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Flavonoids and auxin transport: modulators or regulators?
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TRENDS in Plant Science
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Flavonoids and auxin transport: modulators or regulators? Wendy Ann Peer and Angus S. Murphy Department of Horticulture, 625 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47906, USA
Flavonoids are polyphenolic compounds found in all vascular and non-vascular plants. Although nonessential for plant growth and development, flavonoids have species-specific roles in nodulation, fertility, defense and UV protection. Flavonoids have been shown to modulate transport of the phytohormone auxin in addition to auxin-dependent tropic responses. However, flavonoids are not essential regulators of these processes because transport and tropic responses occur in their absence. Flavonoids modulate the activity of auxin-transporting P-glycoproteins and seem to modulate the activity of regulatory proteins such as phosphatases and kinases. Phylogenetic analysis suggests that auxin transport mechanisms evolved in the presence of flavonoid compounds produced for the scavenging of reactive oxygen species and defense from herbivores and pathogens. Auxin transport inhibitor studies The phytohormone auxin [indole-3-acetic acid (IAA)] is transported from its sites of synthesis in the apices to distal parts of the plant, where it influences growth and development [1]. Historically, auxin transport regulation has been investigated using both artificial and naturally occurring auxin transport inhibitors. One such class of natural inhibitors are flavonoids, of which the most effective subgroups are flavonols and isoflavones [2–4]. Flavonoids are a subgroup of phenylpropanoid compounds whose synthesis is tissue specific, developmentally regulated and dependent on environmental conditions, such as light and temperature [5]. Flavonoids, such as quercetin, kaempferol, apigenin and other aglycone molecules synthesized in the first steps of the flavonoid biosynthetic pathway (Figure 1), have been shown to inhibit polar auxin transport and to enhance consequent localized auxin accumulation in planta. Flavonoids have been shown to displace artificial auxin efflux inhibitors, including 1-N-naphthylphthalamic acid (NPA), from plasma membrane and microsomal binding sites [2,6–8]. NPA displacement from microsomal membranes by flavonoids is biphasic, indicating that there are at least two in planta targets of flavonoid activity [2]. This observation is also consistent with the identification of peripheral membrane actin-associated [9,10] and integral plasma membrane [8] NPA binding sites in Cucurbita pepo. Subsequently, in Arabidopsis, the integral membrane NPA–flavonoid Corresponding author: Peer, W.A. ([email protected]). www.sciencedirect.com
interaction site was shown to be associated with three integral plasma membrane phosphoglycoproteins (PGPs; At2g36910, At2g4700, At3g28860) that transport IAA, whereas a weaker interaction site was associated with at least one peripheral membrane protein, including a membrane-anchored M1 aminopeptidase, (APM1, At4g33090), and the FK506 binding protein immunophilin TWISTED DWARF1 (TWD1, At5g42800) [11,12]. Mutational analyses with transparent testa mutants identify sites of flavonoid action Three Arabidopsis transparent testa (tt) mutants, which lack key enzymes in the flavonoid biosynthetic pathway, were used to determine whether endogenous flavonoids regulate auxin transport in planta: tt4 (a chalcone synthase, CHS, At5g13930, mutant accumulating no flavonoids); tt7 (a flavonoid-30 -hydroxylase, At5g07990, mutant accumulating excess kaempferol); and tt3 (a dihydroflavanol-4-reductase, At5g42800, mutant accumulating excess kaempferol and quercetin) [13,14] (Figure 1). Transport assays are usually performed in five-day-old seedlings, when maximal accumulation of aglycone flavonoids is observed [13,14]. In the tt4 mutant, auxin transport from the shoot to root was found to be elevated compared with the wild-type, whereas the flavonol-overproducing mutants tt7 and tt3 exhibited reduced auxin transport [2–4]. Wild-type auxin transport levels and tissue-specific flavonol accumulation patterns could be restored in tt4 when the lesion in CHS was bypassed by treatment with the downstream product naringenin [2,3] (Figure 1). Furthermore, addition of a naringenin droplet to the shoot apex of wild-type or tt4 seedlings reduced auxin transport levels in shoots and roots to levels similar to those seen in tt7 and tt3 [4]. In addition to auxin transport, lateral root number and length, which are modulated by auxin transport levels, also increased in tt4 seedlings [3]. Together with the tissue-specific accumulation of flavonols in Arabidopsis seedlings that coincided with regions of higher auxin accumulation [2,4], these data suggested that flavonols affect polar auxin transport in apical tissues by modulating the amount of auxin loaded into the long-distance polar auxin transport stream [4]. Manipulation of phenylpropanoid biosynthesis affects auxin transport There are several phenylpropanoid classes, such as cinnamic acids, flavonoids and lignins, that share phenylalanine as a precursor and whose biosynthesis is feedback regulated at various branch points in the metabolic pathway [15]. Recent efforts to use gene silencing to alter floral scent
1360-1385/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2007.10.003
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Figure 1. Phenylpropanoid biosynthesis. This brief outline of the phenylpropanoid pathway includes biosynthetic proteins and products referenced in the text. Double arrows indicate multiple steps. Abbreviations: BPBT, benzoyltransferase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol-4-reductase; F3H, flavone 3-hydroxylase; F30 H, flavonoid 30 -hydroxylase; FLS, flavonol synthase; HCT, hydroxy cinnamoyl transferase. The portion of pathway in the box highlights the only flavonoid compounds (flavanones, flavones and flavonols) observed among bryophytes.
in Petunia x hybrida or lignin production in Arabidopsis had the unintended result of altering auxin transport and phenotypes that are consistent with such alterations [16,17]. Silencing of the gene encoding the volatile benzenoid biosynthetic enzyme benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase (BPBT, AY563157) in Petunia resulted in 4.1-fold accumulation of flavonoids and transcinnamic acid [an early metabolite which might interfere with auxin signalling (Figure 1)] in flowers [16]. The transgenic petunia plants were 1.75-fold larger than wild type, suggesting an auxin-related effect. Enhanced flavonoid accumulation in transgenic Petunia plants was found in seeds and at the seedling root–shoot junction but not in the shoot apex or other tissues where flavonols are most likely to affect auxin transport. However, the BPBT-silenced lines had expanded vascular tissue, perhaps resulting from enhanced auxin movement during embryogenesis, in which auxin is required for vascular differentiation. The observed increases in auxin transport and auxin-dependent growth in mature plants seemed to be a result of this early developmental effect. Such indirect effects on auxin transport have been observed in other cases in which components of cellular trafficking mechanisms (e.g. Vesicle Transport V-snare 11 (VTI11), At5g39510) or auxin-conducting vascular tissues (e.g. Interfascicular Fiberless 1 (IFL1), At5g60690) are mutated [18,19]. By contrast, silencing of the gene encoding the lignin biosynthetic enzyme hydroxy cinnamoyl transferase (HCT, www.sciencedirect.com
At5g48930) increased flavonoid accumulation (mainly kaempferol derivatives), reduced auxin transport, altered vasculature (including reduced lignification) and dwarfed plants in Arabidopsis [17]. When the HCT-silenced lines were crossed with CHS-silenced lines, auxin transport and plant height were restored to wild-type levels. In this case, increased flavonoid accumulation had a greater impact on auxin transport than did changes in the plant vasculature. Increased flavonoid accumulation has been observed even when a genetic modification is not directed at phenylpropanoid manipulation [20]. Altered vascular development, reduced hypocotyl and epicotyl length, and reduced auxin transport were observed in tobacco transformed with a tumorigenic gene 6b (D30626) from Agrobacterium tumefaciens [20]. Induced plants had increased amounts of a kaempferol derivative, suggesting that 6b expression inhibits auxin transport by increasing production of this flavonoid compound [20]. These examples indicate that tissue-specific changes in flavonoid accumulation have a direct effect on auxin transport, whereas modifications in other aspects of phenylpropanoid metabolism that affect vascular architecture indirectly affect auxin transport. Inhibition of auxin transport requires active flavonoids in the correct compartment The activity of flavonoids themselves can be modulated. Sucrose induces expression of CHS and other
