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. 2024 May 29:15:1378418.
doi: 10.3389/fpls.2024.1378418. eCollection 2024.

Integrating GC-MS and comparative transcriptome analysis reveals that TsERF66 promotes the biosynthesis of caryophyllene in Toona sinensis tender leaves

Jianhua Dai  1 , Minyan Wang  1 , Hengfu Yin  1 , Xiaojiao Han  1 , Yanru Fan  1 , Yi Wei  1 , Jie Lin  1 , Jun Liu  1
Affiliations

Integrating GC-MS and comparative transcriptome analysis reveals that TsERF66 promotes the biosynthesis of caryophyllene in Toona sinensis tender leaves

Jianhua Dai et al. Front Plant Sci. .

Abstract

Introduction: The strong aromatic characteristics of the tender leaves of Toona sinensis determine their quality and economic value.

Methods and results: Here, GC-MS analysis revealed that caryophyllene is a key volatile compound in the tender leaves of two different T. sinensis varieties, however, the transcriptional mechanisms controlling its gene expression are unknown. Comparative transcriptome analysis revealed significant enrichment of terpenoid synthesis pathway genes, suggesting that the regulation of terpenoid synthesis-related gene expression is an important factor leading to differences in aroma between the two varieties. Further analysis of expression levels and genetic evolution revealed that TsTPS18 is a caryophyllene synthase, which was confirmed by transient overexpression in T. sinensis and Nicotiana benthamiana leaves. Furthermore, we screened an AP2/ERF transcriptional factor ERF-IX member, TsERF66, for the potential regulation of caryophyllene synthesis. The TsERF66 had a similar expression trend to that of TsTPS18 and was highly expressed in high-aroma varieties and tender leaves. Exogenous spraying of MeJA also induced the expression of TsERF66 and TsTPS18 and promoted the biosynthesis of caryophyllene. Transient overexpression of TsERF66 in T. sinensis significantly promoted TsTPS18 expression and caryophyllene biosynthesis.

Discussion: Our results showed that TsERF66 promoted the expression of TsTPS18 and the biosynthesis of caryophyllene in T. sinensis leaves, providing a strategy for improving the aroma of tender leaves.

Keywords: AP2/ERF; Terpene synthase (TPS); Toona sinensis; caryophyllene; transient overexpression.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Detection of volatile components in tender leaves of two T. sinensis varieties with different aromas. (A) Schematic diagram illustrating the sampling strategy for the two T. sinensis varieties. Upper, middle and lower represent distinct sections of compound leaves. (B) Heat map of the content of the top 20 volatile components.
Figure 2
Figure 2
Comparative transcriptome analysis in different aroma T. sinensis varieties. (A) DEGs in the upper, middle, and lower parts of compound leaves in LJ and WY varieties (P < 0.05). (B) GO enrichment analysis of all the DEGs. (C) KEGG enrichment analysis of all the DEGs. The top 30 enriched GO or KEGG terms are presented. The horizontal axis represents the factors, while the vertical axis represents the GO terms. Counts: number of DEGs.
Figure 3
Figure 3
Identification of caryophyllene gene in T. sinensis. (A) Heat map showing the expression levels of TPS family members in different parts of compound leaves of various T. sinensis varieties. (B) Detection of volatile components in T. sinensis leaves with transient overexpression of TsTPS18. (C) Relative gene expression in T. sinensis leaves with transient overexpression of TsTPS18. ***p < 0.001.
Figure 4
Figure 4
Transient overexpression of the TsTPS18 gene in tobacco leaves. (A) Detection of volatile components in tobacco leaves with transient overexpression of TsTPS18. (B) Relative gene expression in tobacco leaves with transient overexpression of TsTPS18. (C) Detection of caryophyllene components in T. sinensis leaves with transient overexpression of TsTPS18. **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
Expression trend analysis of TsERF66 and TsTPS18 in T. sinensis. (A) Clustering of expression levels of TsAP2/ERF gene family and TsTPS18 gene. (B) Relative expression analysis of TsTPS18 and TsERF66 genes in tender leaves of WY and LJ. (C) Relative expression analysis of TsTPS18 and TsERF66 genes in different tissues of T. sinensis. ***p < 0.001. Symbols a-i represent significant differences between groups.
Figure 6
Figure 6
Exogenous MeJA treatment of caryophyllene content and expression of TsERF66 and TsTPS18 in T. sinensis leaves. (A) T. sinensis leaves treated with MeJA. The leaves were collected at 0, 6, 9, and 18 h after the treatment. (B) Changes in caryophyllene content in T. sinensis leaves after MeJA treatment. (C, D) Relative expression levels of the TsTPS18 and TsERF66 genes in T. sinensis leaves after MeJA treatment. Symbols a-d represent significant differences between groups.
Figure 7
Figure 7
Transient overexpression of TsERF66 gene in T. sinensis. (A) Alterations in volatile substances content, as determined using GC-MS, in T. sinensis tender leaves after transient overexpression of TsERF66. (B) Variations in caryophyllene content in tender leaves of T. sinensis after transient overexpression of TsERF66. (C) qRT-PCR results of the TsERF66 gene in T. sinensis tender leaves after transient overexpression of TsERF66. ***p < 0.001.
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