Measure for measure: determining, inferring and guessing auxin gradients at the root tip.

Physiologia Plantarum 151: 97–111. 2014

© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317

MINIREVIEW

Measure for measure: determining, inferring and guessing auxin gradients at the root tip Wendy Ann Peera,b,∗ , Mark K. Jennessb and Angus S. Murphyb a Department b Department

of Environmental Science and Technology, University of Maryland, College Park, MD, 20742, USA of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, 20742, USA

Correspondence *Corresponding author, e-mail: [email protected] Received 20 December 2013; revised 5 March 2014 doi:10.1111/ppl.12182

The plant hormone auxin is transported from sites of synthesis to sites of action. Auxin responses are mediated by fast (non-transcriptional) and slow (transcriptional; ubiquitinylation) responses, which affect physiological changes at cellular and organismal scales. As such, auxin transport vectors regulate programmed and plastic growth responses to optimize growth and development. Here we address some common problems in extrapolating ‘universal’ understanding of auxin transport streams from analyses of loss-of-function mutants and auxin transport inhibitors. We also discuss the analytical methods and tools used to directly quantify, measure and infer auxin gradients within the plant [DR5:GUS/GFP (beta-glucuronidase/green fluorescent protein), DII-VENUS; surface electrodes, direct quantification]. We discuss the assumptions and limitations of each of these analyses, present comparative summaries of auxin transport methods and assay conditions (diffusion, non-specific transport and relevant assay conditions), and consider what is actually being transported and measured [labeled-indole-3-acetic acid (IAA), IAA metabolites].

Introduction The principle natural plant auxin, indole-3-acetic acid (IAA), is synthesized in meristematic and young tissues, as well as sites of wounding. Auxin is then transported from sites of synthesis to target sites where directed streams maintain programmed development and adaptive response to biotic and abiotic stimuli. At sites of action, SCFTIR1 -Aux/IAA co-receptors and SCFSKP2a mediate transcriptional responses, while Auxin Binding Protein 1 mediates additional non-transcriptional auxin responses (reviewed in Peer 2013). Auxin transporters and carriers functioning at the plasma membrane (PM) and endoplasmic reticulum (ER) are instrumental in regulating the amounts of auxin inside and outside of the cell and in maintaining auxin homeostasis

(reviewed in Sauer et al. 2013, reviewed in Zažímalová et al. 2010). Therefore, auxin transporters and carriers can directly affect auxin signaling and the responses that are measured. Here we examine how auxin gradients, transport and responses are inferred, and discuss what in vivo assays actually measure. We address some common problems in extrapolating ‘universal’ understandings of auxin transport streams from analyses of loss-of-function mutants and use of auxin transport inhibitors. We also discuss the assumptions and limitations of each of these analyses and present comparative summaries of auxin transport methods and assay conditions (diffusion, non-specific transport and relevant assay conditions). We also discuss the analytical methods and tools used to directly quantify,

Abbreviations – 2,4-D, 2,4-dicholorphenoxyacetic acid; ABCB1, ATP-binding cassette subfamily B auxin transporters 1; BY-2, Bright Yellow 2; DII, degron II; DMSO, dimethyl sulphoxide; ER, endoplasmic reticulum; GUS/GFP, beta-glucuronidase/green fluorescent protein; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; LC-MS, liquid chromatography-mass spectrometry; MS, murashige and skoog; NPA, naphthylphthalamic acid; oxIAA, 2-oxindole-3-acetic acid; PIN, PINFORMED; PM, plasma membrane; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TIBA, triiodobenzoic acid; 𝛿-TIP, delta-tonoplast intrinsic protein.

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measure and infer auxin gradients within the plant [DR5:GUS/GFP (beta-glucuronidase/green fluorescent protein), DII-VENUS; surface electrodes, direct quantification]. By examining the methods and the limitations of the methods, we discuss some common assumptions that are inherent in auxin transport estimations.

Avoiding assumptions Polar streams that regulate organogenesis are distinct from long-distance transport Differential auxin gradients play a major role in embryogenesis, organogenesis and amplification of redirected auxin streams in tropic responses. These gradients originate in localized auxin maxima derived from localized synthesis, aggressive auxin uptake from long-distance transport streams mediated by AUX1/LAX transporters, or diversion of long-distance transport streams (reviewed in Peer et al. 2011). Localized gradients are then amplified and propagated by polarized PINFORMED (PIN) efflux carriers in a process described as ‘canalization’ (Sachs 1981, 1968, Sauer et al. 2006). In particular, the localized polar auxin streams directed by the auxin efflux carrier PIN1 are required for normal organ development, as extensive embryonic, vegetative and floral defects are observed in pinformed1 mutants (Gälweiler et al. 1998, Benková et al. 2003, Hay et al. 2006). The long-distance rootward polar transport stream from the shoot apex is often viewed as analogous to localized polar streams, primarily due to the relative ease in measuring long-distance transport (Liu et al. 2011). However, increasing evidence indicates that genetic or pharmacological disruption of long-distance transport does not correspond to altered development apart from loss of apical dominance, decreased lateral root initiation and altered leaf expansion (reviewed in Bandyopadhyay et al. 2007). In addition to its localized function, PIN1 contributes to long-distance rootward auxin transport in the shoot and root, as transport in pin1 hypocotyls and inflorescences is reduced by approximately 30% (Blakeslee et al. 2007; Fig. 1A). This is thought to be a result of PIN1 localization at the basal end of vascular cells resulting in preferential exit of anionic auxin in the direction of the root. However, primary inflorescence length, lateral root formation and gravitropism in pin1 are similar to wild-type. A second carrier, PIN7, is not only apolarly localized in shoot epidermal cells (Blakeslee et al. 2007, Christie et al. 2011), but also overlaps with PIN1 in vascular tissues (Friml et al. 2003). Decreased lateral root number is observed in pin7 (Lewis et al. 2011), and experiments in our lab show that pin1-7 pin7-3 double mutants show a 40 ± 8% reduction of rootward auxin transport in hypocotyls. 98

