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

Review TRENDS in Plant Science Vol.12 No.12

Flavonoids are polyphenolic compounds found in allvascular and non-vascular plants. Although nonessentialfor plant growth and development, flavonoids havespecies-specific roles in nodulation, fertility, defenseand UV protection. Flavonoids have been shown tomodulate transport of the phytohormone auxin inaddition to auxin-dependent tropic responses. However,flavonoids are not essential regulators of these pro-cesses because transport and tropic responses occurin their absence. Flavonoids modulate the activity ofauxin-transporting P-glycoproteins and seem to modu-late the activity of regulatory proteins such as phospha-tases and kinases. Phylogenetic analysis suggests thatauxin transport mechanisms evolved in the presence offlavonoid compounds produced for the scavenging ofreactive oxygen species and defense from herbivoresand pathogens.

Auxin transport inhibitor studiesThe phytohormone auxin [indole-3-acetic acid (IAA)] istransported from its sites of synthesis in the apices todistal parts of the plant, where it influences growth anddevelopment [1]. Historically, auxin transport regulationhas been investigated using both artificial and naturallyoccurring auxin transport inhibitors. One such class ofnatural inhibitors are flavonoids, of which the most effec-tive subgroups are flavonols and isoflavones [2–4].

Flavonoids are a subgroup of phenylpropanoid com-pounds whose synthesis is tissue specific, developmentallyregulated and dependent on environmental conditions,such as light and temperature [5]. Flavonoids, such asquercetin, kaempferol, apigenin and other aglycone mol-ecules synthesized in the first steps of the flavonoid bio-synthetic pathway (Figure 1), have been shown to inhibitpolar auxin transport and to enhance consequent localizedauxin accumulation in planta. Flavonoids have beenshown to displace artificial auxin efflux inhibitors, in-cluding 1-N-naphthylphthalamic acid (NPA), from plasmamembrane and microsomal binding sites [2,6–8]. NPAdisplacement from microsomal membranes by flavonoidsis biphasic, indicating that there are at least two in plantatargets of flavonoid activity [2]. This observation is alsoconsistent with the identification of peripheral membraneactin-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. (peerw@purdue.edu).

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interaction site was shown to be associated with threeintegral plasma membrane phosphoglycoproteins (PGPs;At2g36910, At2g4700, At3g28860) that transport IAA,whereas a weaker interaction site was associated with atleast one peripheral membrane protein, including a mem-brane-anchored M1 aminopeptidase, (APM1, At4g33090),and the FK506 binding protein immunophilin TWISTEDDWARF1 (TWD1, At5g42800) [11,12].

Mutational analyses with transparent testa mutantsidentify sites of flavonoid actionThreeArabidopsis transparent testa (tt)mutants,which lackkey enzymes in the flavonoid biosynthetic pathway, wereused to determine whether endogenous flavonoids regulateauxin transport in planta: tt4 (a chalcone synthase, CHS,At5g13930, mutant accumulating no flavonoids); tt7 (a fla-vonoid-30-hydroxylase, At5g07990, mutant accumulatingexcess kaempferol); and tt3 (a dihydroflavanol-4-reductase,At5g42800, mutant accumulating excess kaempferol andquercetin) [13,14] (Figure 1). Transport assays are usuallyperformed infive-day-old seedlings,whenmaximal accumu-lation of aglycone flavonoids is observed [13,14]. In the tt4mutant, auxin transport from the shoot to root was found tobe elevated compared with the wild-type, whereas the fla-vonol-overproducing mutants tt7 and tt3 exhibited reducedauxin transport [2–4]. Wild-type auxin transport levels andtissue-specific flavonol accumulation patterns could berestored in tt4 when the lesion in CHS was bypassed bytreatment with the downstream product naringenin [2,3](Figure 1). Furthermore, addition of a naringenin droplet tothe shoot apex of wild-type or tt4 seedlings reduced auxintransport levels in shoots and roots to levels similar to thoseseen in tt7 and tt3 [4]. In addition to auxin transport, lateralroot number and length, which are modulated by auxintransport levels, also increased in tt4 seedlings [3]. Togetherwith the tissue-specific accumulation of flavonols in Arabi-dopsis seedlings that coincided with regions of higher auxinaccumulation [2,4], these data suggested that flavonolsaffect polar auxin transport in apical tissues by modulatingthe amount of auxin loaded into the long-distance polarauxin transport stream [4].

