Abstract
Polyamines (PAs) are polycationic compounds found in all living organisms and play crucial roles in growth and survival. We here show the ‘Polyamine and paraquat (PQ) transport assay’ protocol, which can be used to examine the uptake activity of PA/PQ transporters. We have used this protocol to demonstrate that PUT3 in Arabidopsis is a polyamine transporter and is able to take up spermidine and its analog paraquat.
Keywords: Arabidopsis thaliana, Polyamine, Paraquat, Transport, Uptake
Background
PAs are involved in gene regulation by interacting with and modulating the functions of anionic macromolecules such as DNA, RNA and proteins. In living cells, PAs’ contents must be regulated to maintain the cellular hemostasis. In higher plants, three major polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm), are present in either free form or conjugated forms with other molecules (Gill and Tuteja, 2010). In yeast, four plasma membrane polyamine transporters, DUR3, SAM3, GAP1 and AGP2 were identified (Uemura et al., 2007). In Arabidopsis, five putative polyamine uptake transporters (PUT1-PUT5) were identified and PUT1-3 have been experimentally validated as polyamine transporters (Mulangi et al., 2012; Li et al., 2013). Our protocol described below has successfully confirmed that PUT3 is an influx transporter for polyamines and paraquat, and PQ/Spd uptake is impaired in the put3 mutant (Shen et al., 2016).
Materials and Reagents
Equipment
Procedure
Data analysis
The radioactivity (nCi) of each sample was calculated based on the scintillation counter reading (CPM) and the equation of the corresponding standard curve. The total amount of transported PQ/Spd in each sample was then calculated based on the radioactivity and the used nCi per nmol in the transport assay solution. The total protein of each sample was calculated based on the protein concentration and the volume. The PQ/Spd transport rates were presented in the unit of µmole PQ/Spd per gram protein per hour (µmole g-1 h-1). The PQ/Spd transport rate at 0 °C of each sample was considered as non-specific binding of PQ/Spd, and therefore was subtracted from the PQ/Spd transport rates at 25 °C to calculate the adjusted and accurate PQ/Spd transport rates at normal condition. Readers are referred to Shen et al. (2016) for examples of data graphs.
Notes
Recipes
Acknowledgments
This work was partly supported by the US Department of Agriculture National Research Initiative competitive grant 2007-35100-18378 to H.S. and by the National Natural Science Foundation of China (Grant # 31328004) to H.S.
References
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