However, inflorescence length in pin1pin7 is indistinguishable from pin1, and pin1pin7 mutants always have a viable root and some lateral roots, suggesting that residual long-distance auxin transport and localized synthesis are sufficient to maintain the root meristem. In contrast, loss of the ATP-binding cassette subfamily B auxin transporters 1 and 19 (ABCB1 and ABCB19) result in approximately 25 and >50% decrease in rootward auxin transport, respectively (Blakeslee et al. 2007; Fig. 1A), with abcb1abcb19 double mutants showing a decrease of approximately 70% (Blakeslee et al. 2007). Consistent with this decrease in long-distance auxin transport, abcb1abcb19 exhibit epinastic cotyledons, loss of apical dominance, decreased inflorescence height, increased lateral branches, decreased stamen filament elongation and decreased lateral root formation, but no defects in organogenesis (Noh et al. 2001, 2003, Geisler et al. 2005, Lewis et al. 2007). These results suggest that, on their own, decreased rates of long-distance auxin transport should not be accepted as evidence of localized loss-of-function. Compensation for the loss-of-function of one gene may mask effects on auxin transport A related issue is the expectation that loss of single gene function will produce observable and measurable differences in auxin transport, as described above. However, compensatory regulation of transporters and auxin biosynthesis and metabolism is often a factor that must be explored to make sense of transport results. For example, PIN4 transcripts increase during embryogenesis in pin7 compared with wild-type, and PIN1 transcripts increase in the pin2 mutant (Blilou et al. 2005, Vieten et al. 2005). In abcb19, transcripts levels of PIN and other ABCB family members increase approximately 2× (Blakeslee et al. 2007). In general, any mutation that increases auxin delivery to the root decreases PIN1 expression (Peer et al. 2004). As such, evidence of decreased PIN1 signals in the root of a lossor gain-of-function mutant should not be accepted as evidence of a direct impact on PIN1 activity. PIN isoforms are functionally unique Often it seems that what is true for one PIN is extrapolated to all PINs. PIN1 and PIN2 exhibit a high degree of cellular polarity and consistently direct polar auxin flows (reviewed in Peer et al. 2011). PIN1 membrane localization is dynamic, and initially shows non-polar PM localization in the embryo and shoot meristems before aligning with auxin vectors arising from localized gradients (Benková et al. 2003). PIN2 is polarly aligned to direct auxin flows away from the root apex in epidermal


Fig. 1. Effects of mutations, ecotypic background, inhibitors and concentrations on auxin transport. (A) Rootward auxin transport in wild-type (Col-0), pin1, abcb1 and abcb19. Transport was measured after application of 1 μM radiolabeled IAA, 2 μM radiolabeled IAA or 2 μM IAA (50% radiolabeled + 50% unlabeled). Data are means and standard deviations of three independent experiments.* ANOVA followed Student-Newman-Keuls post-hoc analyses (P < 0.001). (B) Rootward auxin transport among ecotypes following application of 1× (1 μM radiolabeled IAA:1 μM unlabeled) or 5× (1 μM radiolabeled IAA:5 μM unlabeled) radiolabeled IAA. Data are means and standard deviations of three independent experiments. * ANOVA followed Student-Newman-Keuls post-hoc analyses (P < 0.05). A 30% change in auxin transport is biologically significant, e.g. pin1 mutant. (C) Rootward auxin transport in wild-type (Col-0) and exo70a. Data are means and standard deviations of three independent experiments. ANOVA followed Dunnett’s post-hoc analyses (P > 0.05). (D) Rootward auxin transport in Col-0 at the upper hypocotyl (UH), root-shoot transition zone (TZ) and root tip with either NPA or TIBA. Data are means and standard deviations of three independent experiments. ANOVA of NPA vs TIBA followed Student-Newman-Keuls post-hoc analyses (P = 0.1).

cells, and pin2 mutants are largely agravitropic, but otherwise are not phenotypically different from wild-type (Luschnig et al. 1998). In the apical root cortex, PIN2 is programmed to direct auxin back into the stele. PIN3 is highly dynamic and exhibits a conditional polar localization in the root, but is more stable and exhibits an apolar distribution in the shoot (Ding et al. 2011). Consistent with these cellular distributions, pin3 mutants exhibit partial gravitropic and phototropic defects in the root and hypocotyl, and etiolated pin3 cannot maintain an apical hook (Friml et al. 2002b, Zádníková et al. 2010, Christie et al. 2011). In contrast, except during embryogenesis, PIN4 and PIN7 exhibit largely non-polar cellular localization (Friml et al. 2002a, Blakeslee et al. 2007, Feraru et al. 2011). pin4 and pin7 mutants exhibit no detectable phenotype apart from slower apical hook


opening, especially in higher order mutants (Zádníková et al. 2010) and slightly reduced lateral root number in pin7 (Lewis et al. 2011). In addition to differential distribution of PINs within specific expression domains (e.g. PIN1 in the stele and PIN2 in epidermal and cortical cells of the root), trafficking of PIN isoforms exhibits cell-specific differential inhibitor sensitivities and dependence on cellular trafficking mechanisms (Shin et al. 2005, Abas et al. 2006, Jaillais et al. 2006). In aggregate, these observed differences call into question some common assumptions regarding PIN function. In particular, the subclade of ‘short’ PINS that lack the central hydrophilic loop must be regarded as particularly distinct from PM-localized full-length PINs. PIN5, PIN6 and PIN8 are localized on the ER and all appear to be involved in auxin homeostasis (Mravec et al. 2009, 99