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phenylpropanoid genes [21] but increasing sucrose concentrations also decreases the levels of early flavonoid products that inhibit auxin transport and increase glycosylated late pathway flavonoids, such as anthocyanins, that have little or no impact on transport [14,21] (Figure 1). UDPglucuronosyltransferase 1 (UGT1, At1g05560) is thought reversibly to catalyze the conversion of a flavonoid from an active aglycone to an inactive glucuronic acid form [22,23]. Glycosylation also reduces the antioxidant activity of quercetin [24]. In addition, flavonoid activity can be modulated through oxidation, as described in a recent review [25]. Compartmentalization in endomembranes or organelles has also been found to modulate active flavonoid levels because the addition of sucrose induces flavonoid accumulation in mutants lacking the peroxisomal COMATOSE (At4g39850) ABCD transporter that is required for flavonoid biosynthesis during germination [26]. Molecular targets of flavonoid regulation Experimental evidence indicates that flavonoid modulation of auxin transport is tissue specific and occurs at the root and shoot apices. Therefore, the molecular targets of flavonoid interaction should also be localized in these regions. Auxin transporters Several targets of flavonols that are involved in auxin transport mechanisms have been proposed [4,27]. Of these, interactions between flavonols and the PGP auxin transporters PGP1, PGP4 and PGP19 (also known as MDR1) are the best characterized. PGPs belong to the ATP-binding cassette subfamily B (ABCB) transporter family, which hydrolyses ATP to transport substrates. Comparisons of in planta auxin transport assays in tt and pgp mutants, PGP gene expression patterns and subcellular localization of PGP proteins [28–32], and flavonoid inhibition of PGPmediated auxin transport in heterologous systems [28–30] suggest that aglycone flavonols modulate PGP1, PGP4 and PGP19 activity at the root and shoot apices [4]. PGP activity in mammals is regulated through phosphorylation, inhibition of ATPase activity or allosteric binding [33] and flavonoid modulation of PGP1, PGP4 and PGP19 activity in planta might target the same sites [11,29]. Flavonoids might also modulate auxin uptake because the root-specific transporter PGP4 seems to be able to mediate cellular auxin uptake or efflux according to the cellular environment [34,35]. In heterologous systems, PGP4 activity is also NPA reversible and flavonoid sensitive [35]. Recently, PGP4, which functions primarily in moving auxin away from the root distal elongation zone [31,34,35], was shown to be epistatic to CHS in pgp4 tt4 double knockouts with regard to gravitropic bending [31] (see section on tropism later), which is consistent with PGP4 being a target of flavonoid modulation. The effects of flavonoids, if any, on the auxin influx protein AUX1 (At2g38120) have not been demonstrated. The effects of flavonoids on members of the PIN-Formed auxin efflux carriers have received less attention. Data so far suggest that changes in PIN1 (At1g73590) and PIN2 (At5g57090) expression and subcellular protein localization observed in flavonoid mutants can be attributed www.sciencedirect.com
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primarily to altered local auxin concentrations, rather than a direct flavonoid interaction [4]. The flavonoiddeficient tt4 mutant exhibits enhanced auxin transport and free IAA levels but changes in PIN1 gene expression and PIN1 protein localization in the mutant can be mimicked by artificially increasing auxin fluxes [4]. Similarly, PIN1 expression and protein localization in tt7 and tt3 were similar to wild-type plants treated with auxin transport inhibitors – again, indicating an indirect flavonoid effect [4]. By contrast, although PIN2 expression and protein signals were slightly enhanced, protein localization was not altered in tt4, and no differences were observed in tt7 or tt3 [4]. Transport assays with PIN proteins heterologously expressed in plant, fungal and mammalian cell systems suggest that flavonoids inhibit the activity of other transporters – for example, PGPs and organic anion transporters (which might be regulated by PINs) – rather than interacting with PINs directly [29,30]. However, flavonoids do seem to affect PIN4 (At2g01420) expression or subcellular protein localization both indirectly by altering localized auxin concentrations and directly by affecting cellular trafficking [4]. Cellular trafficking, auxin transport regulators and phosphorylation Experimental data show that flavonoids also modulate auxin transport through interactions with regulatory proteins. For example, inclusion of exogenous flavonols in a washout solution following treatment with the cellular trafficking inhibitor brefeldin A did not disrupt restoration of PIN1 to the plasma membrane in wild-type Arabidopsis; however, in tt4 cells never exposed to flavonols, after the same treatment, PIN1 was not restored to the wild-type subcellular localization pattern [4]. Micromolar concentrations of flavonols are routinely used as both phosphatase and kinase inhibitors [36,37], so kinases and phosphatases associated with auxin transport are probable targets in apical tissues, in which cellular flavonol levels are high [4,38]. The serine/threonine kinase PINOID (At2g34650) [39,40], which regulates PIN1, PIN2 and PIN4 activity by regulating the polarity of their subcellular localization on the plasma membrane [41], is the most likely candidate, followed by the PINOID-related WAG kinases (At1g53700, At3g14370) [42]. Flavonoid modulation of the protein or lipid phosphorylation state through inhibition of the protein phosphatase 2A subunit ROOTS CURL IN NPA1 (RCN1, At1g25490) [43] or the phosphatidylinositol 3,4,5-trisphosphate kinase (e.g. At1g21980) [44] is also possible. Flavonoids might also influence cellular trafficking by altering membrane fluidity directly because the more hydrophobic flavonoids associate with and intercalate into membranes, making them more rigid or fluid, depending on the sterol content of the membrane, which itself is cell-type specific [27]. In summary, flavonoids modulate auxin transport directly and indirectly by affecting the activity of auxin transporters, proteins that regulate the transporters and trafficking of the components of the auxin transport complex, and by altering the structure of auxin-transporting tissues.
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Flavonoids and physiological responses Fundamental processes, such as auxin transport, gravitropism and phototropism occur in the absence of flavonoids [45]. Therefore, flavonoids are best viewed as modulators of transport processes, rather than as essential regulators. In some cases, flavonoids seem to be synthesized in response to auxin accumulation, presumably to scavenge reactive oxygen species (ROS) generated during auxin catabolism [27]. In the absence of PGP1, PGP4 or PGP19, a localized increase in auxin concentrations occurs, resulting in localized quercetin accumulation similar to that in the wild type after treatment with the auxin transport inhibitor NPA [4,28,35]. Because technical issues make it problematic to identify the exact timing of auxin and flavonol accumulation (Box 1), it is difficult to separate the different roles of localized flavonol accumulation, as seen, for example, in regions of shoot branching [14] or gravitropic bending [46] (Box 2). Shoot and root branching Flavonols are localized in the apical and nodal regions of Arabidopsis inflorescence stems, whereas anthocyanins, which do not inhibit auxin transport, decrease in concentration from the base of the stem towards the apex [14]. Increased auxin transport in tt4 inflorescences might contribute to the increased secondary inflorescence formation observed for one tt4 allele (2YY6) [3]. However, tt4 (2YY6) was subsequently found to harbour a mutation in More Axillary Branches 4 (At4g32810) [47]. MAX1 (At2g26170), a cytochrome P450 that functions downstream of the other MAX genes, is a positive regulator of flavonoid biosynthetic genes and is required for flavonol synthesis in the axillary bud [48]. The MAX genes were identified as regulators of auxin-dependent lateral inflorescence branching [47,49]. The tt4 max4 double knockout exhibits the increased shoot branching observed in max4 [3,47,50], indicating that MAX4 is epistatic to CHS in inflorescences. The max1 phenotype can be rescued by application of kaempferol, quercetin or naringenin, but MAX regulation of auxin transport is dependent on PINs and indirectly on flavonoid activity [47,48]. Flavonols accumulate in regions of lateral root emergence and along the lateral roots themselves in patterns similar to those indicated by auxin reporter genes, including DR5 [14,31]. Although tt4 has an increased number of lateral roots compared with wild type [3], independent of MAX4 [51], this seems to be an indirect effect of increased transport of shoot-derived auxin [52]. The contribution of flavonoids to lateral root development must also be evaluated in the context of macronutrients [53,54]. Flavonoid synthesis is induced two- to four-fold in phosphate-starved plants [55], with a concomitant increase in lateral root number [54]. Under phosphate starvation, tt4 showed an increased number of lateral roots, indicating a role for flavonoid modulation of root architecture [54]. Tropic bending Tropic responses do not require flavonoids because these responses are observed in dark-grown seedlings which are flavonoid deficient. However, flavonoids seem to modulate the rate and extent of gravitropic responses in roots. In www.sciencedirect.com
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Box 1. Visualization of flavonoids The resolution of in situ flavonoid visualization depends on the sensitivity of the microscope used. A low concentration of Triton X100 (a detergent) can be used to move diphenyl boric acid 2aminoethyl ester (DPBA, a fluorescent dye that interacts with flavonoids), into the five-day-old wild-type roots to visualize flavonols using epifluorescence microscopy [14] (Figure Ia). With more-sensitive detection equipment, solubilizing agents are no longer necessary. Using 5 mM DPBA in aqueous solution and spectral scanning with a Zeiss LSM 510 META (405 nm diode laser) confocal microscope and post-processing software, subcellular localization of the flavonols kaemperfol and quercetin can be observed (Figure Ib,c). The subcellular localization can be placed in context with the use of other marker dyes – for example, FM4–64 for plasma membrane labeling and endocytotic uptake (Figure Id–f) and green fluorescent protein fusion marker proteins for various compartments – for example, endoplasmic reticulum, Golgi, an assortment of endosomes and tonoplast. However, using dye for staining has limitations because the dye inactivates flavonols and can take up to two minutes to produce a visible signal. Instantaneous detection that does not interfere with activity is a technical challenge that has yet to be solved for localized concentrations of flavonoids.
