AtAGP18, a lysine-rich arabinogalactan protein in Arabidopsis thaliana,functions in plant growth and development as a putative co-receptor for signal transduction

Arabinogalactan-proteins (AGPs) are a class of hyperglycosylated, hydroxyproline-rich glycoproteins that are widely distributed in the plant kingdom. AtAGP17, 18 and 19 are homologous genes encoding three classical lysine-rich AGPs in Arabidopsis. We observed subcellular localization of AtAGP18 at the plasma membrane by expressing a translational fusion gene construction of AtAGP18 attached to a green fluorescent protein (GFP) tag in Arabidopsis plants. We also overexpressed AtAGP18 without the GFP tag in Arabidopsis plants, and the resulting transgenic plants had a short, bushy phenotype. Here we discuss putative roles of AtAGP18 as a glycosylphosphatidylinositol (GPI)-anchored protein involved in a signal transduction pathway regulating plant growth and development.Key words: Arabidopsis thaliana, arabinogalactan-proteins, co-receptor, glycosylphosphatidylinositol, lipid rafts, overexpressionArabinogalactan-proteins (AGPs) are plant cell surface glycoproteins or proteoglycans which are thought to play important roles in various aspects of plant growth and development, such as somatic embryogenesis, cell proliferation and elongation, pattern formation and hormone signaling.¹ The lysine-rich classical AGP subfamily in Arabidopsis contains three members: AtAGP17, 18 and 19. The subcellular localization of AtAGP17 and AtAGP18 was previously studied in our laboratory by expressing GFP-AtAGP17/18 fusion proteins in tobacco cell cultures.^2,3 In a recent report, we used Arabidopsis plants to overexpress GFP-AtAGP17/18/19 fusion proteins to observe subcellular localization of the lysine-rich AGPs in planta, in contrast to our previous plant cell culture work.⁴ Moreover, the lysine-rich AGPs alone (i.e., AtAGP17/18/19 without the GFP tag) were overexpressed in Arabidopsis plants, and only AtAGP18 overexpressors had a distinctive phenotype. This phenotype included shorter stems, more branches and less seeds, indicating a role for AtAGP18 in plant growth and development.⁴ In this addendum, we further discuss the putative biological role of AtAGP18 on a molecular level and its possible mode of action in cellular signaling.Classical AGPs are frequently predicted to have a glycosylphosphatidylinositol (GPI) anchor, which would allow for the localization of such AGPs to the outer surface of the plasma membrane. Biochemical analyses were carried out to support this hypothesis in tobacco, pear,⁵ rose⁶ and Arabidopsis.⁷ The lysine-rich classical AGPs, AtAGP17 and 18, were predicted to have a GPI anchor.⁸ To test this idea, tobacco cell cultures expressing GFP-AtAGP17/18 fusion proteins were plasmolyzed and GFP fluorescence was observed on the plasma membrane.^2,3 To corroborate this finding in planta, GFP-AtAGP17/18 were expressed in Arabidopsis plants and leaf trichome cells were plasmolyzed. Enhanced GFP fluorescence was observed at the plasma membrane of these transgenic trichome cells, indicating the presence of GFP-AtAGP17/18 at the plasma membrane.⁴ The localization of these lysine-rich classical AGPs at the plasma membrane suggests possible biological roles in sensing extracellular signals. They are likely associated with lipid rafts involved in cell signaling for the following reasons. In plants as well as animals, there are sterol-enriched, detergent-resistant plasma membrane microdomains called lipid rafts. Lipid rafts are known to be involved in signal transduction and are enriched in transmembrane receptors and GPI-anchored proteins, including AGPs.^9–11 The accumulation of these proteins in such microdomains may allow for interactions between these proteins in sensing extracellular signals which lead to various intracellular events. Interestingly, a recent study shows that lipid rafts from hybrid aspen cells contain callose synthase and cellulose synthase, and these enzymes are active since in vitro polysaccharide synthesis by the isolated detergent-resistant membranes was observed. These results demonstrate that lipid rafts are involved in cell wall polysaccharide biosynthesis.¹² In addition, an Arabidopsis pnt mutant study shows GPI-anchored proteins are required in cell wall synthesis and morphogenesis.¹³ These observations, coupled with previous observations that cellulose synthases as well as AGPs interact with microtubules, suggest that AGPs in lipid rafts may have a role in signal events, including those regulating cellulose and/or callose biosynthesis or deposition.^14,15To examine the role of LeAGP-1, a lysine-rich AGP in tomato, transgenic tomato plants were produced which expressed GFP-LeAGP-1 under the control of the cauliflower mosaic virus 35S promoter.¹⁶ The tomato LeAGP-1 overexpressors and Arabidopsis AtAGP18 overexpressors both have a bushy phenotype similar to transgenic tobacco plants overproducing cytokinins.