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Schematic representation of the method. (A) Schematic representation outlining key steps in the preparation and purification of membrane vesicles from E. coli. (B) Schematic representation outlining key steps in transport assay of membrane vesicle preparations using radiolabeled substrates. Please click here to view a larger version of this figure.
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Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologous...
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... Similarly TPO1-5 were characterized as PA excretion proteins in yeast (Aouida et al., 2005;Uemura et al., 2005;Uemura et al., 2007;Sampathkumar and Drouin, 2015). Based on the homology to these antiporters, bi-directional amino acid transporters (BATs) were identified as PA antiporters in Arabidopsis and rice (Ge, 2015;Ariyaratne et al., 2019). In order to demonstrate the exchange of PAs for other amino acids by BATs, individual BAT genes (OsBAT1, AtBAT1.1, and AtBAT1.2) were expressed in the E. coli PotE/CadB-double knockout (DKO) mutant (Ge, 2015;Ariyaratne et al., 2019). ...
... Based on the homology to these antiporters, bi-directional amino acid transporters (BATs) were identified as PA antiporters in Arabidopsis and rice (Ge, 2015;Ariyaratne et al., 2019). In order to demonstrate the exchange of PAs for other amino acids by BATs, individual BAT genes (OsBAT1, AtBAT1.1, and AtBAT1.2) were expressed in the E. coli PotE/CadB-double knockout (DKO) mutant (Ge, 2015;Ariyaratne et al., 2019). Export of polyamines from transformed DKO cells was determined by measuring its uptake into inside-out membrane vesicles of the transformed cells and it was suggested that BATs might act as PA exporters (Ge, 2015;Ariyaratne et al., 2019). ...
... In order to demonstrate the exchange of PAs for other amino acids by BATs, individual BAT genes (OsBAT1, AtBAT1.1, and AtBAT1.2) were expressed in the E. coli PotE/CadB-double knockout (DKO) mutant (Ge, 2015;Ariyaratne et al., 2019). Export of polyamines from transformed DKO cells was determined by measuring its uptake into inside-out membrane vesicles of the transformed cells and it was suggested that BATs might act as PA exporters (Ge, 2015;Ariyaratne et al., 2019). ...
Nitrogen (N) is one of the most expensive nutrients to supply, therefore, improving the efficiency of N use is essential to reduce the cost of commercial fertilization in plant production. Since cells cannot store reduced N as NH3 or NH4 ⁺, polyamines (PAs), the low molecular weight aliphatic nitrogenous bases, are important N storage compounds in plants. Manipulating polyamines may provide a method to increase nitrogen remobilization efficiency. Homeostasis of PAs is maintained by intricate multiple feedback mechanisms at the level of biosynthesis, catabolism, efflux, and uptake. The molecular characterization of the PA uptake transporter (PUT) in most crop plants remains largely unknown, and knowledge of polyamine exporters in plants is lacking. Bi-directional amino acid transporters (BATs) have been recently suggested as possible PAs exporters for Arabidopsis and rice, however, detailed characterization of these genes in crops is missing. This report describes the first systematic study to comprehensively analyze PA transporters in barley (Hordeum vulgare, Hv), specifically the PUT and BAT gene families. Here, seven PUTs (HvPUT1-7) and six BATs (HvBAT1-6) genes were identified as PA transporters in the barley genome and the detailed characterization of these HvPUT and HvBAT genes and proteins is provided. Homology modeling of all studied PA transporters provided 3D structures prediction of the proteins of interest with high accuracy. Moreover, molecular docking studies provided insights into the PA-binding pockets of HvPUTs and HvBATs facilitating improved understanding of the mechanisms and interactions involved in HvPUT/HvBAT-mediated transport of PAs. We also examined the physiochemical characteristics of PA transporters and discuss the function of PA transporters in barley development, and how they help barley respond to stress, with a particular emphasis on leaf senescence. Insights gained here could lead to improved barley production via modulation of polyamine homeostasis.
... However, GABA uptake by mitochondria isolated from the atgabp mutant is not eliminated, suggesting the possible existence of other mitochondrial GABA carriers with overlapping or redundant functions [16]. The substrate preference for AtGABP requires clarification (arginine, Glu and lysine, but not GABA and proline [55]; GABA, but not proline [54]; K m Spd = 55 µM, K m Put = 85 µM, K m arginine = 1.4 mM [56]). ...
Global climate change and associated adverse abiotic and biotic stress conditions affect plant growth and development, and agricultural sustainability in general. Abiotic and biotic stresses reduce respiration and associated energy generation in mitochondria, resulting in the elevated production of reactive oxygen species (ROS), which are employed to transmit cellular signaling information in response to the changing conditions. Excessive ROS accumulation can contribute to cell damage and death. Production of the non-protein amino acid γ-aminobutyrate (GABA) is also stimulated, resulting in partial restoration of respiratory processes and energy production. Accumulated GABA can bind directly to the aluminum-activated malate transporter and the guard cell outward rectifying K+ channel, thereby improving drought and hypoxia tolerance, respectively. Genetic manipulation of GABA metabolism and receptors, respectively, reveal positive relationships between GABA levels and abiotic/biotic stress tolerance, and between malate efflux from the root and heavy metal tolerance. The application of exogenous GABA is associated with lower ROS levels, enhanced membrane stability, changes in the levels of non-enzymatic and enzymatic antioxidants, and crosstalk among phytohormones. Exogenous GABA may be an effective and sustainable tolerance strategy against multiple stresses under field conditions.
Polyamines are low molecular weight amines, with versatile and essential functions during the lifespan of prokaryotes and eukaryotes. Modulation of polyamine metabolism under biotic stress has attracted a great deal of interest; however, the precise functions of these amines in plant defence responses to pathogen attack are still poorly understood. Herein we summarise and review important aspects of the regulation of polyamine biosynthesis, catabolism and conjugation under biotic stress that might contribute to improve present‐day knowledge about these compounds in plant immunity. Particular attention is paid to the intracellular localisation of the proteins/enzymes associated to polyamine metabolism, as well as the impact of pathogen effector proteins on the control of plant polyamine metabolism is discussed.