Ding et al. 2012). PIN8 functions in pollen tube growth, (Dal Bosco et al. 2012, Ding et al. 2012), and PIN6 is required for the development of the two short stamens and nectar production (Bender et al. 2013). The central hydrophilic loop directs the polar localization of PM PIN proteins (Huang et al. 2010), and is thus thought to be unnecessary for short PIN function at the ER. However, it is not known what other motifs in the hydrophilic loop might regulate the auxin efflux function of PINs, particularly if these domains are involved in D6PK activation of PIN efflux activity (Zourelidou et al. 2009, Willige et al. 2013). Altered PIN function is not always a result of altered PIN polarity The timing of auxin transport in physiological events often does not match what is seen with PIN relocalization, and it appears that PIN relocalization follows auxin transport (Ding et al. 2011). Dynamic cycling occurs in cells that are differentiating and expanding. It is easy to observe PIN1 cycling in the root tip, but PIN1 appears to be stably localized on the PM in mature regions of the root. While PIN localization or destabilization on the PM in roots is especially easy to observe under a variety of conditions and treatments which may affect trafficking or stability, that does not necessarily mean that the change in localization is relevant or that relocalization or destabilization of PINs is the exclusive means of regulating polar flows. For example, PIN3 relocalization from one side of the cell to the other side during phototropic bending has been reported (Friml et al. 2002b, Ding et al. 2011). However, auxin redirection and the onset of phototropic bending precede PIN3 relocalization and delayed bending observed in pin3 mutants (Christie et al. 2011, Ding et al. 2011). While PIN3 relocalization may not be the major contributor to phototropism, PIN3 phosphorylation appears to be. PIN3 appears to contribute primarily in auxin movement to the hypocotyl elongation zone after lateral auxin redistribution takes place (Christie et al. 2011, Preuten et al. 2013). D6PK is a protein kinase that activates the transport activity of PIN proteins (Zourelidou et al. 2009, Willige et al. 2013). In d6pk mutants, phototropic bending is only observed at the very top of the hypocotyl, and gradual PIN3 dephosphorylation is observed (Willige et al. 2013). This indicates that PIN3 is activated by phosphorylation at the PM. In another example, the protein phosphatase 2A (PP2A) and the kinase PINOID co-localize with PIN1 and PIN2 on the PM in roots (Michniewicz et al. 2007) and act antagonistically to each other to regulate the phosphorylation status required for proper PIN localization during 100

organogenesis (Cheng et al. 2008, Huang et al. 2010). Therefore, this suggests that the molecular switch of phosphorylation/dephosphorylation at the PM rapidly modulates PIN activity without physical removal of the protein from the PM. Auxin levels and transport vary across ecotypes Attention must be paid to the ecotypic background of each allele that is used, and comparisons across alleles may not be valid based on the ecotypic background. Currently, 1049 accessions of Arabidopsis thaliana have been committed for sequencing in the 1001 genomes project (www.1001genomes.org), and these accessions have adapted to the numerous seasonal temperatures, day lengths, irradiance levels, rain patterns, elevations, soil types, etc., necessary for reproductive success in that ecosystem. Among the differences in stress tolerance and flowering times are differences in auxin transport. For example, in seedling hypocotyls, rates of auxin transport are ranked Mrk-0 > Ws-0 > Col-0 > Ler-2 and results from Mt-0 are very noisy, suggesting heterogeneity in this ecotype [Fig. 1B (1× IAA)]. Tissue-specific free IAA levels also differ among ecotypes. For example, shoot free IAA levels are ranked Ler-2 > Ws-0 > Col-0, while the inverse is true in roots (Novák et al. 2012). However, it is also important to note that free IAA determinations do not always map to auxin transport as one is a measure of transport capacity while the other measures a steady state level that is modulated by multiple metabolic mechanisms (Christie et al. 2011, Dubrovsky et al. 2011). As a general principle, comparisons of genetic lesions are best made in identical genetic backgrounds if meaningful results are to be obtained. Not every mutation or inhibitor tested will affect auxin transport In the >500 separate auxin transport assays of mutants or inhibitor treatments that we have conducted in our lab, we concluded that >60% of the mutants and >75% of the inhibitors tested do not affect long-distance transport. These data usually are never published. For example, a weak allele of exo70a (Synek et al. 2006) shows non-significant changes in auxin transport compared with its Col-0 control (Fig. 1C), although the mutant exhibits a root hair phenotype and Exo70 is involved in secretion, suggesting an altered auxin transport phenotype (Hála et al. 2008). On the other hand, a change in long-distance auxin transport may be an indirect result of a mutation. For example, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mutants vti11 and vti12 show approximately 35% reduction in auxin transport in inflorescence