Figure I. Visualization of flavonoids. (a) quercetin is shown in gold. (b,c) Quercetin is shown in blue, kaempferol in red and the overlap in purple (more quercetin) and magenta (more kaempferol). Note: merged images are presented. (d) FM4–64 is shown in red, (e) quercetin in blue and (f) the overlap in magenta. Size bars, 20 mm. Panel (a): reproduced, with permission, from http://ww.plantphysiol.org).
gravistimulated roots, auxin is redistributed laterally to root cap and elongation zone cells on the lower side of the root, with the result that elongation on the lower side is inhibited and the root bends downward [56]. The initial mechanism of lateral redistribution of auxin in root tips
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Box 2. Split-root system for tropism measurements Analysing flavonoid regulation of lateral auxin redistribution in graviresponding tissues requires monitoring of multiple dynamic processes. Gravistimulation is thought to produce rapid changes in the concentrations of small ions within the lateral root cap and these can be monitored in real-time using dyes and fluorescent protein fusions [29,41,44,60–66]. Changes in flavonol concentrations in Arabidopsis roots can also be visualized, although dynamic imaging remains problematic (Box 1). In situ monitoring of changes in auxin gradients presents an even greater challenge because such visualizations are largely dependent on the use of auxin-responsive transcriptional reporters [78,79]. Use of these reporters is limited by sensitivity of the reporters, the latency of auxin-dependent gene transcription and the persistence of the reporter proteins after auxin levels have subsided. Attempts to create fluorescent auxin reporter proteins that respond instantaneously to changes in auxin concentrations have been hampered by a lack of specificity and aberrant growth resulting from loss of the signalling molecule after it binds to the sensors in vivo. A carbon nanotube auxin-sensing electrode has been used to monitor transport in Arabidopsis roots [34] but this sensor lacks the sensitivity required for lateral auxin redistribution studies at this time. Microscale assays using radiolabeled IAA can be used to track rapid changes in auxin movement, although these assays require
Figure I. Split-root system for tropism measurements.
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destructive sampling techniques. The assays use discontinuous solid support media and microscopically guided precision instruments to place microdroplets (nL) of auxin on discrete tissues and to excise samples (for methods, see http://www.hort.purdue.edu/hort/ research/murphy/protframe.htm). Use of larger (mL) volumes or higher concentrations of auxin produces aberrant or nonspecific results [28,30]. In the example of this type of assay diagrammed here (Figure I), intact Arabidopsis seedling roots were attached to solid support media using surgical glue just before conducting the assay. A 6 nL droplet of 3H-IAA was then placed on the lower columella cells and seedlings were incubated upright or with a 908 rotation. At the time points indicated, the lateral root cap was excised with an obsidian surgical blade and discarded. A 2 mm root section was then excised, divided transversely and collected as two separate sections. Sections from ten seedlings were collected and counted. Using this method, changes in the lateral redistribution of transported auxin could be detected within 30 minutes. Similar results were obtained when a microlaser cutter was used for sectioning. In this case, differences between wild type and tt4 lateral auxin redistribution are not evident until after 75 minutes, suggesting that the initial lateral redistribution of auxin in graviresponding root tips is not regulated by flavonoids.
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has not been elucidated and might occur independently of an auxin gradient [57,58]. However, the rapidity of the response suggests that factors other than transcriptional responses [59] are involved. Calcium, inositol 1,4,5-trisphosphate [44,60], phosphorylation events [61], ROS generation [44,62], pH gradients [63–66], interference with ATPase activity [29] and polar realignment of PIN auxin efflux proteins with the auxin gradient [41,61] have all been implicated in the process. Flavonoid accumulation increases slightly and symmetrically in the root cap and cells of columella gravistimulated roots [46]. Gravitropic bending in the flavonoid-deficient tt4 mutant is delayed compared with wild type [31,46]. However, increases in the auxin content of cells in the elongation zone or the lower side of the gravistimulated roots is seen in both tt4 and wild type [46]. One explanation for this difference was suggested when Lewis et al. [31] demonstrated that pgp4 interaction is epistatic to tt4. The pgp4 mutant exhibits a 30% decrease in auxin transport from the columella [35] and a 50% decrease in auxin transport from the general apical region of the root [31] but also faster rather than slower gravitropic bending [31]. Expression and protein localization studies show that the highest levels of PGP4 abundance are in the elongation zone and epidermis of the mature root [35]. Taken together, these data suggest that PGP4 is a target of flavonoid regulation and functions primarily in the movement of auxin out of the elongation zone. Flavonoids normally partially inhibit PGP4-mediated movement of auxin out of the distal elongation zone, and loss of flavonoids results in a reduction in inhibitory auxin concentrations in this region. An increase in flavonoid levels or loss of PGP4 function results in a more rapid auxin accumulation and enhancement of bending. These results also suggest that flavonoid modulation of auxin transport mediated by PIN and PGP transporters within the lateral root cap is secondary. A split-root assay developed to isolate asymmetric delivery of auxin from the columella and lateral root cap to the elongation zone shows that no difference between wild type and tt4 is seen in the initial asymmetric auxin movement into the elongation zone (Box 2), consistent with a primary flavonoid function in modulating PGP-mediated auxin movement out of the elongation zone. The timing of the events suggests that flavonoids have an additional role that enhances tropic responses. Auxin accumulation can also produce flavonoid accumulation. Gravistimulated roots exhibiting localized auxin accumulation demonstrate increased flavonoid accumulation [46]. Mutations in PGP auxin transport genes result in ectopic flavonoid accumulation [35] that can be mimicked with exogenous auxin application [4,46]. One way that flavonoids might modulate tropic responses is through scavenging ROS because an oxidative burst seems to be an early signal to stimulate gravitropism in the root cap and columella cells [44,62], and the flavonol quercetin, an excellent ROS scavenger, increases in the columella response to auxin accumulation [4]. After the ROS signal is initiated, flavonoids might quench this signal so that it does not continue to stimulate a response. Oxidative catabolism of laterally accumulated IAA trapped in the elongation zone [28,35] also seems to generate ROS [27,67,68]. Flavonoid www.sciencedirect.com
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quenching of ROS generated in this manner would prevent cellular oxidative damage [25]. Phosphorylation might also have a role in graviresponses [69]. In addition to modulating the phosphorylation of PGPs, flavonoids might also mediate the serine/ threonine kinases PINOID and WAG. PIN2 reorientation is observed in the PID overexpressor mutants [41]. Although PIN2 localization was not affected by protein phosphatase inhibitors [61] or in tt4 [4], PIN2-mediated gravitropism is regulated by protein phosphatases that are biochemically similar to RCN1 [61]. The role of flavonoids in gravitropism is most probably a combination of factors: modulation of PGP activity, ROS quenching and retardation of phosphorylation events. Flavonoid modulation of physiological responses is tissue and process specific, involving mechanisms that evolved over time. Systems biology approaches utilizing mutants and precise studies of auxin movement out of the lateral root cap (such as those shown in Box 2) are required to develop a more complete model. Evolution of flavonoid modulation of auxin transport Flavonoid synthesis is observed in extant members of the first land plants but not in algae [70]. Unlike angiosperms, gymnosperms and pteridophytes, bryophytes only accumulate flavonoids synthesized by the biosynthetic genes early in the pathway (Figure 1), and the moss (Physcomitrella patens) genome does not seem to contain the genes required for the later steps in flavonoid synthesis, despite the presence of 19 CHS-like genes [71,72]. Bryophytes exhibit polar growth and