Activity of purified and reconstituted NtPDR1, a plant ABC transporter, is stimulated by diterpenes and sesquiterpenes involved in constitutive and induced defense
(2017)
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- Authors
- Supervisors
- Boutry, Marc
- Abstract
- In plants, members of the ATP-Binding Cassette (ABC) superfamily of proteins play a wide range of essential functions, including hormone transport or defense against (a)biotic stresses. Despite their physiological importance, very little information is available about their biochemical properties. Our laboratory previously demonstrated that Nicotiana tabacum NtPDR1, a full-size ABCG transporter is involved in constitutive defense against pathogens. In vivo experiments suggested that the protein could secrete cembrene and sclareol (toxic cyclic diterpenes) out of tobacco trichomes. Yet, direct evidence associating NtPDR1 with its putative substrates was lacking. Our project aimed at providing such data as well as enzymatic and structural information about NtPDR1. To do so, we first expressed the protein provided with a purification tag in N. tabacum Bright Yellow 2 (BY-2) suspension cells under the control of a strong constitutive transcriptional promoter. We then optimized the solubilization and purification of NtPDR1 and obtained purified enzyme with sufficient yields to perform biochemical studies. Purified NtPDR1 was monodispersed and monomeric as observed by gel filtration and electron microscopy followed by single particle analysis. In addition, purified NtPDR1 retained vanadate sensitive ATP hydrolytic activity that was stimulated by both sclareol and cembrene. The transporter was further reconstituted in asolectin liposomes. Upon reconstitution, NtPDR1 ATPase activity was increased from 21 to 38 nmol Pi.mg-1.min-1 and the reconstituted transporter was again stimulated by sclareol and cembrene. Interestingly, capsidiol, a sesquiterpene involved in induced defense against pathogens, was also found to stimulate NtPDR1 ATPase activity. In contrast, other terpenoids (geranylgeraniol and eucalyptol) did not stimulate the transporter. In conclusion, our work provides direct evidence of the interaction between a plant ABC transporter and its substrates. We demonstrated in vitro that the activity of purified NtPDR1 is specifically stimulated by both cyclic di- and sesqui-terpenes involved in constitutive and induced plant defense. In addition, this project demonstrates that N. tabacum BY-2 cells are a convenient host for plant ABC transporters expression, purification, reconstitution, and biochemical characterization.
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Citations
Pierman, B. (2017). Activity of purified and reconstituted NtPDR1, a plant ABC transporter, is stimulated by diterpenes and sesquiterpenes involved in constitutive and induced defense. https://hdl.handle.net/2078.5/177514