^4,16,17 Cytokinins are an important class of plant hormones involved in many plant growth and development processes, such as cell growth and division, differentiation and other physiological processes.¹⁸ Therefore, Sun et al. proposed that LeAGP-1 might function in concert with the cytokinin signal transduction pathway.¹⁶ Since the overexpression phenotypes of AtAGP18 are similar to those of LeAGP-1, AtAGP18 is also likely associated with the cytokinin signal transduction pathway. The prevailing model for cytokinin signaling in Arabidopsis is similar to the two-component system in bacteria and yeast. In this model, the cytokinin receptor contains an extracellular domain, a kinase domain and a receiver domain. When the cytokinin receptor senses cytokinin signals, it auto-phosphorylates at a His residue in the kinase domain. The phosphoryl group is then transferred to an Asp residue in the receiver domain. Subsequently, the phosphoryl group is transferred to a His residue in the histidine phosphotransfer protein (Hpt) and the Hpt translocates to the nucleus and transfers the phosphoryl group to an Asp residue in a downstream response regulator to activate it.¹⁹ This model is consistent with our hypothesis since the cytokinin receptor in this model is a receptor kinase located in the plasma membrane with an extra-cellular domain that can potentially interact with AtAGP18. AtAGP18 may function as a co-receptor that first binds to cytokinins, then either directly interacts with cytokinin receptors or brings the cytokinins to cytokinin receptors in the plasma membrane. The first scenario is analogous to the interaction of contactin and contactin-associated protein (Caspr) in neurons. In this model, contactin is a GPI-anchored protein on the cell surface that binds to signal molecules and interacts with the transmembrane receptor Caspr to transmit signals to the cell interior.²⁰ The second scenario is analogous to fibroblast growth factor (FGF) signal activation in which heparan sulfate proteoglycans bind to FGF molecules and bring them to the FGF receptor.²¹Based on all the above observations and findings, a hypothetical model for AtAGP18 function is proposed in Figure 1. The model shows AtAGP18 located on the outer surface of the plasma membrane in lipid rafts where it could act as a co-receptor to sense extracellular signals (such as cytokinin) and interact with transmembrane proteins, possibly receptor kinases or ion channels, in the lipid rafts to initiate signaling by triggering various intracellular events. Interestingly, receptor tyrosine kinases and ion channels are known to be present in lipid rafts.^9,22 Moreover, AGPs are likely associated with ion channels since addition of the AGP-binding reagent Yariv phenylglycoside resulted in elevated cytoplasmic calcium concentrations in tobacco cells and lily pollen tubes.^15,23,24 Clearly, additional work will be required to verify such a model, and to better understand how AtAGP18 might sense extracellular signals and interact with the transmembrane proteins in the lipid rafts.Open in a separate windowFigure 1Model for atAGP18 functioning in cellular signaling to control plant growth and development. In this model, lipid rafts are enriched in glycosphingolipids, sterols, transmembrane proteins (such as receptors, receptor kinases and ion channel proteins) and GPI-anchored proteins including AtAGP18. (a) AtAGP18 acts as a co-receptor by binding to signaling molecules and directly interacting with transmembrane proteins in the lipid rafts. (B) AtAGP18 acts as a co-receptor by binding to signaling molecules and bringing the signaling molecules to transmembrane proteins in the lipid rafts. Upon activation by the extracellular signals, the transmembrane proteins initiate signaling and lead to various intracellular events (e.g., phosphorylation similar to the two-component signaling system, influx of calcium ions). The different components of the AtAGP18 molecule and the various lipid components of lipid rafts and plasma membrane are shown in the boxed inset. Hpt, histidine phosphotransfer protein.     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.¹ Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.^2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.^4,5 PPR proteins are known to have roles in RNA processing⁶ and the SMR domain of GUN1 has been shown to bind DNA,⁴ but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,⁷ redox,⁴ ABA1,⁴ and sucrose signaling,^1,4,8 as well as light quality and intensity sensing pathways.^9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.^1,11     

Protein interactors of acyl-CoA-binding protein ACBP2 mediate cadmium tolerance in Arabidopsis