Manipulation of phenylpropanoid biosynthesis affectsauxin transportThere are several phenylpropanoid classes, such ascinnamic acids, flavonoids and lignins, that share phenyl-alanine as a precursor and whose biosynthesis is feedbackregulatedatvariousbranchpoints in themetabolic pathway[15]. Recent efforts to use gene silencing to alter floral scent

d. doi:10.1016/j.tplants.2007.10.003

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; F30H, 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.

Review TRENDS in Plant Science Vol.12 No.12 557

inPetunia x hybrida or lignin production inArabidopsishadthe unintended result of altering auxin transport and phe-notypes that are consistent with such alterations [16,17].Silencing of the gene encoding the volatile benzenoid bio-synthetic enzyme benzoyl-CoA:benzyl alcohol/phenyletha-nol benzoyltransferase (BPBT, AY563157) in Petuniaresulted in 4.1-fold accumulation of flavonoids and trans-cinnamic acid [an early metabolite which might interferewith auxin signalling (Figure 1)] in flowers [16]. The trans-genic petunia plants were 1.75-fold larger than wild type,suggesting an auxin-related effect. Enhanced flavonoidaccumulation in transgenic Petunia plants was found inseeds and at the seedling root–shoot junction but not inthe shoot apex or other tissues where flavonols are mostlikely toaffectauxintransport.However, theBPBT-silencedlines had expanded vascular tissue, perhaps resulting fromenhanced auxinmovement during embryogenesis, in whichauxin is required for vascular differentiation. The observedincreases inauxin transport andauxin-dependentgrowth inmature plants seemed to be a result of this early develop-mental effect. Such indirect effects on auxin transport havebeen observed in other cases inwhich components of cellulartrafficking mechanisms (e.g. Vesicle Transport V-snare 11(VTI11), At5g39510) or auxin-conducting vascular tissues(e.g. Interfascicular Fiberless 1 (IFL1), At5g60690) aremutated [18,19].

By contrast, silencing of the gene encoding the ligninbiosynthetic enzyme hydroxy cinnamoyl transferase (HCT,

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At5g48930) increased flavonoid accumulation (mainlykaempferol derivatives), reduced auxin transport, alteredvasculature (including reduced lignification) and dwarfedplants in Arabidopsis [17]. When the HCT-silenced lineswere crossed with CHS-silenced lines, auxin transport andplant height were restored to wild-type levels. In this case,increased flavonoid accumulation had a greater impact onauxin transport than did changes in the plant vasculature.

Increased flavonoid accumulation has been observedeven when a genetic modification is not directed at phe-nylpropanoid manipulation [20]. Altered vascular devel-opment, reduced hypocotyl and epicotyl length, andreduced auxin transport were observed in tobacco trans-formed with a tumorigenic gene 6b (D30626) from Agro-bacterium tumefaciens [20]. Induced plants had increasedamounts of a kaempferol derivative, suggesting that 6bexpression inhibits auxin transport by increasing pro-duction of this flavonoid compound [20]. These examplesindicate that tissue-specific changes in flavonoid accumu-lation have a direct effect on auxin transport, whereasmodifications in other aspects of phenylpropanoid metab-olism that affect vascular architecture indirectly affectauxin transport.

