Activity of the purified plant ABC transporter NtPDR1 is stimulated by diterpenes and sesquiterpenes involved in constitutive and induced defenses.

Within the plant ATP-binding cassette transporter family, pleiotropic drug resistance (PDR) transporters play essential functions, such as in hormone transport or defense against biotic and abiotic stresses. NtPDR1 from Nicotiana tabacum has been shown to be involved in the constitutive defense against pathogens through the secretion of toxic cyclic diterpenes, such as the antimicrobial substrates cembrene and sclareol from the leaf hairs (trichomes). However, direct evidence of an interaction between NtPDR1 and terpenes is lacking. Here, we stably expressed NtPDR1 in N. tabacum BY-2 suspension cells. NtPDR1 was purified as an active monomer glycosylated at a single site in the third external loop. NtPDR1 reconstitution in proteoliposomes stimulated its basal ATPase activity from 21 to 38 nmol of Pi·mg-1·min-1, and ATPase activity was further stimulated by the NtPDR1 substrates cembrene and sclareol, providing direct evidence of an interaction between NtPDR1 and its two substrates. Interestingly, NtPDR1 was also stimulated by capsidiol, a sesquiterpene produced by N. tabacum upon pathogen attack. We also monitored the transcriptional activity from the NtPDR1 promoter in situ with a reporter gene and found that, although NtPDR1 expression was limited to trichomes under normal conditions, addition of methyl jasmonate, a biotic stress hormone, induced expression in all leaf tissues. This finding indicated that NtPDR1 is involved not only in constitutive but also in induced plant defenses. In conclusion, we provide direct evidence of an interaction between the NtPDR1 transporter and its substrates and that NtPDR1 transports compounds involved in both constitutive (diterpenes) and induced (sesquiterpenes) plant defenses.

Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter.

Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission.

Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed in Nicotiana tabacum BY-2 suspension cells.

Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia, which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min-1 mg-1) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters.