Abstract
Phytohormones play a major role in plant growth and development. They are in most cases not synthesized in their target location and hence need to be transported to the site of action, by for instance ATP-binding cassette transporters. Within the ATP-binding cassette transporter family, Pleiotropic Drug Resistance transporters are known to be involved in phytohormone transport. Interestingly, PDRs are only present in plants and fungi. In contrast to fungi, there are few biochemical studies of plant PDRs and one major reason is that suitable overexpression systems have not been identified. In this study, we evaluate the expression system Pichia pastoris for heterologous overexpression of PDR genes of the model plant Arabidopsis thaliana. We successfully cloned and expressed the potential phytohormone transporters PDR2 and PDR8 in P. pastoris. Sucrose gradient centrifugation confirmed that the overexpressed proteins were correctly targeted to the plasma membrane of P. pastoris and initial functional studies demonstrated ATPase activity for WBC1. However, difficulties in cloning and heterologous overexpression might be particular obstacles of the PDR family, since cloning and overexpression of White Brown Complex 1, a half-size transporter of the same ABCG subfamily with comparable domain organization, was more easily achieved. We present strategies and highlight critical factors to successfully clone plant PDR genes and heterologously expressed in P. pastoris.
Highlights
Phytohormones are signal molecules which enable plants to adapt growth and development to internal and external stimuli [1]
We evaluate the P. pastoris expression system for heterologous expression of plant ATP-binding cassette (ABC) transporters belonging to the Pleiotropic Drug Resistance (PDR) subfamily and employed P. pastoris as a host for this protein family
Sequence data in this article can be found in The Arabidopsis Information Resource (TAIR) database under accession numbers AT4G15230 (AtABCG30/PDR2), AT2G26910
Summary
Phytohormones (plant hormones) are signal molecules which enable plants to adapt growth and development to internal and external stimuli [1] They comprise various molecule classes like abscisic acid (ABA), auxin, cytokinin, gibberellin, jasmonic acid, ethylene, strigolactones or salicylic acid [2]. ABC transporters are ubiquitous in all kingdoms of life and facilitate the transport of a large variety of substrates across membranes using ATP hydrolysis as energy source [6, 7] They are composed of four core domains: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). NBDs comprise all the sequence motifs required for ATP hydrolysis, while the TMDs are composed of 4 to 10 α-helices and form the substrate translocation pathway across the lipid bilayer. These four domains can be either encoded on a single gene (fullsize transporter), or one NBD and one TMD are fused on one gene (half-size transporter), or all the domains are encoded by separate genes [8,9,10,11,12]
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