The Blinin Accumulation Promoted by CbMYB32 Involved in Conyza blinii Resistance to Nocturnal Low Temperature

Abstract

Blinin, a unique terpenoid from Conyza blinii (C. blinii), benefits our health even though this is not its primary function. Physiological and ecological studies have found that the great secondary metabolites participate in important biological processes and relate to species evolution, environmental adaptation, and so on. Moreover, our previous studies have shown that the metabolism and accumulation of blinin has a close correspondence with nocturnal low temperature (NLT). To find out the transcriptional regulation linker in the crosstalk between blinin and NLT, RNA-seq, comparative analysis, and co-expression network were performed. The results indicated that CbMYB32 is located in a nucleus without independent transcriptional activation activity and is probably involved in the metabolism of blinin. Furthermore, we compared the silence and overexpression of CbMYB32 with wild C. blinii. Compared with the overexpression and the wildtype, the CbMYB32 silence line lost more than half of the blinin and detected more peroxide under NLT. Finally, as a characteristic secret of C. blinii, it is reasonable to infer that blinin participates in the NLT adaptation mechanism and has contributed to the systematic evolution of C. blinii.

Keywords: Conyza blinii; nocturnal low temperature resistance; transcriptional regulation; unique terpenoid blinin; virus-induced gene silencing.

Figure 1

Conyza blinii transcriptome under NLT. The number of differentially expressed genes in S2W, S5W, and S8W. The intersecting part represents the common genes (A). All clusters were divided into four subclusters according to the expression trend. Above the red line is the up-regulated gene, and below the red line is the down-regulated gene (B). Gene expression of key enzymes in MEP and MVA metabolism pathways. Red represents high expression and blue represents low expression (C).

Figure 2

Gene-terpenoid network of C. blinii in NLT. Co-expression modules of terpenoid metabolism genes clustered. The different colors represent different gene modules. Green modules represent transcription factors, and other modules represent genes regulated by transcription factors. Potentially related genes are connected by gray lines.

Figure 3

Phylogenetic analysis. Phylogenetic tree with TFs of C. blinii and other TFs have been reported involved in terpenoid metabolism within Artemis iaannua, Salvia miltiorrhiza, Scutellaria baicalensis, etc (A). Motif and phylogenetic tree with CbMYB32 and other MYB TFs. Different colors show different motifs (B).

Figure 4

Expression pattern analysis of CbMYB32. Colocalization of p35S-CbMYB32-GFP in tobacco epidermal cells (A). The relative expression of CbMYB32 in NLT and different plant tissues (B). Three independent biological repeats were set for each group. Asterisk indicates significant differences between each group (*** p < 0.001). Transcriptional activation assays of full length CbMYB32 fused with the GAL4 DNA-binding domain (GAL4DB) in yeast (C). (-/Trp) indicates selective medium lacking Trp, (-Trp/-His/) indicates selective medium lacking Trp, His. Yeast growing on SD-Trp/-His/ soiled medium dyed by X-β-gal.

Figure 5

Agrobacterium-mediated transient transformation of C. blinii. Development of CbPDS transgenic leaves. DAI: days after infected. Leaves will bleach when the CbPDS is silenced. Bar = 1 cm. (A). CbPDS-VIGS leaf phenotype. Three samples were randomly selected to detect changes in expression after CbPDS was silenced. The pTRV₁ + pTRV₂ group indicating the empty vector control (B). The relative expression of CbMYB32 treated with OE (overexpression) and VIGS under NLT for 3 days. Changes of blinin content after OE and VIGS under NLT for 3 days. The pCambia1300 indicating the empty vector control (C). Determination of hydrogen peroxide content under NLT (D). Determination of hydroxyl radical content under NLT (E). Determination of Malondialdehyde (MAD) content under NLT (F). Three independent biological repeats were set for each group. Asterisk indicates significant differences between CK and experimental groups at the same time (*** p < 0.05). Different letters indicate significant differences at the p < 0.05 level when comparing different experimental groups according to a one-way ANOVA.

Figure 6

A model for NLS in C. blinii mediated by CbMYB32. Under NLS, CbMYB32 can enhance the terpenoid metabolism activity of C. blinii by activating blinin biosynthesis in the MEP pathway, which may connect with the SA signal pathway. The solid line represents the upstream stress signal transduction. The dashed line indicates that there may be an activation reaction.