In our recent paper in the Plant Journal, we reported that Arabidopsis thaliana lysophospholipase 2 (lysoPL2) binds acyl-CoA-binding protein 2 (ACBP2) to mediate cadmium [Cd(II)] tolerance in transgenic Arabidopsis. ACBP2 contains ankyrin repeats that have been previously shown to mediate protein-protein interactions with an ethylene-responsive element binding protein (AtEBP) and a farnesylated protein 6 (AtFP6). Transgenic Arabidopsis ACBP2-overexpressors, lysoPL2-overexpressors and AtFP6-overexpressors all display enhanced Cd(II) tolerance, in comparison to wild type, suggesting that ACBP2 and its protein partners work together to mediate Cd(II) tolerance. Given that recombinant ACBP2 and AtFP6 can independently bind Cd(II) in vitro, they may be able to participate in Cd(II) translocation. The binding of recombinant ACBP2 to [¹⁴C]linoleoyl-CoA and [¹⁴C]linolenoyl-CoA implies its role in phospholipid repair. In conclusion, ACBP2 can mediate tolerance to Cd(II)-induced oxidative stress by interacting with two protein partners, AtFP6 and lysoPL2. Observations that ACBP2 also binds lysophosphatidylcholine (lysoPC) in vitro and that recombinant lysoPL2 degrades lysoPC, further confirm an interactive role for ACBP2 and lysoPL2 in overcoming Cd(II)-induced stress.Key words: acyl-CoA-binding protein, cadmium, hydrogen peroxide, lysophospholipase, oxidative stressAcyl-CoA-binding proteins (ACBP1 to ACBP6) are encoded by a multigene family in Arabidopsis thaliana.¹ These ACBP proteins are well studied in Arabidopsis in comparison to other organisms,^1–4 and are located in various subcellular compartments.¹ Plasma membranelocalized ACBP1 and ACBP2 contain ankyrin repeats that have been shown to function in protein-protein interactions.^5,6 ACBP1 and ACBP2 which share 76.9% amino acid identity also confer tolerance in transgenic Arabidopsis to lead [Pb(II)] and Cd(II), respectively.^1,5,7 Since recombinant ACBP1 and ACBP2 bind linolenoyl-CoA and linoleoyl-CoA in vitro, they may possibly be involved in phospholipid repair in response to heavy metal stress at the plasma membrane.^5,7 In contrast, ACBP3 is an extracellularly-localized protein⁸ while ACBP4, ACBP5 and ACBP6 are localized to cytosol.^9,10 ACBP1 and ACBP6 have recently been shown to be involved in freezing stress.^9,11 ACBP4 and ACBP5 bind oleoyl-CoA ester and their mRNA expressions are lightregulated.^12,13 Besides acyl-CoA esters, some ACBPs also bind phospholipids.^9,11,13 To investigate the biological function of ACBP2, we have proceeded to establish its interactors at the ankyrin repeats, including AtFP6,⁵ AtEBP⁶ and now lysoPL2 in the Plant Journal paper. While the significance in the interaction of ACBP2 with AtEBP awaits further investigations, some parallels can be drawn between those of ACBP2 with AtFP6 and with lysoPL2.     

Regulation of MAPK signaling and cell death by MAPK phosphatase MKP2

Mitogen-activated protein kinase (MAPK) pathways play crucial roles in developmental and adaptive responses. Depending on the stimulus, MAPK activation regulates a wide variety of plant cell responses, such as proliferation, differentiation and cell death, which normally require precise spatial and temporal control. In this context, protein phosphatases play important roles by regulating the duration and magnitude of MAPK activities. During infection by non-host and incompatible host microorganisms, MAPK activity can promote a local cell death mechanism called hypersensitive response (HR), which is part of the plant defence response. HR-like responses require sustained MAPK activity and correlate with oxidative burst. We recently showed that MAPK phosphatase MKP2 positively controls biotic and abiotic stress responses in Arabidopsis. MKP2 interacts with MPK6 in HR-like responses triggered by fungal elicitors, suggesting that MKP2 protein is part of the mechanism involved in MAPK regulation during HR. Here we discuss the interplay of MAPK and MKP2 phosphatase signaling during cell death responses elicited by host-pathogen interactions.Key words: Arabidopsis, hypersensitive response (HR), MAPK, MPK6, MKP2, ROSDifferent studies have identified conserved components of MAPK pathways in plants and have provided evidence that MAPK signaling regulates a wide variety of plant biological responses.¹ For example, MAPK signaling is required for the regulation of stomatal functions,^2–4 hormone signaling^5,6 and innate immunity responses.^7–9 An increasing number of reports indicate that plant MAPKs, in particular tobacco SIPK/Ntf4 and WIPK and their Arabidopsis orthologs, MPK6 and MPK3, are converging points for signals elicited by different pathogens and play regulatory roles in disease responses.¹⁰One of the most efficient and immediate immune responses dependent on MAPK signaling is a mechanism of cell death called hypersensitive response (HR). HR is a rapid, localized cell death process at the site of pathogen infection, which is associated with specific molecular effects such as the generation of reactive oxygen species (ROS) and protein phosphorylation.¹¹ The best evidence implicating MAPK activity in HR comes from gain-of-function studies overexpressing SIPK/Ntf4 and WIPK in tobacco leaves. In these experiments, activation of SIPK/Ntf4 kinases efficiently induces HR-like cell death,^12,13 but the absence of endogenous WIPK function causes delayed induction of this HR phenotype, suggesting that WIPK activity facilitates or potentiates the SIPK signal.