Inhibition of auxin transport requires activeflavonoids in the correct compartmentThe activity of flavonoids themselves can be modulated.Sucrose induces expression of CHS and other

558 Review TRENDS in Plant Science Vol.12 No.12

phenylpropanoid genes [21] but increasing sucroseconcentrations also decreases the levels of early flavonoidproducts that inhibit auxin transport and increase glycosy-lated late pathway flavonoids, such as anthocyanins, thathave little orno impact on transport [14,21] (Figure1).UDP-glucuronosyltransferase 1 (UGT1, At1g05560) is thoughtreversibly to catalyze the conversion of a flavonoid from anactive aglycone to an inactive glucuronic acid form [22,23].Glycosylation also reduces the antioxidant activity of quer-cetin [24]. In addition, flavonoid activity can be modulatedthrough oxidation, as described in a recent review [25].Compartmentalization in endomembranes or organelleshas also been found to modulate active flavonoid levelsbecause the addition of sucrose induces flavonoid accumu-lation in mutants lacking the peroxisomal COMATOSE(At4g39850) ABCD transporter that is required for flavo-noid biosynthesis during germination [26].

Molecular targets of flavonoid regulationExperimental evidence indicates that flavonoid modu-lation of auxin transport is tissue specific and occurs atthe root and shoot apices. Therefore, the molecular targetsof flavonoid interaction should also be localized in theseregions.

Auxin transporters

Several targets of flavonols that are involved in auxintransport mechanisms have been proposed [4,27]. Of these,interactions between flavonols and the PGP auxin trans-porters PGP1, PGP4 and PGP19 (also known asMDR1) arethe best characterized. PGPs belong to the ATP-bindingcassette subfamily B (ABCB) transporter family, whichhydrolyses ATP to transport substrates. Comparisons ofin planta auxin transport assays in tt and pgp mutants,PGP gene expression patterns and subcellular localizationof PGP proteins [28–32], and flavonoid inhibition of PGP-mediated auxin transport in heterologous systems [28–30]suggest that aglycone flavonols modulate PGP1, PGP4 andPGP19 activity at the root and shoot apices [4]. PGPactivity in mammals is regulated through phosphoryl-ation, inhibition of ATPase activity or allosteric binding[33] and flavonoid modulation of PGP1, PGP4 and PGP19activity in planta might target the same sites [11,29].

Flavonoids might also modulate auxin uptake becausethe root-specific transporter PGP4 seems to be able tomediate cellular auxin uptake or efflux according to thecellular environment [34,35]. In heterologous systems,PGP4 activity is also NPA reversible and flavonoid sensi-tive [35]. Recently, PGP4, which functions primarily inmoving auxin away from the root distal elongation zone[31,34,35], was shown to be epistatic to CHS in pgp4 tt4double knockouts with regard to gravitropic bending [31](see section on tropism later), which is consistent withPGP4 being a target of flavonoid modulation. The effectsof flavonoids, if any, on the auxin influx protein AUX1(At2g38120) have not been demonstrated.

The effects of flavonoids onmembers of the PIN-Formedauxin efflux carriers have received less attention. Data sofar suggest that changes in PIN1 (At1g73590) and PIN2(At5g57090) expression and subcellular protein localiz-ation observed in flavonoid mutants can be attributed

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primarily to altered local auxin concentrations, ratherthan a direct flavonoid interaction [4]. The flavonoid-deficient tt4 mutant exhibits enhanced auxin transportand free IAA levels but changes in PIN1 gene expressionand PIN1 protein localization in the mutant can bemimicked by artificially increasing auxin fluxes [4]. Sim-ilarly, PIN1 expression and protein localization in tt7 andtt3 were similar to wild-type plants treated with auxintransport inhibitors – again, indicating an indirect flavo-noid effect [4]. By contrast, although PIN2 expression andprotein signals were slightly enhanced, protein localizationwas not altered in tt4, and no differences were observed intt7 or tt3 [4]. Transport assays with PIN proteins hetero-logously expressed in plant, fungal and mammalian cellsystems suggest that flavonoids inhibit the activity of othertransporters – for example, PGPs and organic anion trans-porters (which might be regulated by PINs) – rather thaninteracting with PINs directly [29,30]. However, flavonoidsdo seem to affect PIN4 (At2g01420) expression or subcel-lular protein localization both indirectly by altering loca-lized auxin concentrations and directly by affecting cellulartrafficking [4].

