Friday, July 3, 2015
Identification and characterization of plant hormone transporters by using receptor complexes as sensors
RIKEN Center for Sustainable Resource Science
Plant hormones regulate various physiological processes during life cycles. Physiological responses induced by hormones often associated with the changes in endogenous hormone levels. Many genes involved in the biosynthesis and catabolism (inactivation) of hormones have been identified. Although the transport is one of the important factors that determine endogenous hormone levels in association with biosynthesis and catabolism, its regulatory mechanisms remain largely unknown. We developed a functional screening system to identify abscisic acid (ABA) transporters based on their activities. The yeast two-hybrid system with the ABA receptor and PP2C protein phosphatases was used to detect ABA transport activities of proteins expressed in the yeast cells. We screened Arabidopsis cDNAs capable of inducing the interactions between the receptor and PP2C under low ABA concentrations, and identified a member of NRT1/PTR FAMILY (NPF) proteins, NPF4.6, as a candidate ABA importer. Mutants defective in NPF4.6 (npf4.6) was less sensitive to exogenously applied ABA during germination compared to wild type whereas overexpression of NPF4.6 resulted in ABA hypersensitivity. Furthermore, npf4.6 had lower surface temperature with more open stomata in inflorescence stems, suggesting that NPF4.6 functions as an ABA importer in vivo. Interestingly, NPF4.6 was identical to the previously characterized low affinity nitrate transporter NRT1.2. Km value of NPF4.6 for nitrate was approximately 5.9 mM. On the other hand, Km value of NPF4.6 for ABA was around 5 μM. In addition, nitrate did not inhibit the ABA transport activities of NPF4.6. These results suggest that NPF4.6 is a high affinity ABA transporter. We also developed yeast two-hybrid systems with the receptor complexes of gibberellin (GA) and jasmonoyl isoleucine (JA-Ile) to determine the activities of NPFs to transport these hormones. Interestingly, some NPF proteins transported GA and/or JA-Ile, suggesting their involvement in hormone transport. Screening of GA and JA-Ile transporters other than NPFs is now underway using the yeast two-hybrid systems with the receptor complexes.
Contact : Tou Cheu Xiong