have different auxin transport mechanisms within each bryophyte division, ranging from NPA-insensitive diffusion to NPA-sensitive polar auxin transport [73,74]. Poli et al. [73] conclude that auxin transport developed independently in each extant bryophyte lineage. Because polar IAA transport in some bryophytes is NPA sensitive, one hypothesis is that polar auxin transport and auxin transporters, such as the PINs and, to a lesser extent, PGPs, evolved in the presence of flavonols because all bryophytes examined accumulate flavonols. The activity of PGPs is inhibited by flavonoids; however, plant PGP activity is less sensitive to flavonoids than are mammalian transporters because flavonoids are endogenous in plants [28,29,75]. PINs evolved more recently than PGPs [76,77] and exhibit less apparent sensitivity to flavonoids, perhaps as a result of undergoing much of their evolution into auxin transporters in the presence of flavonoids. By contrast, PGPs are part of the ancient ABC transporter lineage [77] and flavonoids inhibit their essential ATPase activity [28,29]. Therefore, it is unsurprising that PGPs are more sensitive to flavonoids than are PINs. Thus, we might regard auxin transport proteins and their regulators as cellular components that have been adapted to function with greater efficiency in a cellular environment that includes flavonoid compounds, rather than as specific targets of flavonoid regulation. Concluding remarks and future perspectives Flavonoids modulate auxin transport through direct and indirect interactions with cellular transport and regulatory mechanisms. Developmental regulation of flavonoid
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biosynthesis also determines where and when these interactions take place. This makes it difficult to ascertain when flavonoids are functioning primarily as ROS scavengers, defense compounds, regulators of phosphorylation, enzyme inhibitors or modulators of membrane fluidity. An evolutionary approach using natural variation among the bryophytes could provide a context for assessing the specificity of flavonoid regulation. It seems that as the complexity of flavonoid biosynthesis increased during plant evolution, flavonoid function also expanded. A systems biology approach comparing bryophytes and tracheophytes could be used to identify which flavonoid functions are the most fundamental and which are the most divergent. When compared with the evolution of auxin transport mechanisms, those processes that evolved in the presence of flavonoids could then be differentiated from those that are more specifically regulated by these compounds. Acknowledgements This work was supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council (BBSRC). We thank Simon Gilroy and John Courage for suggesting the Box 2 experiment.
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18 Surpin, M. et al. (2003) The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways. Plant Cell 15, 2885–2899 19 Zhong, R.Q. and Ye, Z.H. (2001) Alteration of auxin polar transport in the Arabidopsis ifl1 mutants. Plant Physiol. 126, 549–563 20 Kakiuchi, Y. et al. (2006) Reduction of polar auxin transport in tobacco by the tumorigenic Agrobacterium tumefaciens AK-6b gene. Planta 223, 237–247 21 Solfanelli, C. et al. (2006) Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis. Plant Physiol. 140, 637–646 22 Woo, H.H. et al. (1999) Meristem-localized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development in pea and alfalfa. Plant Cell 11, 2303–2315 23 Woo, H. et al. (2005) Flavonoids: from cell cycle regulation to biotechnology. Biotechnol. Lett. 27, 365–374 24 Brown, J.E. et al. (1998) Structural dependence of flavonoid interactions with Cu2+ ions: implications for their antioxidant properties. Biochem. J. 330, 1173–1178 25 Pourcel, L. et al. (2007) Flavonoid oxidation in plants: from biochemical properties to physiological functions. Trends Plant Sci. 12, 29–36 26 Carrera, E. et al. (2007) Gene expression profiling reveals defined functions of the ATP-binding cassette transporter COMATOSE late in phase II of germination. Plant Physiol. 143, 1669–1679 27 Peer, W. and Murphy, A. (2006) Flavonoids as signal molecules. In The Science of Flavonoids (Grotewold, E., ed.), pp. 239–268, Springer 28 Geisler, M. et al. (2005) Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1. Plant J. 44, 179–194 29 Bouchard, R. et al. (2006) Immunophilin-like TWISTED DWARF1 modulates auxin efflux activities of Arabidopsis P-glycoproteins. J. Biol. Chem. 281, 30603–30612 30 Blakeslee, J.J. et al. (2007) Interactions among PIN-FORMED and Pglycoprotein auxin transporters in Arabidopsis. Plant Cell 19, 131–147 31 Lewis, D.R. et al. (2007) Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes. Plant Cell 19, 1838– 1850 32 Wu, G. et al. (2007) Mutations in Arabidopsis multidrug resistance-like ABC transporters separate the roles of acropetal and basipetal auxin transport in lateral root development. Plant Cell 19, 1826–1837 33 Szabo, K. et al. (1997) Phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated ATPase activity. J. Biol. Chem. 272, 23165–23171 34 Santelia, D. et al. (2005) MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development. FEBS Lett. 579, 5399–5406 35 Terasaka, K. et al. (2005) PGP4, an ATP binding cassette Pglycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots. Plant Cell 17, 2922–2939 36 Holder, S. et al. (2007) Characterization of a potent and selective smallmolecule inhibitor of the PIM1 kinase. Mol. Cancer Ther. 6, 163–172 37 Russo, M. et al. (2003) Flavonoid quercetin sensitizes a CD95-resistant cell line to apoptosis by activating protein kinase Calpha. Oncogene 22, 3330–3342 38 Sheahan, J.J. and Rechnitz, G.A. (1992) Flavonoid-specific staining of Arabidopsis thaliana. Biotechniques 13, 880–883 39 Benjamins, R. et al. (2001) The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. Development 128, 4057–4067 40 Christensen, S.K. et al. (2000) Regulation of auxin response by the protein kinase PINOID. Cell 100, 469–478 41 Friml, J. et al. (2004) A PINOID-dependent binary switch in apicalbasal PIN polar targeting directs auxin efflux. Science 306, 862–865 42 Santner, A.A. and Watson, J.C. (2006) The WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis. Plant J. 45, 752–764 43 Deruere, J. et al. (1999) The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo. Plant J. 20, 389–399 44 Joo, J.H. et al. (2005) Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase. FEBS Lett. 579, 1243–1248 45 Winkel-Shirley, B. (2002) Biosynthesis of flavonoids and effects of stress. Curr. Opin. Plant Biol. 5, 218–223
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46 Buer, C.S. and Muday, G.K. (2004) The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light. Plant Cell 16, 1191–1205 47 Bennett, T. et al. (2006) The Arabidopsis MAX pathway controls shoot branching by regulating auxin transport. Curr. Biol. 16, 553–563 48 Lazar, G. and Goodman, H.M. (2006) MAX1, a regulator of the flavonoid pathway, controls vegetative axillary bud outgrowth in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 103, 472–476 49 Booker, J. et al. (2005) MAX1 encodes a cytochrome P450 family member that acts downstream of MAX3/4 to produce a carotenoidderived branch-inhibiting hormone. Dev. Cell 8, 443–449 50 Sorefan, K. et al. (2003) MAX4 and RMS1 are orthologous dioxygenaselike genes that regulate shoot branching in Arabidopsis and pea. Genes Dev. 17, 1469–1474 51 Buer, C.S. et al. (2006) Ethylene modulates flavonoid accumulation and gravitropic responses in roots of Arabidopsis. Plant Physiol. 140, 1384– 1396 52 Rashotte, A.M. et al. (2000) Basipetal auxin transport is required for gravitropism in roots of Arabidopsis. Plant Physiol. 122, 481–490 53 Malamy, J.E. and Ryan, K.S. (2001) Environmental regulation of lateral root initiation in Arabidopsis. Plant Physiol. 127, 899–909 54 Jain, A. et al. (2007) Differential effects of sucrose and auxin on localized phosphate deficiency-induced modulation of different traits of root system architecture in Arabidopsis. Plant Physiol. 144, 232– 247 55 Misson, J. et al. (2005) A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc. Natl. Acad. Sci. U. S. A. 102, 11934–11939 56 Perrin, R.M. et al. (2005) Gravity signal transduction in primary roots. Ann. Bot. (Lond.) 96, 737–743 57 Wolverton, C. et al. (2002) Root gravitropism in response to a signal originating outside of the cap. Planta 215, 153–157 58 Boonsirichai, K. et al. (2002) Root gravitropism: an experimental tool to investigate basic cellular and molecular processes underlying mechanosensing and signal transmission in plants. Annu. Rev. Plant Biol. 53, 421–447 59 Kimbrough, J.M. et al. (2004) The fast and transient transcriptional network of gravity and mechanical stimulation in the Arabidopsis root apex. Plant Physiol. 136, 2790–2805 60 Perera, I.Y. et al. (2006) A universal role for inositol 1,4,5trisphosphate-mediated signaling in plant gravitropism. Plant Physiol. 140, 746–760 61 Shin, H. et al. (2005) Complex regulation of Arabidopsis AGR1/PIN2mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases. Plant J. 42, 188–200