¹⁴ Similarly, overexpression analyses of Arabidopsis MPK3 and MPK6 proteins, either alone or co-expressed with activated upstream regulators (MKK proteins), also triggers a cell death phenotype,¹⁵ suggesting a coordinated role of MKK/MAPK signaling modules in HR.¹⁵ Thus, the involvement of MAPK activities such as SIPK/MPK6 in HR cell death responses is supported by different studies; however their regulation by phosphatases remains less understood.The main regulators of MAPKs are specific phosphatases belonging to various families, including PP2C Ser/Thr phosphatases, Tyr phosphatases (PTPs) or dual specificity phosphatases (DSPs) such as the MAPK phosphatase (MKP) subgroup.^16,17 In general, dephosphorylation of MAPKs inactivates their function in many metabolic, developmental or adaptive responses. In the context of HR, we have recently shown that Arabidopsis MKP phosphatase MKP2 interacts with MPK6 in the response triggered by fungal elicitors. In particular, co-expression of MPK6 and MKP2 proteins in infected tobacco leaves significantly attenuates the cell death phenotype produced by expressing MPK6 alone, suggesting that MKP2 negatively regulates MAPK activities in this process.¹⁸     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Evaluating the function of putative hormone transporters

Hormones typically serve as long distance signaling molecules. To reach their site of action, hormones need to be transported from the sites of synthesis. Many plant hormones are mobile, thus requiring specific transport systems for the export from their source cells as well as subsequent import into target cells. Hormone transport in general is still poorly understood. Auxin is probably the most intensively studied plant hormone concerning transport in the moment. To advance our understanding of hormone transport we need two principal data sets: information on the properties of the transport systems including substrate specificity and kinetics, and we need to identify candidate genes for the respective transporters. Physiological transport data can provide an important basis for identifying and characterizing candidate transporters and to define their in vivo role. A recent publication in Plant Physiology highlights how kinetic and specificity studies may help to identify cytokinin transporters.¹Key words: kinetin, zeatin, adenine, phytohormone, transportBy definition, hormones are compounds that interact at low concentrations with cellular receptors to modulate signal transduction pathways. A comparison of the chemical structures of animal and plant hormones suggests potential common origins. Peptide hormones are found in both kingdoms and share common processing mechanisms (e.g., TRH, vasopressin and kinins in animals; systemins, phytosulfokines, self incompatibility peptides in plants).^2,3 Steroid hormones derived from cholesterol such as testosterone, cortisol and calcitriol regulate development in mammals; the steroid hormone brassinolide is essential for plant development.⁴ Glutamate can serve as metabolite and signal in both plants and animals.^5,6 Finally, lipid and phospholipid-derived signaling compounds such as linoleic acid and arachidonic acid also function in both plants and animals; with phospholipid-derived prostaglandins and eicosanoids bearing similarities to the plant defense compound jasmonic acid.⁷Other signaling compounds present in animals have yet to be shown to function in plants, e.g., glycoprotein hormones such as luteinizing hormone, follicle-stimulating hormone or thyroid-stimulating hormone have been not been described to exist in plants.⁸ Compounds structurally similar to animal amine-derived hormones derived from tyrosine and tryptophan (such as catecholamines and thyroxine) are also present in plants, but appear to function primarily in herbivore defense.⁹The best characterized, and arguably most important plant hormones, bear little similarity to animal hormones and are mechanistically distinct. These include auxins, cytokinins, gibberellins, abscisic acid, ethylene and an apparent carotenoid-derivative, the MAX-dependent regulator of auxin signaling.^10,11 Arguably, the stress response compound salicylic acid, which functions in stress, wounding and defense responses could also be considered a plant hormone.¹²Hormonal signaling mechanisms can be categorized as autocrine (acting at the site of biosynthesis), paracrine (acting in adjacent or proximal cells), and endocrine (acting in cells distal to the site of production). In both, plants and animals, paracrine and endocrine hormone action is mediated and influenced by multiple long distance delivery systems. Hormones move primarily through the circulatory system in animals, but, in plants, are mobilized by transpiration and source-sink flows, which can be directed by chemisomotically-driven cellular uptake and efflux. However, the mechanisms driving uptake and efflux at the cellular level, as well as the proteins that mediate this movement, are surprisingly similar in plants and animals, despite the dissimilarities of plant and animal cell structure (central vacuoles, cell walls and H⁺ versus K⁺/Na²⁺ in/out gradients).Surprisingly little is known about plant hormone transport. Most hormones have autocrine activity, but in order to act at a distance or to even act on adjacent cells they must be transported across membranes. The existence of cellular export and import mechanisms are suggested by the presence of multiple hormones in the phloem sap^13,14 and the well documented polar long distance movement of auxin.