stems. However, this reflects a loss of cellular organization and identity in the mutants that impacts cellular integrity and a slight effect on ABCB19 trafficking (Yang et al. 2013) rather than altered trafficking or localization of PIN1 or PIN3 (Surpin et al. 2003). Inhibitor experiments are especially subject to experimental conditions. Many of the inhibitors that were once characterized as specific for auxin transport have been shown to have pleiotropic targets or concentration-dependent activities. For example, naphthylphthalamic acid (NPA) and triiodobenzoic acid (TIBA) both inhibit rootward auxin transport, but TIBA has little inhibitory effect at the shoot apex (approximately wild-type) compared with NPA (approximately 80% wild-type) (Fig. 1D). This may be a result of differential modes of actions, as TIBA is a weak auxin that competes with IAA for transport sites (Katekar and Geissler 1980), while NPA can inhibit the transport activity of ABCBs and disrupt interactions between ABCBs and TWISTED DWARF 1 (TWD1/FKBP42) (Noh et al. 2001, Murphy et al. 2002, Geisler et al. 2003, Bouchard et al. 2006). However, at higher concentrations, NPA inhibits trafficking and interferes with the activity of other enzymes (Peer et al. 2009, Hosein et al. 2010, McLamore et al. 2010), and TIBA has been shown to stabilize the actin cytoskeleton at higher concentrations (Dhonukshe et al. 2008). The same appears to be true for newer auxin transport inhibitors. Gravacin not only inhibits auxin transport, specifically targeting the region surrounding glutamate 1174 in ABCB19, but also affects tonoplast biogenesis and trafficking of delta-tonoplast intrinsic protein (𝛿-TIP) (Surpin et al. 2005, Rojas-Pierce et al. 2007). Another example is the chemical inhibitor endosidin/prieurianin, which appears to affect auxin-dependent growth and reduces both actin rearrangements and vesicle mobility, but does not affect auxin transport (Robert et al. 2008, Tóth et al. 2012). Transport in heterologous systems does not always reflect native auxin transport activity In addition to studies in seedlings and mature plants, heterologous systems are commonly used to characterize auxin transporter activity. Plant-based and non-plant systems have advantages and disadvantages which need to be considered when analyzing the transport data. For example, Bright Yellow 2 (BY-2) tobacco cells are an easily transformed plant-based system (reviewed in Nagata et al. 2006). BY-2 cell cultures are maintained in media containing the synthetic auxin 2,4-dicholorphenoxyacetic acid (2,4-D), as it stimulates cell division (Nagata et al. 1992, Campanoni and Nic 2005). However, 2,4-D has been shown to alter PIN,


ABCB and AUX/LAX expression and activity (Terasaka et al. 2005, Yang and Murphy 2009, Kubeš et al. 2012) making it difficult to interpret transport data. In addition, IAA and NAA are rapidly catabolized in BY-2 cells (Delbarre et al. 1994, 1996), which is thought to be a consequence of 2,4-D in the culture media. While plant-based systems provide a more ideal membrane environment for auxin transport studies, it may be difficult to separate the endogenous auxin transport activity from that of the auxin transporter being characterized (Titapiwatanakun et al. 2009). Non-plant systems include mammalian HeLa cells, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Xenopus laevis oocytes. Xenopus laevis oocytes are large, easy to manipulate and can efficiently translate injected mRNA. However, not all transporters are properly targeted to the correct compartment, as additional plant-specific components appear to be needed in some cases (Péret et al. 2012). HeLa cells contain sterol enriched PM microdomains that are required for transporter functionality (Belli et al. 2009, Titapiwatanakun et al. 2009); however, differences in sterol composition between plants and animals may effect protein stabilization and/or transport activity. While S. cerevisiae provides a more plant-like environment (Worrall et al. 2003), its glycosylation mechanism causes mislocalization of some auxin transporters (Noh et al. 2001, Geisler et al. 2005). Schizosaccharomyces pombe has a plant-like membrane composition, an N-glycosylation mechanism and a cell wall, and has been the only system to date to compare the functionality of ABCBs, PINs and AUX/LAX proteins in a single heterologous expression system (Yang and Murphy 2009). In our experience, radiotracer assays with S. pombe must be kept short (12–15 min) as transport activity becomes highly variable as cellular health degrades. Other heterologous systems may be implemented but the message remains the same: no system is perfect. A prime example of how different heterologous systems can lead to differing results is the case of the conditional uptake/efflux transporter AtABCB4. Previous results from expression in S. cerevisiae showed that ABCB4 exhibited IAA uptake activity (Santelia et al. 2005). Seemingly conflicting results from expression in BY-2 tobacco cells reported that ABCB4 had enhanced NAA efflux (Cho et al. 2007). When expressed in HeLa and S. pombe cells, ABCB4 was shown to have IAA uptake activity at low intracellular IAA concentrations, and IAA efflux activity at higher intracellular IAA concentrations (Terasaka et al. 2005, Yang and Murphy 2009, Kubeš et al. 2012). These results highlight the fact that no one system is appropriate for all auxin transporter characterizations. 101

Analyses of auxin transporters in heterologous systems allows for an expanded and more thorough investigation of activity, often beyond what is possible in seedlings and mature plants. The results they provide, however, must be interpreted carefully as different systems may provide conflicting, masked or non-physiologically relevant results.