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62 Joo, J.H. et al. (2001) Role of auxin-induced reactive oxygen species in root gravitropism. Plant Physiol. 126, 1055–1060 63 Boonsirichai, K. et al. (2003) ALTERED RESPONSE TO GRAVITY is a peripheral membrane protein that modulates gravity-induced cytoplasmic alkalinization and lateral auxin transport in plant statocytes. Plant Cell 15, 2612–2625 64 Fasano, J.M. et al. (2001) Changes in root cap pH are required for the gravity response of the Arabidopsis root. Plant Cell 13, 907–921 65 Hou, G.C. et al. (2004) The promotion of gravitropism in Arabidopsis roots upon actin disruption is coupled with the extended alkalinization of the columella cytoplasm and a persistent lateral auxin gradient. Plant J. 39, 113–125 66 Li, J.S. et al. (2005) Arabidopsis H+-PPase AVP1 regulates auxinmediated organ development. Science 310, 121–125 67 Ostin, A. et al. (1998) Metabolism of indole-3-acetic acid in Arabidopsis. Plant Physiol. 118, 285–296 68 Kai, K. et al. (2007) Three oxidative metabolites of indole-3-acetic acid from Arabidopsis thaliana. Phytochemistry 68, 1651–1663 69 Delbarre, A. et al. (1998) Short-lived and phosphorylated proteins contribute to carrier-mediated efflux, but not to influx, of auxin in suspension-cultured tobacco cells. Plant Physiol. 116, 833–844 70 Rausher, M. (2006) The evolution of flavonoids and their genes. In The Science of Flavonoids (Grotewold, E., ed.), pp. 175–212, Springer 71 Markham, K. (1988) Distribution of flavonoids in the lower plants and its evolutionary significance. In The Flavonoids: Advances in Research Since 1980 (Harbourne, J., ed.), pp. 427–468, Chapman and Hall 72 Jiang, C.G. et al. (2006) Cloning and characterization of chalcone synthase from the moss, Physcomitrella patens. Phytochemistry 67, 2531–2540 73 Poli, D.B. et al. (2003) Auxin regulation of axial growth in bryophyte sporophytes: Its potential significance for the evolution of early land plants. Am. J. Bot. 90, 1405–1415 74 Cooke, T.J. et al. (2002) Evolutionary patterns in auxin action. Plant Mol. Biol. 49, 319–338 75 Petrasek, J. et al. (2006) PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312, 914–918 76 Zazimalova, E. et al. (2007) Polar transport of the plant hormone auxin – the role of PIN-FORMED (PIN) proteins. Cell. Mol. Life Sci. 64, 1621– 1637 77 Martinoia, E. et al. (2002) Multifunctionality of plant ABC transporters – more than just detoxifiers. Planta 214, 345–355 78 Ulmasov, T. et al. (1997) Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9, 1963–1971 79 Ottenschlager, I. et al. (2003) Gravity-regulated differential auxin transport from columella to lateral root cap cells. Proc. Natl. Acad. Sci. U. S. A. 100, 2987–2991
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Flavonoids and auxin transport: modulators or regulators? Wendy Ann Peer and Angus S. Murphy Department of Horticulture, 625 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47906, USA
Flavonoids are polyphenolic compounds found in all vascular and non-vascular plants. Although nonessential for plant growth and development, flavonoids have species-specific roles in nodulation, fertility, defense and UV protection. Flavonoids have been shown to modulate transport of the phytohormone auxin in addition to auxin-dependent tropic responses. However, flavonoids are not essential regulators of these processes because transport and tropic responses occur in their absence. Flavonoids modulate the activity of auxin-transporting P-glycoproteins and seem to modulate the activity of regulatory proteins such as phosphatases and kinases. Phylogenetic analysis suggests that auxin transport mechanisms evolved in the presence of flavonoid compounds produced for the scavenging of reactive oxygen species and defense from herbivores and pathogens. Auxin transport inhibitor studies The phytohormone auxin [indole-3-acetic acid (IAA)] is transported from its sites of synthesis in the apices to distal parts of the plant, where it influences growth and development [1]. Historically, auxin transport regulation has been investigated using both artificial and naturally occurring auxin transport inhibitors. One such class of natural inhibitors are flavonoids, of which the most effective subgroups are flavonols and isoflavones [2–4]. Flavonoids are a subgroup of phenylpropanoid compounds whose synthesis is tissue specific, developmentally regulated and dependent on environmental conditions, such as light and temperature [5]. Flavonoids, such as quercetin, kaempferol, apigenin and other aglycone molecules synthesized in the first steps of the flavonoid biosynthetic pathway (Figure 1), have been shown to inhibit polar auxin transport and to enhance consequent localized auxin accumulation in planta. Flavonoids have been shown to displace artificial auxin efflux inhibitors, including 1-N-naphthylphthalamic acid (NPA), from plasma membrane and microsomal binding sites [2,6–8]. NPA displacement from microsomal membranes by flavonoids is biphasic, indicating that there are at least two in planta targets of flavonoid activity [2]. This observation is also consistent with the identification of peripheral membrane actin-associated [9,10] and integral plasma membrane [8] NPA binding sites in Cucurbita pepo. Subsequently, in Arabidopsis, the integral membrane NPA–flavonoid Corresponding author: Peer, W.A. ([email protected]). www.sciencedirect.com
interaction site was shown to be associated with three integral plasma membrane phosphoglycoproteins (PGPs; At2g36910, At2g4700, At3g28860) that transport IAA, whereas a weaker interaction site was associated with at least one peripheral membrane protein, including a membrane-anchored M1 aminopeptidase, (APM1, At4g33090), and the FK506 binding protein immunophilin TWISTED DWARF1 (TWD1, At5g42800) [11,12]. Mutational analyses with transparent testa mutants identify sites of flavonoid action Three Arabidopsis transparent testa (tt) mutants, which lack key enzymes in the flavonoid biosynthetic pathway, were used to determine whether endogenous flavonoids regulate auxin transport in planta: tt4 (a chalcone synthase, CHS, At5g13930, mutant accumulating no flavonoids); tt7 (a flavonoid-30 -hydroxylase, At5g07990, mutant accumulating excess kaempferol); and tt3 (a dihydroflavanol-4-reductase, At5g42800, mutant accumulating excess kaempferol and quercetin) [13,14] (Figure 1). Transport assays are usually performed in five-day-old seedlings, when maximal accumulation of aglycone flavonoids is observed [13,14]. In the tt4 mutant, auxin transport from the shoot to root was found to be elevated compared with the wild-type, whereas the flavonol-overproducing mutants tt7 and tt3 exhibited reduced auxin transport [2–4]. Wild-type auxin transport levels and tissue-specific flavonol accumulation patterns could be restored in tt4 when the lesion in CHS was bypassed by treatment with the downstream product naringenin [2,3] (Figure 1). Furthermore, addition of a naringenin droplet to the shoot apex of wild-type or tt4 seedlings reduced auxin transport levels in shoots and roots to levels similar to those seen in tt7 and tt3 [4]. In addition to auxin transport, lateral root number and length, which are modulated by auxin transport levels, also increased in tt4 seedlings [3]. Together with the tissue-specific accumulation of flavonols in Arabidopsis seedlings that coincided with regions of higher auxin accumulation [2,4], these data suggested that flavonols affect polar auxin transport in apical tissues by modulating the amount of auxin loaded into the long-distance polar auxin transport stream [4]. Manipulation of phenylpropanoid biosynthesis affects auxin transport There are several phenylpropanoid classes, such as cinnamic acids, flavonoids and lignins, that share phenylalanine as a precursor and whose biosynthesis is feedback regulated at various branch points in the metabolic pathway [15]. Recent efforts to use gene silencing to alter floral scent
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Figure 1. Phenylpropanoid biosynthesis. This brief outline of the phenylpropanoid pathway includes biosynthetic proteins and products referenced in the text. Double arrows indicate multiple steps. Abbreviations: BPBT, benzoyltransferase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol-4-reductase; F3H, flavone 3-hydroxylase; F30 H, flavonoid 30 -hydroxylase; FLS, flavonol synthase; HCT, hydroxy cinnamoyl transferase. The portion of pathway in the box highlights the only flavonoid compounds (flavanones, flavones and flavonols) observed among bryophytes.
