Molecular Cloning and Functional Characterization of CpMYC2 and CpBHLH13 Transcription Factors from Wintersweet (Chimonanthus praecox L.)

Abstract

Wintersweet (Chimonanthus praecox L.) is an ornamental and economically significant shrub known for its unique flowering characteristics, especially the emission of abundant floral volatile organic compounds. Thus, an understanding of the molecular mechanism of the production of these compounds is necessary to create new breeds with high volatile production. In this study, two bHLH transcription factors (CpMYC2 and CpbHLH13) of Wintersweet H29 were functionally characterized to illustrate their possible role in the production of volatile compounds. The qRT-PCR results showed that the expression of CpMYC2 and CpbHLH13 increased from the flower budding to full bloom stage, indicating that these two genes may play an essential role in blooming and aroma production in wintersweet. Gas chromatography-mass spectroscopy (GC-MS) analysis revealed that the overexpression of CpMYC2 in arabidopsis (Arabidopsis thaliana) AtMYC2-2 mutant (Salk_083483) and tobacco (Nicotiana tabaccum) genotype Petit Havana SR1 significantly increased floral volatile monoterpene, especially linalool, while the overexpression of CpbHLH13 in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and tobacco genotype SR1 increased floral sesquiterpene β-caryophyllene production in both types of transgenic plants respectively. High expression of terpene synthase (TPS) genes in transgenic A. thaliana along with high expression of CpMYC2 and CpbHLH13 in transgenic plants was also observed. The application of a combination of methyl jasmonic acid (MeJA) and gibberellic acid (GA3) showed an increment in linalool production in CpMYC2-overexpressing arabidopsis plants, and the high transcript level of TPS genes also suggested the involvement of CpMYC2 in the jasmonic acid (JA) signaling pathway. These results indicate that both the CpMYC2 and CpbHLH13 transcription factors of wintersweet are possibly involved in the positive regulation and biosynthesis of monoterpene (linalool) and sesquiterpene (β-caryophyllene) in transgenic plants. This study also indicates the potential application of wintersweet as a valuable genomic material for the genetic modification of floral scent in other flowering plants that produce less volatile compounds.

Keywords: bHLH transcription factors; functional analysis; molecular cloning; terpene production; volatile production; wintersweet (Chimonanthus praecox L.).

Figure 1

Developing stages of the flower of the Wintersweet (Chimonanthus praecox) H29 (Huazhong 29) genotype. Abbreviations: FB, flower bud; DP, display petal; POF, partially-open flower; OF, open flower stage; SF, senescing flower. Scale bar = 1 cm.

Figure 2

Multiple sequence alignment of the wintersweet CpMYC2 protein sequence with its homologous proteins from other plant species: Cinnamomum micranthum CmMYC2 (RWR86802.1), Nicotiana tabacum NtMYC2 (XP_016500373.1), Solanum lycopersicum SlMYC2 (NP_001311412.1), Vitis vinifera VvMYC2 (XP_002280253.2), Arabidopsis thaliana AtMYC2 (At1g32640), and Zea mays ZmMYC2 (PWZ55921.1). The red box indicates the basic helix-loop-helix DNA-binding domain common in plants.

Figure 3

Multiple sequence alignment of the Wintersweet bHLH13 protein sequence with its homologous proteins from other plant species: Cinnamomum micranthum CmbHLH13 (RWR96436.1), Vitis vinifera VvbHLH13 (RVW95465.1), Malus domestica MdbHLH13 (XP_028955372.1), Nicotiana tabacum NtbHLH13 (XP_016459177.1), Solanum lycopersicum SlbHLH13 (XP_004229991.1), and Arabidopsis thaliana AtbHLH13 (AT1G01260). The red box indicates the basic helix-loop-helix DNA-binding domain common in plants.

Figure 4

The transcriptomic expression pattern of (A) CpMYC2 and (B) CpbHLH13 during flower developing stages of H29. The stages are FB: flower bud, DP: display petal, POF: partially-open flower, OF: open flower, SF: senescing flower. Tissue-specific analysis of (C) CpMYC2 and (D) CpbHLH13 expression in wintersweet. The different letters on the bars show significance between the treatments by using least significant difference (LSD) at p < 0.05.

Figure 5

Expression of (A) the monoterpene synthase genes At1g61680 and At3g25810 in arabidopsis wild type (WT-Col-0), mutant (Salk_083483), and mutant Salk_083483 transformed by the empty vector pCAMBIA2300S (2300S-Sk83) and overexpressed CpMYC2 gene (35S::CpMYC2) plants and (B) the sesquiterpene synthase genes At5g23960 and At5g44630 in arabidopsis wild type (WT-Col-0), wild type transformed by the empty vector pCAMBIA2300S (2300S-Col-0) and overexpressed CpbHLH13 gene (35S::CpbHLH13) plants measured by qRT-PCR. The different letters on the bars show significance between the treatments by using least significant difference (LSD) at p < 0.05.