Measurement of auxin transport Inferred transport DR5:GUS/GFP and DII-VENUS are auxin-responsive reporters. Therefore, these tools report auxin signaling events, and changes in auxin transport can be inferred from direct quantitation of the DR5 and DII-VENUS signals. DR5 is an auxin-responsive promoter with modified auxin response elements from the promoter of GH3, encoding an aminotransferase gene product. In other words, DR5 drives expression of GUS or GFP in cell types when auxin conjugation to amino acids is necessary. DR5 responsiveness is dose-dependent between 10–1000 nM IAA (Nakamura et al. 2003), and relative DR5 signals can be more or less when compared with wild-type. The gain or loss of signal can infer a change in transport and relative auxin amounts, but it does not give a quantitative measure of auxin in the cells. A reduced signal in the root tip indicates that long-distance auxin transport has been reduced (Geisler et al. 2005), while an increased signal after inhibitor treatment or in a mutant suggests that auxin transport is reduced due to auxin pooling in the tissue (Casimiro et al. 2001, Peer et al. 2009). Global treatments of auxin or auxin transport inhibitors are difficult to interpret. A recurring problem with DR5:GUS is over-staining. Long staining times and/or high substrate concentrations can result in saturation and non-specific staining which will mask the changes in auxin maxima and transport and may lead to misinterpretation of the data. DII-VENUS is a nuclear localized auxin-responsive reporter comprised of degron II (DII) of the AUX/IAA proteins fused to the fast maturing form of yellow fluorescent protein VENUS (Brunoud et al. 2012). Upon perception of auxin the SCFTIR1 proteosome rapidly degrades DII-VENUS. Therefore, the loss of VENUS signal represents enhanced auxin transport through that tissue (Christie et al. 2011, Vernoux et al. 2011, Brunoud et al. 2012). As DII-VENUS relies on degradation of the reporter rather than production, as with DR5:GUS/GFP, relative auxin abundance can be visualized more rapidly. This is important in experiments where changes in auxin occur within short timeframes, such as during phototropic bending (Han et al. 2014). 102

Direct measurement of transport Auxin can be directly measured using a ‘selective’ electrode (Mancuso et al. 2005, McLamore et al. 2010). Specificity for IAA can be achieved using integrated enhanced surface microflux sensors combined with acquired signal decoupling and integrated flux analyses which can provide direct quantification of real-time endogenous IAA movement. While this technique has the advantage of being non-invasive, auxin transport can only be measured along the root surface, so only the IAA that is lost from the system is recorded, as the apoplast is continuous with the rhizosphere (medium). There is also a requirement for the addition of IAA, a ‘doping effect’, to measure the signal (Mancuso et al. 2005, McLamore et al. 2010). Transport capacity When we think about long-distance auxin transport, we are usually thinking about transport capacity. Transport capacity is what is measured in radiotracer studies, and in any study where auxin deposition (not global auxin treatment) is followed by auxin measurement or visualization of DR5 activity and DII-VENUS degradation. A number of factors must be considered when measuring transport capacity: Diffusion Due to surface cuticular waxes, diffusion is generally not a problem with auxin transport assays utilizing small quantities of radiolabeled auxin in the shoot. However, diffusion is a problem in the root, where the cortical and epidermal apoplast is essentially a continuum with the rhizosphere. This problem is overcome by the use of discontinuous media for root transport assays (Blakeslee et al. 2007, Peer and Murphy 2007). In these assays, the media supporting the root material to be sampled is discontinuous with the site of application, and nanodroplets of auxin deposited at the root apex can only move in or along the root. Surface diffusion becomes a greater concern when transport assays are conducted on continuous gel media (Lewis and Muday 2009). When a nanodroplet of radiolabeled auxin is deposited on 1% agar with a pH of 6.0, diffusion is very limited and non-directional (Fig. 2). When a 1.5 mm nylon filament (approximately the same diameter as an Arabidopsis root) is placed on the agar with one end at the site of radiolabeled auxin deposition, the auxin diffuses more directionally and farther than is seen without the filament. If the filament is replaced with a Col-0 seedling root placed in the same orientation, there is a similar increase in directional auxin diffusion.


If the seedling is first killed with liquid nitrogen, diffusion still occurs along the surface vector provided by the seedling root when the agar is sampled after seedling removal. On discontinuous media, shootward transport of radiolabeled auxin in the twd1 mutant is severely limited. However, when assayed on continuous gel media, shootward transport in twd1 appears to be enhanced. Removal of the seedling and sampling of the gel media shows that this is a surface wicking effect resulting from the twisted surface of the twd1 root. This demonstrates that surface diffusion is a real factor that can impact root auxin transport assays. Non-specific transport Non-specific transport can and does occur in assays. Therefore, there must be a control for this baseline activity. Benzoic acid is a good control for auxin transport assays because its acid dissociation constant (pKa ) of 4.2 is similar to IAA, and it has an aromatic ring and carboxylic acid group (Fig. 3A). Therefore, it can be used as a measure of the amount of non-specific transport/diffusion in assays. Timing The time when the sample is collected is critical to interpreting data. The auxin transport assays must be calibrated to when the auxin front reaches the sampling point (Dubrovsky et al. 2011). This may not be the time that the maximum or pulse of radiolabeled auxin reaches the root apex. Figure 3A, B shows the progress of radiolabeled auxin from the shoot to the root. In this example, the peak of the pulse of auxin is at the root–shoot transition zone (tr), and the front of radiolabeled auxin can be detected at the root apex (r-6). The optimal sample collection time is when the bulk of the labeled auxin is at the root–shoot transition zone because the root apex sample only includes unidirectional transport of the labeled IAA. If the sampling time point occurs when the bulk of the labeled auxin reaches the root apex, then the transport from the root apex towards the shoot and subsequent reuptake into the rootward transport stream would be measured as well, and therefore obscure the results (Fig. 3C). Assay conditions In order to obtain physiologically relevant data, auxin transport assay conditions must be relevant to gene function. In other words, the seedlings must be grown and assayed under growth conditions and at developmental stages which are appropriate to test the hypothesis. For example, members of the Phytochrome Kinase


Fig. 2. Surface diffusion impacts root auxin transport assays. Movement of radiolabeled auxin applied directly to the media upon which a nylon filament was laid, or on a root tip laid on the media. Samples were taken at 45 degree intervals at 5 and 10 mm from the site of application after 3 h. On continuous media, increased diffusion is observed at pH 6.0 compared with pH 4.5. Rapid movement was also observed in wild-type (Col-0) seedlings that were freeze-thawed, and in twd1. Reduced movement was observed in twd1 on discontinuous media. A heat map corresponds to the amount of radiolabeled auxin measured. Individual data points pooled from three independent experiments are presented.

