Characterization of Cytokinin and Adenine Transport in Arabidopsis Cell Cultures

Abstract

Cytokinins are distributed through the vascular system and trigger responses of target cells via receptor-mediated signal transduction. Perception and transduction of the signal can occur at the plasma membrane or in the cytosol. The signal is terminated by the action of extra-or intracellular cytokinin oxidases. While radiotracer studies have been used to study transport and metabolism of cytokinins in plants, little is known about the kinetic properties of cytokinin transport. To provide a reference dataset, radiolabeled trans-zeatin (tZ) was used for uptake studies in Arabidopsis (Arabidopsis thaliana) cell culture. Uptake kinetics of tZ are multiphasic, indicating the presence of both low-and high-affinity transport systems. The protonophore carbonyl cyanide m-chlorophenylhydrazone is an effective inhibitor of cytokinin uptake, consistent with H + -mediated uptake. Other physiological cytokinins, such as isopentenyl adenine and benzylaminopurine, are effective competitors of tZ uptake, whereas allantoin has no inhibitory effect. Adenine competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. Comparison of adenine and tZ uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties, as well as substrate specificity determined in cell cultures, are compatible with the hypothesis that members of the plant-specific purine permease family play a role in adenine transport for scavenging extracellular adenine and may, in addition, be involved in low-affinity cytokinin uptake.Due to their immobile nature, plants require highly efficient mechanisms for acclimation to rapidly changing environmental conditions and for communication between the distal organs of the plant. Besides classical hormones, such as steroids, oligopeptides, and eicosanoid-like compounds, plants have developed a specific set of phytohormones. Most phytohormones are synthesized by few conversions from common metabolic intermediates. The phytohormone cytokinin is mainly generated by isopentylation of AMP, ADP, or ATP by adenosine-phosphate-isopentenyl transferase (IPT), resulting in the production of corresponding isopentenyl adenosine-5#-phosphates. These isopentenyl adenine (iP) nucleotides are converted to trans-zeatin (tZ) derivatives by trans-hydroxylases CYP735A1 and CYP735A2 (Takei et al

Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism

“…However, NUP1 and CjMDR1 differ in how the alkaloid uptake is energized. Based on similarity to AtPUP1 (29,31), NUP1 most likely uses proton symport for the uptake of nicotine, whereas CjMDR1 uses the hydrolysis of ATP to energize the transport of berberine into the cytoplasm (28). Both tobacco and C. japonica use the antiport of protons from the vacuole in conjunction with MATE-type membrane transporters to move cytoplasmic alkaloids into the vacuole (25,26,42).…”

“…DR752036) contains a cDNA fragment encoding a predicted polypeptide with similarity to AtPUP1 (BLASTX e-value = 1.35 e −22 ). AtPUP1 and AtPUP2 are responsible for the proton-mediated uptake of purines and cytokinins in Arabidopsis (29)(30)(31). Uptake of purines into yeast cells expressing the recombinant AtPUP1 transporter is inhibited 35% in the presence of nicotine, suggesting that nicotine might be partially recognized by AtPUP1 (29).…”

Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis

“…Two transporter families are unique to plants and include the purine permease (PUP) with 21 members and ureide permease (UPS) with eight members. Characterized Arabidopsis PUP proteins transport adenine, cytosine or secondary compounds such as cytokinins and caffeine [8][9][10]. Two transporters in the Arabidopsis UPS family transport uracil, allantoin and the purines xanthine and hypoxanthine [11][12][13], and an UPS from French bean transports allantoin [14].…”

The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility

“…In Arabidopsis, the genome contains loci that encode for five distinct nucleobase transporter families. Two nucleobase transporter families unique to plants include the twenty onemember purine permeases (PUP) that transport adenine, cytosine, and cytokinins (Gillissen et al 2000;Bürkle et al 2003;Cedzich et al 2008;Jelesko 2012;Szydlowski et al 2013) and the five-member ureide permeases (UPS) facilitating the movement of allantoin, uracil, uric acid, and xanthine (Desimone et al 2002;Schmidt et al 2004Schmidt et al , 2006. The remaining three nucleobase transporter families have orthologues among prokaryotes and eukaryotes.…”