Characterization of Cytokinin and Adenine Transport in Arabidopsis Cell Cultures

Center for Plant Molecular Biology, University of Tuebingen, D-72071 Tuebingen, Germany.
Plant physiology (Impact Factor: 6.84). 11/2008; 148(4):1857-67. DOI: 10.1104/pp.108.128454
Source: PubMed


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.

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    • ", inhibiting CK degradation with the specific CKX inhibitor, INCYDE, resulted in slightly elevated levels of the CK bases iP and tZ and the riboside iPR, compounds known to be efficiently degraded by CKXs (Gajdosová et al., 2011; Köllmer et al., 2014). Treatment with adenine (Figure 1B), a known cytokinin transport antagonist (Bürkle et al., 2003; Cedzich et al., 2008), caused a slight decrease of cZ and tZ bases and increased levels of their respective ribosides. This is in accordance with the H + -coupled high-affinity purine uptake transporter system shared by the structurally similar CK bases and adenine. "
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    ABSTRACT: Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific. © 2015 American Society of Plant Biologists. All rights reserved.
    The Plant Cell 07/2015; 27(7):1955–1967. DOI:10.1105/tpc.15.00176 · 9.34 Impact Factor
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    • "Three members of the AtPUP gene family (AtPUP1, AtPUP2, and AtPUP3) have been thoroughly studied (Gillissen et al. 2000; Bürkle et al. 2003; Cedzich et al. 2008). The function of AtPUP1 and AtPUP2 as CK transporters was proven by yeast direct uptake experiments, but AtPUP3 had no detectable transporting activity in yeast. "
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    ABSTRACT: In this study, PUP-type cytokinin transporter genes were identified in rice (Oryza sativa L.). The OsPUP (Oryza sativa purine permease) family has 12 members that show similar predicted protein sequences with AtPUPs. To reveal the functions of OsPUP genes, we searched the T-DNA mutant library of rice and found one mutant for the member OsPUP7. The T-DNA insertion caused a new transcript that encodes a protein with 26 amino acids different from the native OsPUP7 at the C-terminus. The mutant showed multiple phenotypic changes including increased plant height, big seeds, and delayed flowering. The mutant also showed increased sensitivity to drought and salt stresses and treatments with kinetin and abscisic acid. OsPUP7 is expressed mainly in the vascular bundle, pistil, and stamens. The measurement of CKs showed that CK content in the mutant spikelets accumulated higher than that in the wild type. Moreover, uptake experiment in the yeast fcy2 mutant suggested that OsPUP7 has the ability to transport caffeine, a CK derivative. Our results indicate that the PUP transport system also exists in rice, and OsPUP7 has an important role in the transport of CK, thus affecting developmental process and stress responses.
    Journal of Integrative Plant Biology 08/2013; 55(11). DOI:10.1111/jipb.12101 · 3.34 Impact Factor
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    • "Multiscale models have addressed the transport of several hormones, including auxin (Swarup et al., 2005; Grieneisen et al., 2007), gibberellin (Band et al., 2012a), and cytokinin (Chavarria-Krauser et al., 2005). These models of gibberellin and cytokinin have supposed passive diffusion between adjacent cells, taking the simplest assumption, which may require revision as biological knowledge increases (Cedzich et al., 2008). By contrast, auxin has been shown to move through root tissues in a polar manner due to the spatial distributions of specialized auxin influx (i.e., AUX1 or LAX) and/or efflux proteins (i.e., PIN) present on the cell membranes (Kramer and Bennett, 2006). "
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    ABSTRACT: Over recent decades, we have gained detailed knowledge of many processes involved in root growth and development. However, with this knowledge come increasing complexity and an increasing need for mechanistic modeling to understand how those individual processes interact. One major challenge is in relating genotypes to phenotypes, requiring us to move beyond the network and cellular scales, to use multiscale modeling to predict emergent dynamics at the tissue and organ levels. In this review, we highlight recent developments in multiscale modeling, illustrating how these are generating new mechanistic insights into the regulation of root growth and development. We consider how these models are motivating new biological data analysis and explore directions for future research. This modeling progress will be crucial as we move from a qualitative to an increasingly quantitative understanding of root biology, generating predictive tools that accelerate the development of improved crop varieties.
    The Plant Cell 10/2012; 24(10). DOI:10.1105/tpc.112.101550 · 9.34 Impact Factor
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