Substrate (PKS) family function during early phototropic responses in low light and in the dark (Fankhauser et al. 1999, Lariguet et al. 2006, Schepens et al. 2008, de Carbonnel et al. 2010, Kami et al. 2014). If auxin transport assays are conducted under standard conditions (i.e. 5-day light-grown seedlings), no difference can be observed between single pks isoform mutants and the 103

Fig. 3. Non-specific transport, timing and diffusion out of the stele. (A) Rootward auxin or benzoic acid (BA) transport in the shoot (s), root-shoot transition zone (tr) and root segments (r-1 through r-6). Root segments are 2 mm with r-6 containing the root tip. The majority of the labeled auxin travels as a pulse, but non-specific transport substrates (e.g. BA) do not. Data are means and standard deviations of three independent experiments. (B) In rootward auxin transport assays, the site of radiolabeled IAA application at the shoot tip is shown (red dot), and IAA is transported polarly toward the root tip (red line). (C) When radiolabeled IAA reaches the root tip, some of the labeled IAA is transported shootward as in the reflux model. Therefore, sampling times occur when the front reaches the root tip and before the maximum radiolabeled signal arrives at the root tip. (D) Rootward auxin transport in wild-type, abcb1, abcb19 and lax3. For the +IAA data, 1 μM IAA was added to a strip of paper at the upper root at the beginning of the experiment. Data are means and standard deviations of three independent experiments. * ANOVA followed Dunnett’s post-hoc analyses (P < 0.05).

wild-type, despite clear differences observed in the light responses of the mutants (Kami et al. 2014). Therefore, assays conditions used must be physiologically relevant to the gene function being assessed. As another example, assays studying phot1-mediated phototropism must be conducted under conditions where phot1 is in its ground state, which is in etiolated and dark-acclimated seedlings (Christie et al. 2011), as phot1 is not thought to be active in light-grown seedlings. Consequently, assays with light-grown seedlings will not reflect the full phot1-mediated phototropic bending response and inherent auxin transport. The media used before and during assays can also affect auxin concentration and transport. To account for 104

CO2 utilization, in vitro sucrose is often used as an additional carbon source for seedlings as it is the main transportable sugar in plants. Increasing sucrose concentrations (up to approximately 1%) in the media does not change the amount of IAA in seedlings (Stokes et al. 2013) but the amount of oxIAA-Glc and auxin transport activity are increased. For our standard assay conditions, we use 0.5% sucrose. At this concentration enough exogenous carbon is provided to achieve proper seedling development without inducing sucrose stress responses or alterations in auxin transport and homeostasis. The temperature at which seedlings are grown and assays are conducted can have significant effects on auxin production and sensitivity. In Gray et al. (1998),


free IAA was shown to increase approximately 1.75× in Arabidopsis grown at 29∘ C compared with 20∘ C. Additionally, in our lab, the time for radiolabeled IAA placed at the shoot apex to reach the root tip is decreased by 1 h in assays conducted at 25∘ C compared with those done at 20∘ C. In order to obtain physiologically relevant data multiple assay conditions need to be accounted for when attempting to approximate transport capacity. Unfortunately, it is not practical to assay all mutants under all conditions. Solvents As stated above, inhibitors must be used with care as many have concentration-dependent effects and pleotropic targets that are not specific to auxin transport. Additionally, when applying exogenous compounds (inhibitor, auxins, etc.) choosing the right solvent for the assay is also important as some solvents themselves can affect cellular auxin concentrations and/or auxin transport. For example, when ≥5% dimethyl sulphoxide (DMSO) is applied to DII-VENUS hypocotyls, an increase in DII-VENUS signal is observed indicating a decrease in cellular auxin. While the specific mechanism is unknown, this can be attributed to leaking of auxin from the seedling as an increase in radiolabeled IAA is detected in the media proximal to the site of DMSO application during hypocotyl transport assays. DMSO has also been shown to affect the PM localization of the auxin transporter ABCB4 in root epidermal cells (Kubeš et al. 2012). After treatment with 0.05% DMSO internalizations of N- and C-terminal GFP fusions of ABCB4 are observed. These results highlight the importance of choosing the proper solvent, solvent concentration and the use of solvent controls in assays. Auxin concentration The amount of auxin used can dramatically alter observed results. ‘Doping’ of radiolabeled IAA with cold IAA can enhance IAA uptake and transport (Blakeslee et al. 2007), and addition of IAA at the shoot apex alters PIN1 abundance in the root (Peer et al. 2004). Ecotype-dependent concentration effects can also be observed. In the assays shown in Fig. 1B, results from Col-0 and Mt-0, but not Ws-0, become noisier when auxin application increases 5×. A similar effect can be observed in rootward transport assays. Although we can generally assume that auxin stays within the stele except during lateral root emergence, an increase in auxin application can alter auxin retention. For example, in radiolabeled auxin transport assays on discontinuous media (our standard assay conditions), if 1 μM IAA