Cellular trafficking, auxin transport regulators and

phosphorylation

Experimental data show that flavonoids also modulateauxin transport through interactions with regulatoryproteins. For example, inclusion of exogenous flavonolsin a washout solution following treatment with the cellulartrafficking inhibitor brefeldin A did not disrupt restorationof PIN1 to the plasmamembrane in wild-type Arabidopsis;however, in tt4 cells never exposed to flavonols, after thesame treatment, PIN1 was not restored to the wild-typesubcellular localization pattern [4]. Micromolar concen-trations of flavonols are routinely used as both phospha-tase and kinase inhibitors [36,37], so kinases andphosphatases associatedwith auxin transport are probabletargets in apical tissues, in which cellular flavonol levelsare high [4,38].

The serine/threonine kinase PINOID (At2g34650)[39,40], which regulates PIN1, PIN2 and PIN4 activityby regulating the polarity of their subcellular localiz-ation on the plasma membrane [41], is the most likelycandidate, followed by the PINOID-related WAG kinases(At1g53700, At3g14370) [42]. Flavonoid modulationof the protein or lipid phosphorylation state throughinhibition of the protein phosphatase 2A subunit ROOTSCURL IN NPA1 (RCN1, At1g25490) [43] or thephosphatidylinositol 3,4,5-trisphosphate kinase (e.g.At1g21980) [44] is also possible. Flavonoids might alsoinfluence cellular trafficking by altering membrane fluid-ity directly because the more hydrophobic flavonoidsassociate with and intercalate into membranes, makingthem more rigid or fluid, depending on the sterol contentof the membrane, which itself is cell-type specific [27]. Insummary, flavonoids modulate auxin transport directlyand indirectly by affecting the activity of auxin trans-porters, proteins that regulate the transporters andtrafficking of the components of the auxin transportcomplex, and by altering the structure of auxin-trans-porting tissues.

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 X-

100 (a detergent) can be used to move diphenyl boric acid 2-

aminoethyl 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 endo-

somes and tonoplast. However, using dye for staining has limita-

tions 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).

Review TRENDS in Plant Science Vol.12 No.12 559

Flavonoids and physiological responsesFundamental processes, such as auxin transport,gravitropism and phototropism occur in the absence offlavonoids [45]. Therefore, flavonoids are best viewed asmodulators of transport processes, rather than as essentialregulators. In some cases, flavonoids seem to be synthes-ized in response to auxin accumulation, presumably toscavenge reactive oxygen species (ROS) generated duringauxin catabolism [27]. In the absence of PGP1, PGP4 orPGP19, a localized increase in auxin concentrations occurs,resulting in localized quercetin accumulation similar tothat in the wild type after treatment with the auxintransport inhibitor NPA [4,28,35]. Because technicalissues make it problematic to identify the exact timingof auxin and flavonol accumulation (Box 1), it is difficult toseparate the different roles of localized flavonol accumu-lation, 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 ofArabidopsis inflorescence stems, whereas anthocyanins,which do not inhibit auxin transport, decrease in concen-tration from the base of the stem towards the apex [14].Increased auxin transport in tt4 inflorescences might con-tribute to the increased secondary inflorescence formationobserved for one tt4 allele (2YY6) [3]. However, tt4 (2YY6)was subsequently found to harbour a mutation in MoreAxillary Branches 4 (At4g32810) [47]. MAX1 (At2g26170),a cytochrome P450 that functions downstream of the otherMAX genes, is a positive regulator of flavonoid biosyntheticgenes and is required for flavonol synthesis in the axillarybud [48]. The MAX genes were identified as regulators ofauxin-dependent lateral inflorescence branching [47,49].The tt4 max4 double knockout exhibits the increased shootbranching observed in max4 [3,47,50], indicating thatMAX4 is epistatic to CHS in inflorescences. The max1phenotype can be rescued by application of kaempferol,quercetin or naringenin, but MAX regulation of auxintransport is dependent on PINs and indirectly on flavonoidactivity [47,48].