Figure 6

The concentration of emitted linalool (ng g-1 FW h-1) from (A) arabidopsis wild type (WT-Col-0), mutant (Salk_083483), mutant Salk_083483 transformed by the empty vector pCAMBIA2300S (2300S-Sk83) and overexpressed CpMYC2 gene (35S::CpMYC2), and (B) tobacco wild type SR1, transformed by the empty vector pCAMBIA2300S (2300S-SR1) and overexpressed CpMYC2 gene (35S::CpMYC2) plants. Concentration of emitted β-caryophyllene (ng g-1 FW h-1) from (C) arabidopsis wild type (WT-Col-0), wild type transformed by empty vector pCAMBIA2300S (2300S-Col-0) and overexpressed CpbHLH13 gene (35S::CpbHLH13), and (D) tobacco wild type SR1, transformed by the empty vector pCAMBIA2300S (2300S-SR1) and overexpressed CpbHLH13 gene (35S::CpbHLH13) plants. The different letters on the bars show significance between the treatments by using least significant difference (LSD) at p < 0.05.

Figure 7

GC-MS analysis of linalool emitted from the flowers of (A) arabidopsis wild type (WT-Col-0), mutant Salk-083483, transformed by the empty vector pCAMBIA2300S (2300S-Sk83) and overexpressed CpMYC2 gene (35S::CpMYC2) plants, and (B) tobacco wild type SR1, transformed by the empty vector pCAMBIA2300S (2300S-SR1) and overexpressed CpMYC2 gene (35S::CpMYC2) plants. (a) GC-MS chromatogram comparison between different plants. (b) Mass spectrum of the product. (c) Product confirmation in the NIST database according to retention index and mass spectrum.

Figure 8

GC-MS analysis of β-caryophyllene emitted from the flowers of (A) tobacco wild type SR1, transformed by the empty vector pCAMBIA2300S (2300S-SR1) and overexpressed CpbHLH13 gene (35S::CpbHLH13) plants; (B) arabidopsis wild type (WT-Col-0), transformed by the empty vector pCAMBIA2300S (2300S-SR1) and overexpressed CpbHLH13 gene (35S:CpbHLH13) plants. (a) GC-MS chromatogram comparison between different plants. (b) Mass spectrum of the product. (c) Product confirmation in the NIST database according to retention index and mass spectrum.

Figure 9

The concentration of emitted linalool (ng g-1 FW h-1) after the application of methyl jasmonic acid (MeJA) and gibberellic acid (GA3) from arabidopsis wild type (WT-Col-0), mutant (Salk_083483), mutant Salk_083483 transformed by the empty vector pCAMBIA2300S (2300S-Sk83), and CpMYC2 gene (35S::CpMYC2) plants. The different letters on the bars show significance between the treatments by using least significant difference (LSD) at p < 0.05.

Figure 10

GC-MS analysis of linalool emitted after the application of MeJA and GA3 from the flowers of arabidopsis wild type (WT-Col-0), mutant (Salk_083483), mutant Salk_083483 transformed by the empty vector pCAMBIA2300S (2300S-Sk83), and CpMYC2 gene (35S::CpMYC2) plants. (a) GC-MS chromatogram comparison between different plants. (b) Mass spectrum of the product. (c) Product confirmation in the NIST database according to retention index and mass spectrum.

Figure 11

Expression of the monoterpene synthase genes At1g61680 and At3g25810 in arabidopsis plants by qRT-PCR after the application of MeJA and GA3 in arabidopsis wild type (WT-Col-0), mutant (Salk_083483), mutant Salk_083483 transformed by the empty vector pCAMBIA2300S (2300S-Sk83), and CpMYC2 gene (35S::CpMYC2) plants measured by qRT-PCR. The different letters on the bars show significance between the treatments by using least significant difference (LSD) at p < 0.05.

Figure 12

Anthocyanin accumulation from the flowers of (A) overexpressed CpbHLH13 tobacco plants (35S:CpbHLH13), wild type tobacco SR1 (Wild Type-SR1), and tobacco plants transformed by the empty vector pCAMBIA2300S (2300S-SR1). (B) Anthocyanin detection from the described plants. (C) Quantitative anthocyanin concentration in tobacco plants. Scale bar = 1 cm. The different letters on the bars show significance between the treatments by using least significant difference (LSD) at p < 0.05.