in Petunia x hybrida or lignin production in Arabidopsis had the unintended result of altering auxin transport and phenotypes that are consistent with such alterations [16,17]. Silencing of the gene encoding the volatile benzenoid biosynthetic enzyme benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase (BPBT, AY563157) in Petunia resulted in 4.1-fold accumulation of flavonoids and transcinnamic acid [an early metabolite which might interfere with auxin signalling (Figure 1)] in flowers [16]. The transgenic petunia plants were 1.75-fold larger than wild type, suggesting an auxin-related effect. Enhanced flavonoid accumulation in transgenic Petunia plants was found in seeds and at the seedling root–shoot junction but not in the shoot apex or other tissues where flavonols are most likely to affect auxin transport. However, the BPBT-silenced lines had expanded vascular tissue, perhaps resulting from enhanced auxin movement during embryogenesis, in which auxin is required for vascular differentiation. The observed increases in auxin transport and auxin-dependent growth in mature plants seemed to be a result of this early developmental effect. Such indirect effects on auxin transport have been observed in other cases in which components of cellular trafficking mechanisms (e.g. Vesicle Transport V-snare 11 (VTI11), At5g39510) or auxin-conducting vascular tissues (e.g. Interfascicular Fiberless 1 (IFL1), At5g60690) are mutated [18,19]. By contrast, silencing of the gene encoding the lignin biosynthetic enzyme hydroxy cinnamoyl transferase (HCT, www.sciencedirect.com
At5g48930) increased flavonoid accumulation (mainly kaempferol derivatives), reduced auxin transport, altered vasculature (including reduced lignification) and dwarfed plants in Arabidopsis [17]. When the HCT-silenced lines were crossed with CHS-silenced lines, auxin transport and plant height were restored to wild-type levels. In this case, increased flavonoid accumulation had a greater impact on auxin transport than did changes in the plant vasculature. Increased flavonoid accumulation has been observed even when a genetic modification is not directed at phenylpropanoid manipulation [20]. Altered vascular development, reduced hypocotyl and epicotyl length, and reduced auxin transport were observed in tobacco transformed with a tumorigenic gene 6b (D30626) from Agrobacterium tumefaciens [20]. Induced plants had increased amounts of a kaempferol derivative, suggesting that 6b expression inhibits auxin transport by increasing production of this flavonoid compound [20]. These examples indicate that tissue-specific changes in flavonoid accumulation have a direct effect on auxin transport, whereas modifications in other aspects of phenylpropanoid metabolism that affect vascular architecture indirectly affect auxin transport. Inhibition of auxin transport requires active flavonoids in the correct compartment The activity of flavonoids themselves can be modulated. Sucrose induces expression of CHS and other
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phenylpropanoid genes [21] but increasing sucrose concentrations also decreases the levels of early flavonoid products that inhibit auxin transport and increase glycosylated late pathway flavonoids, such as anthocyanins, that have little or no impact on transport [14,21] (Figure 1). UDPglucuronosyltransferase 1 (UGT1, At1g05560) is thought reversibly to catalyze the conversion of a flavonoid from an active aglycone to an inactive glucuronic acid form [22,23]. Glycosylation also reduces the antioxidant activity of quercetin [24]. In addition, flavonoid activity can be modulated through oxidation, as described in a recent review [25]. Compartmentalization in endomembranes or organelles has also been found to modulate active flavonoid levels because the addition of sucrose induces flavonoid accumulation in mutants lacking the peroxisomal COMATOSE (At4g39850) ABCD transporter that is required for flavonoid biosynthesis during germination [26]. Molecular targets of flavonoid regulation Experimental evidence indicates that flavonoid modulation of auxin transport is tissue specific and occurs at the root and shoot apices. Therefore, the molecular targets of flavonoid interaction should also be localized in these regions. Auxin transporters Several targets of flavonols that are involved in auxin transport mechanisms have been proposed [4,27]. Of these, interactions between flavonols and the PGP auxin transporters PGP1, PGP4 and PGP19 (also known as MDR1) are the best characterized. PGPs belong to the ATP-binding cassette subfamily B (ABCB) transporter family, which hydrolyses ATP to transport substrates. Comparisons of in planta auxin transport assays in tt and pgp mutants, PGP gene expression patterns and subcellular localization of PGP proteins [28–32], and flavonoid inhibition of PGPmediated auxin transport in heterologous systems [28–30] suggest that aglycone flavonols modulate PGP1, PGP4 and PGP19 activity at the root and shoot apices [4]. PGP activity in mammals is regulated through phosphorylation, inhibition of ATPase activity or allosteric binding [33] and flavonoid modulation of PGP1, PGP4 and PGP19 activity in planta might target the same sites [11,29]. Flavonoids might also modulate auxin uptake because the root-specific transporter PGP4 seems to be able to mediate cellular auxin uptake or efflux according to the cellular environment [34,35]. In heterologous systems, PGP4 activity is also NPA reversible and flavonoid sensitive [35]. Recently, PGP4, which functions primarily in moving auxin away from the root distal elongation zone [31,34,35], was shown to be epistatic to CHS in pgp4 tt4 double knockouts with regard to gravitropic bending [31] (see section on tropism later), which is consistent with PGP4 being a target of flavonoid modulation. The effects of flavonoids, if any, on the auxin influx protein AUX1 (At2g38120) have not been demonstrated. The effects of flavonoids on members of the PIN-Formed auxin efflux carriers have received less attention. Data so far suggest that changes in PIN1 (At1g73590) and PIN2 (At5g57090) expression and subcellular protein localization observed in flavonoid mutants can be attributed www.sciencedirect.com
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primarily to altered local auxin concentrations, rather than a direct flavonoid interaction [4]. The flavonoiddeficient tt4 mutant exhibits enhanced auxin transport and free IAA levels but changes in PIN1 gene expression and PIN1 protein localization in the mutant can be mimicked by artificially increasing auxin fluxes [4]. Similarly, PIN1 expression and protein localization in tt7 and tt3 were similar to wild-type plants treated with auxin transport inhibitors – again, indicating an indirect flavonoid effect [4]. By contrast, although PIN2 expression and protein signals were slightly enhanced, protein localization was not altered in tt4, and no differences were observed in tt7 or tt3 [4]. Transport assays with PIN proteins heterologously expressed in plant, fungal and mammalian cell systems suggest that flavonoids inhibit the activity of other transporters – for example, PGPs and organic anion transporters (which might be regulated by PINs) – rather than interacting with PINs directly [29,30]. However, flavonoids do seem to affect PIN4 (At2g01420) expression or subcellular protein localization both indirectly by altering localized auxin concentrations and directly by affecting cellular trafficking [4]. Cellular trafficking, auxin transport regulators and phosphorylation Experimental data show that flavonoids also modulate auxin transport through interactions with regulatory proteins. For example, inclusion of exogenous flavonols in a washout solution following treatment with the cellular trafficking inhibitor brefeldin A did not disrupt restoration of PIN1 to the plasma membrane in wild-type Arabidopsis; however, in tt4 cells never exposed to flavonols, after the same treatment, PIN1 was not restored to the wild-type subcellular localization pattern [4]. Micromolar concentrations of flavonols are routinely used as both phosphatase and kinase inhibitors [36,37], so kinases and phosphatases associated with auxin transport are probable targets in apical tissues, in which cellular flavonol levels are high [4,38]. The serine/threonine kinase PINOID (At2g34650) [39,40], which regulates PIN1, PIN2 and PIN4 activity by regulating the polarity of their subcellular localization on the plasma membrane [41], is the most likely candidate, followed by the PINOID-related WAG kinases (At1g53700, At3g14370) [42]. Flavonoid modulation of the protein or lipid phosphorylation state through inhibition of the protein phosphatase 2A subunit ROOTS CURL IN NPA1 (RCN1, At1g25490) [43] or the phosphatidylinositol 3,4,5-trisphosphate kinase (e.g. At1g21980) [44] is also possible. Flavonoids might also influence cellular trafficking by altering membrane fluidity directly because the more hydrophobic flavonoids associate with and intercalate into membranes, making them more rigid or fluid, depending on the sterol content of the membrane, which itself is cell-type specific [27]. In summary, flavonoids modulate auxin transport directly and indirectly by affecting the activity of auxin transporters, proteins that regulate the transporters and trafficking of the components of the auxin transport complex, and by altering the structure of auxin-transporting tissues.
