Identification of the glucosinolate transporter complement

Intra- and intercellular transport of metabolites is key for nutrient acquisition, hormonal growth, cellular homeostasis, and for communication and coordination of responses in multi-cellular organisms. The highly diverse array of metabolites has driven the evolution of numerous importer and exporter proteins that facilitate transport of these compounds between organelles, cells and organs. The spatial and temporal expression of the transporters requires high levels of coordination to ensure efficient and precise delivery and to prevent clogging. The full transporter complement of any metabolite in a multi-cellular organism needs to be identified to study all of the above in coordination.

Identification of all the players in the GLS transport complement in A. thaliana provides an excellent model system for studying how fine-tuned coordination of the spatial and temporal expression of different transporters ensures that the metabolites are transported within and between organs.

 

Glucosinolates are known to be transported around in the plant, e.g. from silique walls to the seeds. Upon long-distance transport, glucosinolates have to cross multiple membrane barriers as they exit source tissues, are loaded into the phloem 'transport highway', exit this and enter sink tissues, which in the case of seeds represent another individual. At the subcellular level, additional transporters are involved. Our goal is to identify (sub)cellular importers and exporters of glucosinolates. This will identify critical barriers along the source/sink route and advance our understanding of the driving force behind sink and source strength at the molecular level.

 

Recently, we have taken advantage of post-genomic tools including predictive genome annotations and full-length cDNA databases to develop a functional genomics approach for screening plant transport proteins based on expression cloning in Xenopus oocytes. Based on a search between a database of predicted Arabidopsis membrane proteins and full-length cDNA databases, a normalized library of all available cDNAs of Arabidopsis organic solute transporters was generated. The method can be utilized to isolate transporters, for which no sequence knowledge has been obtained.

 

Screening of the library with glucosinolates has identified transporters capable of transporting glucosinolates. We are currently analyzing knockout mutants to show if the transporters are responsible for the transport of glucosinolates in vivo. The transporter cDNA library has great potential for assignment of function to the many uncharacterized Arabidopsis transporters, which is an important prerequisite for implementation of transport engineering to specifically eliminate anti-nutritional natural products from high-value crop tissues.

 

Screen of transporter cDNA library in Xenopus oocytes.