is added to a strip of paper at the upper root at the beginning of the experiment, then IAA leakage from the nonstelar apoplast in the root can be induced. In fact, more IAA leakage is induced in the lax3 mutant when compared with wild-type, abcb1 or abcb19 (Fig. 3D). The message is clear: to perturb the system as little as possible, auxin must be used in discrete applications, and at low doses to minimize the change in activity. Sinks are important Auxin sinks are as important as transporters, and the sink-source dynamics of long-distance auxin transport are important and need to be taken into consideration when characterizing mutants. Roots are often thought of as the auxin sink. Mutations in auxin transporters, symporters, and carriers that are primarily expressed in the root, aux1, pin4, abcb4, affect auxin transport from the shoot to the root by altering the sink. We usually think of AUX1 functioning as an auxin permease in the root. However, AUX1 also functions as a sink in the shoot, and aux1-7 (strong allele) and aux1-2 (weak allele) (Swarup et al. 2004) have altered auxin transport in the shoot. Auxin transport in aux1-7 is faster at the shoot apex compared with aux1-2 and wild-type, and the auxin transport in the two alleles become similar in the root. Auxin metabolism: conversion, oxidation and conjugation Finally, radiolabeled auxin applied in transport assays may be transformed by metabolic processes. In radiotracer assays, the identity of the transported label is just as important as what radiolabel is applied and where the label is. Radiotracer studies typically use 3 H-IBA and 3 H-IAA to examine indole-3-butyric acid (IBA) and auxin homeostasis and transport. For example, in abcg37/pis1 3 H-IBA applied to the root apex is converted to 3 H-IAA by the time that the label reaches 2.4–4 mm above the root apex (Ru˚ žiˇcka et al. 2010). Therefore, IBA is quickly metabolized to IAA locally, and IAA is the transported species. Auxin oxidation and conjugation also occur under natural conditions (Normanly 2010, Peer et al. 2013), and the degree to which they occur under assay conditions depends upon how much auxin is applied. Just as it is important to determine the species of the transported radiolabel, it is also important to determine the fate of the radiolabeled IAA applied. For example, as the amount of IAA applied to the shoot tip in long-distance transport assays increases, so does the amount of 2-oxindole-3-acetic acid (oxIAA) and glucosylated oxIAA (oxIAA-Glc) that is formed at the root tip (Kubeš 105

et al. 2012). This results in local auxin accumulation which produces the reactive oxygen species leading to auxin oxidation to oxIAA and oxIAA-Glc (Peer et al. 2013). Previously, it was common practice to add 10× cold IAA to assays to enhance auxin transport. The addition of the excess cold IAA results in the production of metabolites, such as oxIAA, that can increase transport (Geisler et al. 2005).

Conclusions Determination of auxin/metabolite levels, radiotracer assays and ‘specific’ electrode assays continue to be important analytical approaches to confirm phenotypic and molecular reporter analyses of auxin-dependent development. As developmental research increasingly seeks to leverage information derived from model systems to elucidate mechanisms in non-model species, these analytical tools will become even more valuable. Use of uniform genetic backgrounds, standardization of methodologies and clear documentation of metadata are essential to these efforts.

Materials and methods for unpublished assays described in this paper Plant materials and growth conditions Arabidopsis thaliana seeds were obtained from Arabidopsis Biological Resource Center, The Ohio State University, Columbus, OH, Nottingham Arabidopsis Stock Centre, Nottingham, England and Lehle Seeds, Round Rock TX. The ecotypes used in the study were Col-0 (Columbia, MO), Cvi (Cape Verde Islands), Ler-2 (Landsberg erecta, Wageningen University, The Netherlands), Mrk-0 (Märk, Germany), Mt-0 (Martuba, Libya), Ws-0 (Wassilewskija, Vasil’yevka, Belarus) and 1602 (N1602, Ws-0). The mutants used in the study were pin1, abcb1, abcb19, twd1 and lax3. Seeds were grown as previously described (Peer et al. 2009). Briefly, seedlings were grown on 1% phytagar plates, containing quarter-strength Murashige and Skoog (MS) basal salts, pH 5.5, at 22∘ C, 14 h at 100 μmol m−2 s−1 except as indicated for specific treatments. Auxin transport assays Auxin transport assays were as previously described (Geisler et al. 2005, Peer et al. 2009, Dubrovsky et al. 2011). Briefly, for root assays, Arabidopsis seedlings were grown on 1/2 MS media, constant light for 4 days after germination. Seedlings were then grown under dim light for 12–24 h to provide for hypocotyl elongation. The duration of this treatment was varied to equalize 106