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Flavonoids and physiological responses Fundamental processes, such as auxin transport, gravitropism and phototropism occur in the absence of flavonoids [45]. Therefore, flavonoids are best viewed as modulators of transport processes, rather than as essential regulators. In some cases, flavonoids seem to be synthesized in response to auxin accumulation, presumably to scavenge reactive oxygen species (ROS) generated during auxin catabolism [27]. In the absence of PGP1, PGP4 or PGP19, a localized increase in auxin concentrations occurs, resulting in localized quercetin accumulation similar to that in the wild type after treatment with the auxin transport inhibitor NPA [4,28,35]. Because technical issues make it problematic to identify the exact timing of auxin and flavonol accumulation (Box 1), it is difficult to separate the different roles of localized flavonol accumulation, as seen, for example, in regions of shoot branching [14] or gravitropic bending [46] (Box 2). Shoot and root branching Flavonols are localized in the apical and nodal regions of Arabidopsis inflorescence stems, whereas anthocyanins, which do not inhibit auxin transport, decrease in concentration from the base of the stem towards the apex [14]. Increased auxin transport in tt4 inflorescences might contribute to the increased secondary inflorescence formation observed for one tt4 allele (2YY6) [3]. However, tt4 (2YY6) was subsequently found to harbour a mutation in More Axillary Branches 4 (At4g32810) [47]. MAX1 (At2g26170), a cytochrome P450 that functions downstream of the other MAX genes, is a positive regulator of flavonoid biosynthetic genes and is required for flavonol synthesis in the axillary bud [48]. The MAX genes were identified as regulators of auxin-dependent lateral inflorescence branching [47,49]. The tt4 max4 double knockout exhibits the increased shoot branching observed in max4 [3,47,50], indicating that MAX4 is epistatic to CHS in inflorescences. The max1 phenotype can be rescued by application of kaempferol, quercetin or naringenin, but MAX regulation of auxin transport is dependent on PINs and indirectly on flavonoid activity [47,48]. Flavonols accumulate in regions of lateral root emergence and along the lateral roots themselves in patterns similar to those indicated by auxin reporter genes, including DR5 [14,31]. Although tt4 has an increased number of lateral roots compared with wild type [3], independent of MAX4 [51], this seems to be an indirect effect of increased transport of shoot-derived auxin [52]. The contribution of flavonoids to lateral root development must also be evaluated in the context of macronutrients [53,54]. Flavonoid synthesis is induced two- to four-fold in phosphate-starved plants [55], with a concomitant increase in lateral root number [54]. Under phosphate starvation, tt4 showed an increased number of lateral roots, indicating a role for flavonoid modulation of root architecture [54]. Tropic bending Tropic responses do not require flavonoids because these responses are observed in dark-grown seedlings which are flavonoid deficient. However, flavonoids seem to modulate the rate and extent of gravitropic responses in roots. In www.sciencedirect.com
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Box 1. Visualization of flavonoids The resolution of in situ flavonoid visualization depends on the sensitivity of the microscope used. A low concentration of Triton X100 (a detergent) can be used to move diphenyl boric acid 2aminoethyl ester (DPBA, a fluorescent dye that interacts with flavonoids), into the five-day-old wild-type roots to visualize flavonols using epifluorescence microscopy [14] (Figure Ia). With more-sensitive detection equipment, solubilizing agents are no longer necessary. Using 5 mM DPBA in aqueous solution and spectral scanning with a Zeiss LSM 510 META (405 nm diode laser) confocal microscope and post-processing software, subcellular localization of the flavonols kaemperfol and quercetin can be observed (Figure Ib,c). The subcellular localization can be placed in context with the use of other marker dyes – for example, FM4–64 for plasma membrane labeling and endocytotic uptake (Figure Id–f) and green fluorescent protein fusion marker proteins for various compartments – for example, endoplasmic reticulum, Golgi, an assortment of endosomes and tonoplast. However, using dye for staining has limitations because the dye inactivates flavonols and can take up to two minutes to produce a visible signal. Instantaneous detection that does not interfere with activity is a technical challenge that has yet to be solved for localized concentrations of flavonoids.
Figure I. Visualization of flavonoids. (a) quercetin is shown in gold. (b,c) Quercetin is shown in blue, kaempferol in red and the overlap in purple (more quercetin) and magenta (more kaempferol). Note: merged images are presented. (d) FM4–64 is shown in red, (e) quercetin in blue and (f) the overlap in magenta. Size bars, 20 mm. Panel (a): reproduced, with permission, from http://ww.plantphysiol.org).
gravistimulated roots, auxin is redistributed laterally to root cap and elongation zone cells on the lower side of the root, with the result that elongation on the lower side is inhibited and the root bends downward [56]. The initial mechanism of lateral redistribution of auxin in root tips
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Box 2. Split-root system for tropism measurements Analysing flavonoid regulation of lateral auxin redistribution in graviresponding tissues requires monitoring of multiple dynamic processes. Gravistimulation is thought to produce rapid changes in the concentrations of small ions within the lateral root cap and these can be monitored in real-time using dyes and fluorescent protein fusions [29,41,44,60–66]. Changes in flavonol concentrations in Arabidopsis roots can also be visualized, although dynamic imaging remains problematic (Box 1). In situ monitoring of changes in auxin gradients presents an even greater challenge because such visualizations are largely dependent on the use of auxin-responsive transcriptional reporters [78,79]. Use of these reporters is limited by sensitivity of the reporters, the latency of auxin-dependent gene transcription and the persistence of the reporter proteins after auxin levels have subsided. Attempts to create fluorescent auxin reporter proteins that respond instantaneously to changes in auxin concentrations have been hampered by a lack of specificity and aberrant growth resulting from loss of the signalling molecule after it binds to the sensors in vivo. A carbon nanotube auxin-sensing electrode has been used to monitor transport in Arabidopsis roots [34] but this sensor lacks the sensitivity required for lateral auxin redistribution studies at this time. Microscale assays using radiolabeled IAA can be used to track rapid changes in auxin movement, although these assays require
Figure I. Split-root system for tropism measurements.