¹⁵ Brassinosteroid receptors have been demonstrated as integral plasma membrane proteins which receive the hormone signal from outside the cell.¹⁶ This suggests a need for the hormone to first move into the apoplasm after biosynthesis. However, until recently, only the cellular auxin transport mechanisms mediated by the AUX/LAX, PIN and AtABCB/PGP proteins has been well characterized (reviewed in ref. ¹⁷).The study of these transporters has benefited from the use of plant, yeast and animal expression systems to characterize the proteins involved. Analyses of auxin transport proteins have capitalized on earlier suppression cloning and radiotracer uptake studies used successfully to characterize ion and metabolite transporters in yeast.^18–21 In cases where yeast systems have proven intractable for analysis of auxin transport proteins, heterologous systems based on mammalian cell systems have proven to be highly effective for radiotracer uptake studies.^18–23 Xenopus oocyte expression has been successfully utilized to characterize the AUX/LAX family of auxin influx symporters.^24,25 Plant cell culture systems have also been used to characterize transport proteins. This can however be problematic when endogenous substrates are metabolized by the cells, as is the case with IAA in tobacco BY-2 and Arabidopsis cell cultures.¹⁹ It is also difficult to assess the function of plant proteins in undifferentiated cell cultures, which may differ from the native function in phloem or xylem parenchyma cells.A recent article describes the use of a heterologous expression system based on the fission yeast S. pombe to express and characterize the PIN1 auxin efflux protein after knock-out of the endogenous yeast PIN-like gene AEL1.²¹ Previously, PIN1 had only been functionally expressed in plant cell systems and was nonfunctional when expressed in baker''s yeast or mammalian cells.^19,22 This report suggests that PIN1, interacts synergistically with the AtABCB19/PGP19 auxin efflux transporter, but appears to also mediate auxin efflux on its own, consistent with the distant phylogenetic similarity of the auxin efflux transporter protein family to major facilitator proteins.Subsequent work in the Murphy lab has shown that S. pombe can be used for comparisons of all known auxin transporters in a single system in which all ABC transporters and a solitary AUX1-like gene had been knocked out (Yang and Murphy, unpublished). This system also allows for the more detailed analyses of substrate specificity, transport kinetics and coupling mechanisms (primary and secondary active transport, uniport, cotransport antiport) necessary for functional assignment of auxin transport proteins. This system may also provide an attractive alternative to baker''s yeast when functional expression of a plant protein in Saccharomyces cerevisiae proves unsuccessful.Similar efforts are required for characterizing the transport of all other plant hormones including cytokinin. Arabidopsis transporters mediating both trans-zeatin and adenine uptake had been identified using yeast as an expression system.²⁶ Recently, the Schulz and Frommer labs provided a reference data set for trans-zeatin uptake by characterizing radiolabeled trans-zeatin uptake in Arabidopsis cell cultures.¹ The data show that the uptake kinetics of trans-zeatin are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore CCCP is an effective inhibitor of cytokinin uptake, consistent with H⁺-mediated uptake. Other physiologically active cytokinins such as isopentenyladenine and benzylaminopurine are effective competitors of trans-zeatin uptake, whereas allantoin had no inhibitory effect. Adenine competes for zeatin uptake indicating that degradation products of cytokinin oxidases can be transported by the same systems. Comparison of adenine and trans-zeatin 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 PUP transporter family may play a role in adenine transport to scavenge extracellular adenine. In addition, the findings are also compatible with the hypothesis that this class of transporters may be involved at least in low affinity (µM range) cytokinin uptake. PUPs are encoded by a large gene family of 21 members, so it is conceivable that other members of the family may be involved in high affinity transport. Systematic analyses of single knock outs in Arabidopsis and combinations thereof my help to shed more light on the role of PUPs in cytokinin transport.