In plants, transport processes are important for the reallocation of defence compounds to protect tissues of high value, as demonstrated in the plant model Arabidopsis, in which the major defence compounds, glucosinolates, are translocated to seeds on maturation. The molecular basis for long-distance transport of glucosinolates and other defence compounds, however, remains unknown. Here we identify and characterize two members of the nitrate/peptide transporter family, GTR1 and GTR2, as high-affinity, proton-dependent glucosinolate-specific transporters. The gtr1 gtr2 double mutant did not accumulate glucosinolates in seeds and had more than tenfold over-accumulation in source tissues such as leaves and silique walls, indicating that both plasma membrane-localized transporters are essential for long-distance transport of glucosinolates. We propose that GTR1 and GTR2 control the loading of glucosinolates from the apoplasm into the phloem. Identification of the glucosinolate transporters has agricultural potential as a means to control allocation of defence compounds in a tissue-specific manner.

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.

Although it is essential for plant survival to synthesize and transport defense compounds, little is known about the coordination of these processes. Here, we investigate the above-and belowground source-sink relationship of the defense compounds glucosinolates in vegetative Arabidopsis thaliana. In vivo feeding experiments demonstrate that the glucosinolate transporters1 and 2 (GTR1 and GTR2), which are essential for accumulation of glucosinolates in seeds, are likely to also be involved in bidirectional distribution of glucosinolates between the roots and rosettes, indicating phloem and xylem as their transport pathways. Grafting of wild-type, biosynthetic, and transport mutants show that both the rosette and roots are able to synthesize aliphatic and indole glucosinolates. While rosettes constitute the major source and storage site for short-chained aliphatic glucosinolates, long-chained aliphatic glucosinolates are synthesized both in roots and rosettes with roots as the major storage site. Our grafting experiments thus indicate that in vegetative Arabidopsis, GTR1 and GTR2 are involved in bidirectional longdistance transport of aliphatic but not indole glucosinolates. Our data further suggest that the distinct rosette and root glucosinolate profiles in Arabidopsis are shaped by long-distance transport and spatially separated biosynthesis, suggesting that integration of these processes is critical for plant fitness in complex natural environments.

Gibberellins (GAs) are plant hormones that promote a wide range of developmental processes. While GA signalling is well understood, little is known about how GA is transported or how GA distribution is regulated. Here we utilize fluorescently labelled GAs (GA-Fl) to screen for Arabidopsis mutants deficient in GA transport. We show that the NPF3 transporter efficiently transports GA across cell membranes in vitro and GA-Fl in vivo. NPF3 is expressed in root endodermis and repressed by GA. NPF3 is targeted to the plasma membrane and subject to rapid BFA-dependent recycling. We show that abscisic acid (ABA), an antagonist of GA, is also transported by NPF3 in vitro. ABA promotes NPF3 expression and GA-Fl uptake in plants. On the basis of these results, we propose that GA distribution and activity in Arabidopsis is partly regulated by NPF3 acting as an influx carrier and that GA–ABA interaction may occur at the level of transport.