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destructive sampling techniques. The assays use discontinuous solid support media and microscopically guided precision instruments to place microdroplets (nL) of auxin on discrete tissues and to excise samples (for methods, see http://www.hort.purdue.edu/hort/ research/murphy/protframe.htm). Use of larger (mL) volumes or higher concentrations of auxin produces aberrant or nonspecific results [28,30]. In the example of this type of assay diagrammed here (Figure I), intact Arabidopsis seedling roots were attached to solid support media using surgical glue just before conducting the assay. A 6 nL droplet of 3H-IAA was then placed on the lower columella cells and seedlings were incubated upright or with a 908 rotation. At the time points indicated, the lateral root cap was excised with an obsidian surgical blade and discarded. A 2 mm root section was then excised, divided transversely and collected as two separate sections. Sections from ten seedlings were collected and counted. Using this method, changes in the lateral redistribution of transported auxin could be detected within 30 minutes. Similar results were obtained when a microlaser cutter was used for sectioning. In this case, differences between wild type and tt4 lateral auxin redistribution are not evident until after 75 minutes, suggesting that the initial lateral redistribution of auxin in graviresponding root tips is not regulated by flavonoids.
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has not been elucidated and might occur independently of an auxin gradient [57,58]. However, the rapidity of the response suggests that factors other than transcriptional responses [59] are involved. Calcium, inositol 1,4,5-trisphosphate [44,60], phosphorylation events [61], ROS generation [44,62], pH gradients [63–66], interference with ATPase activity [29] and polar realignment of PIN auxin efflux proteins with the auxin gradient [41,61] have all been implicated in the process. Flavonoid accumulation increases slightly and symmetrically in the root cap and cells of columella gravistimulated roots [46]. Gravitropic bending in the flavonoid-deficient tt4 mutant is delayed compared with wild type [31,46]. However, increases in the auxin content of cells in the elongation zone or the lower side of the gravistimulated roots is seen in both tt4 and wild type [46]. One explanation for this difference was suggested when Lewis et al. [31] demonstrated that pgp4 interaction is epistatic to tt4. The pgp4 mutant exhibits a 30% decrease in auxin transport from the columella [35] and a 50% decrease in auxin transport from the general apical region of the root [31] but also faster rather than slower gravitropic bending [31]. Expression and protein localization studies show that the highest levels of PGP4 abundance are in the elongation zone and epidermis of the mature root [35]. Taken together, these data suggest that PGP4 is a target of flavonoid regulation and functions primarily in the movement of auxin out of the elongation zone. Flavonoids normally partially inhibit PGP4-mediated movement of auxin out of the distal elongation zone, and loss of flavonoids results in a reduction in inhibitory auxin concentrations in this region. An increase in flavonoid levels or loss of PGP4 function results in a more rapid auxin accumulation and enhancement of bending. These results also suggest that flavonoid modulation of auxin transport mediated by PIN and PGP transporters within the lateral root cap is secondary. A split-root assay developed to isolate asymmetric delivery of auxin from the columella and lateral root cap to the elongation zone shows that no difference between wild type and tt4 is seen in the initial asymmetric auxin movement into the elongation zone (Box 2), consistent with a primary flavonoid function in modulating PGP-mediated auxin movement out of the elongation zone. The timing of the events suggests that flavonoids have an additional role that enhances tropic responses. Auxin accumulation can also produce flavonoid accumulation. Gravistimulated roots exhibiting localized auxin accumulation demonstrate increased flavonoid accumulation [46]. Mutations in PGP auxin transport genes result in ectopic flavonoid accumulation [35] that can be mimicked with exogenous auxin application [4,46]. One way that flavonoids might modulate tropic responses is through scavenging ROS because an oxidative burst seems to be an early signal to stimulate gravitropism in the root cap and columella cells [44,62], and the flavonol quercetin, an excellent ROS scavenger, increases in the columella response to auxin accumulation [4]. After the ROS signal is initiated, flavonoids might quench this signal so that it does not continue to stimulate a response. Oxidative catabolism of laterally accumulated IAA trapped in the elongation zone [28,35] also seems to generate ROS [27,67,68]. Flavonoid www.sciencedirect.com
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quenching of ROS generated in this manner would prevent cellular oxidative damage [25]. Phosphorylation might also have a role in graviresponses [69]. In addition to modulating the phosphorylation of PGPs, flavonoids might also mediate the serine/ threonine kinases PINOID and WAG. PIN2 reorientation is observed in the PID overexpressor mutants [41]. Although PIN2 localization was not affected by protein phosphatase inhibitors [61] or in tt4 [4], PIN2-mediated gravitropism is regulated by protein phosphatases that are biochemically similar to RCN1 [61]. The role of flavonoids in gravitropism is most probably a combination of factors: modulation of PGP activity, ROS quenching and retardation of phosphorylation events. Flavonoid modulation of physiological responses is tissue and process specific, involving mechanisms that evolved over time. Systems biology approaches utilizing mutants and precise studies of auxin movement out of the lateral root cap (such as those shown in Box 2) are required to develop a more complete model. Evolution of flavonoid modulation of auxin transport Flavonoid synthesis is observed in extant members of the first land plants but not in algae [70]. Unlike angiosperms, gymnosperms and pteridophytes, bryophytes only accumulate flavonoids synthesized by the biosynthetic genes early in the pathway (Figure 1), and the moss (Physcomitrella patens) genome does not seem to contain the genes required for the later steps in flavonoid synthesis, despite the presence of 19 CHS-like genes [71,72]. Bryophytes exhibit polar growth and have different auxin transport mechanisms within each bryophyte division, ranging from NPA-insensitive diffusion to NPA-sensitive polar auxin transport [73,74]. Poli et al. [73] conclude that auxin transport developed independently in each extant bryophyte lineage. Because polar IAA transport in some bryophytes is NPA sensitive, one hypothesis is that polar auxin transport and auxin transporters, such as the PINs and, to a lesser extent, PGPs, evolved in the presence of flavonols because all bryophytes examined accumulate flavonols. The activity of PGPs is inhibited by flavonoids; however, plant PGP activity is less sensitive to flavonoids than are mammalian transporters because flavonoids are endogenous in plants [28,29,75]. PINs evolved more recently than PGPs [76,77] and exhibit less apparent sensitivity to flavonoids, perhaps as a result of undergoing much of their evolution into auxin transporters in the presence of flavonoids. By contrast, PGPs are part of the ancient ABC transporter lineage [77] and flavonoids inhibit their essential ATPase activity [28,29]. Therefore, it is unsurprising that PGPs are more sensitive to flavonoids than are PINs. Thus, we might regard auxin transport proteins and their regulators as cellular components that have been adapted to function with greater efficiency in a cellular environment that includes flavonoid compounds, rather than as specific targets of flavonoid regulation. Concluding remarks and future perspectives Flavonoids modulate auxin transport through direct and indirect interactions with cellular transport and regulatory mechanisms. Developmental regulation of flavonoid
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biosynthesis also determines where and when these interactions take place. This makes it difficult to ascertain when flavonoids are functioning primarily as ROS scavengers, defense compounds, regulators of phosphorylation, enzyme inhibitors or modulators of membrane fluidity. An evolutionary approach using natural variation among the bryophytes could provide a context for assessing the specificity of flavonoid regulation. It seems that as the complexity of flavonoid biosynthesis increased during plant evolution, flavonoid function also expanded. A systems biology approach comparing bryophytes and tracheophytes could be used to identify which flavonoid functions are the most fundamental and which are the most divergent. When compared with the evolution of auxin transport mechanisms, those processes that evolved in the presence of flavonoids could then be differentiated from those that are more specifically regulated by these compounds. Acknowledgements This work was supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council (BBSRC). We thank Simon Gilroy and John Courage for suggesting the Box 2 experiment.
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