acid within C. blinii tissues was built using Pearson ((A), Supplemental Tables S1 and S2). The red line is the positive correlation, while the blue line is the negative correlation. Meanwhile, The thicker and clearer the line, the stronger the correlation and the greater the p value. Subsequently, exogenous SA and ABA with their inhibitors were utilized to verify their function in terpenoid metabolism (B). Three independent strains were selected. Different letters indicate significant differences between CK and experimental groups at the same time (a, b, p < 0.05).

Figure 5

Changes in gene expression related to ABA and SA signaling pathways in LTS. (A) SA signaling pathway diagram and changes in expression of genes related to SA signal transduction pathway in C. blinii. (B) The schematic diagram of the ABA signaling pathway and the change in gene expression related to the ABA signal transduction pathway in C. blinii. CK: LTS treatment 0 weeks, S2W: LTS treatment 2 weeks, S5W: LTS treatment 5 weeks, S8W: LTS treatment 8 weeks. Three independent strains were selected. Letters indicate significant differences between CK and experimental groups at the same time (a, p < 0.01).

Figure 6

The effects of different LTS sources on blinin, oleanolic acid, SA, and ABA in leaves. In this study, the LTS source was divided into ground LTS (Glt), underground LTS (Ult), and whole LTS (Wlt) for 48 h, and we monitored the emergency trends of blinin and oleanolic acid to represent terpenoid metabolism (A) in leaves, as well as that of SA and ABA to represent ABA-SA crosstalk (B). Three independent strains were selected. Different letters indicate significant differences between CK and experimental groups at the same time (a, b, c, p < 0.05).

Figure 7

The potential mode of ABA-SA crosstalk involved in terpenoid metabolism regulation when C. blinii subjected to LTS.