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

Muhammad Zeshan Aslam,Xiang Lin,Xiang Li,Longqing Chen,Nan Yang

doi:10.3390/plants9060785

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.

Highlights

Plants produce a flower aroma that plays a significant role in their economic value and aesthetic properties [1]
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 and sesquiterpene (β-caryophyllene) in transgenic plants
The results indicated that the expression levels of CpMYC2 and CpbHLH13 increased from the budding stage of the flower to the fully-open flower stage and gradually decreased in the senescing stage (Figure 4a,b)

Summary

Introduction

Plants produce a flower aroma that plays a significant role in their economic value and aesthetic properties [1]. Each plant has its own unique floral aroma, consisting of volatile organic compounds, which are mainly derivatives of fatty acids, terpenoids, and phenylpropanoids or benzoids. Plants 2020, 9, 785 an understanding of the mechanism associated with the formation of these floral aromas is necessary to create new breeds, mainly in plants that produce less volatile compounds [2,3]. The MVA pathway is involved in the biosynthesis of sesquiterpenes, which accounts for 28% of all floral terpenoids. The MEP pathway is mainly involved in the biosynthesis of monoterpene and diterpene, producing approximately 53% and 1% of the total floral terpenoids, respectively [4]. Many flower-specific terpene syntheses have been isolated and characterized, such as linalool in Arabidopsis thaliana, Osmanthus fragrans, Antirrhinum majus, Clarkia breweri, and Hedychium coronarium [4,5,6,7]; myrcene in Alstroemeria peruviana and A. majus [8]; 1,8-cineole in Nicotiana suaveolens, Citrus unshiu, and H. coronarium [9,10]; E-(β)-ocimine in A. majus and H. coronarium [11,12,13]; sesquiterpene α-farnesene in Actinidia deliciosa and H. coronarium [14,15]; germacrene D in Rosa hybrid, A. deliciosa, and Vitus vinifera [13,16]; nerolidol in A. chinensis and

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Conclusion