hypocotyl length of the genotypes assayed. Prior to the assay, 10 seedlings were transferred to vertically arranged discontinuous filter paper strips saturated in 1/4 MS and allowed to equilibrate for 2 h under yellow light. Auxin solutions for transport measurements were in 0.25% (w/v) agarose, 2% (v/v) DMSO, 25 mM MES, pH 5.2. Under yellow light, a 0.1-μl microdroplet containing 500 nM unlabeled IAA and 500 nM 3 H-IAA (specific activity 25 Ci mmol−1 ; American Radiolabeled Chemicals, St. Louis, MO) was placed on the columella of the root tip of seedlings using a nanopipettor (WPI, Sarasota, FL). Seedlings were incubated in yellow light for 2 h, and then 2 mm segments were excised as indicated and placed in scintillation vials. Where indicated, filter paper sections were harvested separately and placed in scintillation vials: three 2-mm sections of filter paper with 2-mm segment of tissue containing the 2–4, 4–6 and 6–8 mm from root tip, as well as a 4-mm filter paper strip section containing the remaining part of the seedlings; 5 ml EcoLite scintillation fluid was added to each vial, vortexed for 10 s, incubated for 48 h, vortexed for 10 s again, and DPM were measured in a scintillation counter (Perkin Elmer, Danbury, CT). For shoot assays, radiolabeled IAA was deposited at the shoot apex, seedlings were incubated for 5 h on discontinuous horizontal strips of filter paper, and the radioactivity of the cotyledons, upper hypocotyls and root-shoot transition zone was counted along with the 2 mm filter paper strips supporting those tissues. Auxin diffusion assays All experiments were conducted as above with the following exceptions. A 0.2 μl drop of 0.3% agarose, 20 Ci mmol−1 3 H-IAA from Gilson pipettor was pipetted onto media or on the root apex. Samples were collected at 3 h at 5 and 10 mm, at 45 degree intervals (or compass points) from the point of application; 180 degrees (or ‘North’) was aligned with the root or filament. For the filament diffusion assays, a 1.5 mm nylon filament, laid from application point to 180 degrees, and samples were collected from the media underneath the filament or seedling after lifting them from the media. For the root diffusion assays, Col-0 seedlings were live or killed-frozen in liquid nitrogen and thawed prior to diffusion assays, and twd1 seedlings were on either continuous or discontinuous media. All experiments were replicated independently three times. Auxin quantitations and mass spectroscopy determinations Auxin quantitations were conducted by mass spectroscopy as previously described (Kim et al. 2007, Kubeš


et al. 2012). Three sets of sampled sections, biological replicates, were assayed for each auxin determination. Briefly, the tissue was homogenized in liquid nitrogen, 800 μl 0.05 M sodium phosphate, pH 7.0, 0.02% (w/v) sodium diethyldithiocarbamate, combined with 4 ng 13 C-IAA working standard was added. Samples were shaken for 15 min at 4∘ C, and then combined with 40 μl of 1 M HCl (depending on starting weight of plant material) to a final pH of 2.7. Samples were passed through a 0.45-μm syringe filter and applied to an Isolute C8-EC (500 mg/3 ml; no. 291–0050-B) solid-phase extraction column preconditioned by methanol/acetic acid. The sample was washed with 2 ml 10% MeOH/1% AcOH, vacuumed to remove water phase (without drying), and eluted into derivatization vials with 1 ml 70% methanol/1% acetic acid. The samples were vacuum evaporated to dryness at 30∘ C and methylated by adding 200 μl methanol, 1 ml dichloromethane and 5 μl 2 M trimethylsilyl-diazomethane (in hexanes), followed by incubation at 42∘ C for 30 min. After neutralization with 5 μl of 2 M acetic acid/hexane to destroy excess diazomethane, samples were evaporated to dryness and resuspended in acetonitrile. Samples analyzed by gas chromatography–mass spectrometry were analyzed as described by Ljung et al. (2005), except that an Agilent/LECO gas chromatographer-mass spectrometer was used with a split injection volume of 5 μl, a transfer port temperature of 260∘ C, separation through a DB-5, 10-m × 0.18-mm × 0.20-μm column with helium carrier flow at 1 ml min−1 . The temperature program was 80∘ C for 2 min, 20∘ C min−1 to 260∘ C, 260∘ C for 2 min, and mass ranges were monitored from 70 to 200 mass-to-charge ratio. Liquid chromatography-mass spectrometry (LC-MS) determinations were made with underivatized samples using an Agilent 9460 LC-MS (Santa Clara, CA) in multiple reaction monitoring mode. Quantitations are based on comparisons of IAA peaks to 13 C-IAA standards normalized to fresh weight of original sample. Acknowledgements – We thank Yan Cheng for assistance with assays. This work was supported by DOE #DE-FG02-06ER15804 (for work related to ABCB transporters) and NSF # IOS-0820648 (all other work) to A. S. M. and by the University of Maryland.

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The finger-tip unit--a new practical measure.

Root system patterning: auxin synthesis at the root periphery.

Measure for measure: the cost of improving quality.

"Measure Your Gradient": a new way to measure gradients in high performance liquid chromatography by mass spectrometric or absorbance detection.

Arabidopsis Myrosinase Genes AtTGG4 and AtTGG5 Are Root-Tip Specific and Contribute to Auxin Biosynthesis and Root-Growth Regulation.

The fear of food measure: a novel measure for use in exposure therapy for eating disorders.

Karyotype asymmetry: again, how to measure and what to measure?

Photopic Negative Response Obtained Using a Handheld Electroretinogram Device: Determining the Optimal Measure and Repeatability.

Auxin gradients across wood-instructive or incidental?

The measure of laterality.

The measure of a physician, the measure of our practices: what we measure reflects what we believe.

Diffusion-assisted selective dynamical recoupling: a new approach to measure background gradients in magnetic resonance.

Local potential gradients as a unifying measure for thresholds of stimulation, standstill, tachyarrhythmia and fibrillation appearing after strong capacitor discharges.

A mix-and-measure assay for determining the activation status of endogenous Cdc42 in cytokine-stimulated macrophage cell lysates.

The measure-unit of evolution.

How do we measure blood pressure at home?

Measure for Measure: How Proficiency-Based Accountability Systems Affect Inequality in Academic Achievement.

Made to measure - keeping Rho kinase at a distance.

The measure of a surgeon.

The Circumscribed Fear Measure: Development and initial validation of a trans-stimulus phobia measure.

Comparison of a Timing-Based Measure of Unintended Pregnancy and the London Measure of Unplanned Pregnancy.

The spinal cord independence measure.

London Measure of Unplanned Pregnancy: guidance for its use as an outcome measure.

The measure of research merit.