Flavonols accumulate in regions of lateral root emer-gence and along the lateral roots themselves in patternssimilar to those indicated by auxin reporter genes, in-cludingDR5 [14,31]. Although tt4 has an increased numberof lateral roots compared with wild type [3], independent ofMAX4 [51], this seems to be an indirect effect of increasedtransport of shoot-derived auxin [52]. The contribution offlavonoids to lateral root development must also be eval-uated in the context of macronutrients [53,54]. Flavonoidsynthesis is induced two- to four-fold in phosphate-starvedplants [55], with a concomitant increase in lateral rootnumber [54]. Under phosphate starvation, tt4 showed anincreased number of lateral roots, indicating a role forflavonoid modulation of root architecture [54].

Tropic bending

Tropic responses do not require flavonoids because theseresponses are observed in dark-grown seedlings which areflavonoid deficient. However, flavonoids seem to modulatethe rate and extent of gravitropic responses in roots. In

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gravistimulated roots, auxin is redistributed laterally toroot cap and elongation zone cells on the lower side of theroot, with the result that elongation on the lower side isinhibited and the root bends downward [56]. The initialmechanism of lateral redistribution of auxin in root tips

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 visualiza-

tions are largely dependent on the use of auxin-responsive transcrip-

tional 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

destructive sampling techniques. The assays use discontinuous

solid support media and microscopically guided precision instru-

ments 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.

Figure I. Split-root system for tropism measurements.

560 Review TRENDS in Plant Science Vol.12 No.12

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has not been elucidated and might occur independently ofan auxin gradient [57,58]. However, the rapidity of theresponse suggests that factors other than transcriptionalresponses [59] are involved. Calcium, inositol 1,4,5-tri-sphosphate [44,60], phosphorylation events [61], ROSgeneration [44,62], pH gradients [63–66], interference withATPase activity [29] and polar realignment of PIN auxinefflux proteins with the auxin gradient [41,61] have allbeen implicated in the process.

Flavonoid accumulation increases slightly and symme-trically in the root capand cells of columella gravistimulatedroots [46]. Gravitropic bending in the flavonoid-deficient tt4mutant is delayed compared with wild type [31,46]. How-ever, increases in the auxin content of cells in the elongationzone or the lower side of the gravistimulated roots is seen inboth tt4 and wild type [46]. One explanation for this differ-ence was suggested when Lewis et al. [31] demonstratedthat pgp4 interaction is epistatic to tt4. The pgp4 mutantexhibits a 30% decrease in auxin transport from the colu-mella [35] and a 50% decrease in auxin transport from thegeneral apical region of the root [31] but also faster ratherthan slower gravitropic bending [31]. Expression andprotein localization studies show that the highest levels ofPGP4 abundance are in the elongation zone and epidermisof the mature root [35]. Taken together, these data suggestthat PGP4 is a target of flavonoid regulation and functionsprimarily in the movement of auxin out of the elongationzone. Flavonoids normally partially inhibit PGP4-mediatedmovementof auxinout of thedistal elongationzone, and lossof flavonoids results in a reduction in inhibitory auxinconcentrations in this region. An increase in flavonoid levelsor loss of PGP4 function results in a more rapid auxinaccumulation and enhancement of bending.

These results also suggest that flavonoid modulation ofauxin transport mediated by PIN and PGP transporterswithin the lateral root cap is secondary. A split-root assaydeveloped to isolate asymmetric delivery of auxin from thecolumella and lateral root cap to the elongation zone showsthat no difference between wild type and tt4 is seen in theinitial asymmetric auxin movement into the elongationzone (Box 2), consistent with a primary flavonoid functionin modulating PGP-mediated auxin movement out of theelongation zone.

The timing of the events suggests that flavonoids havean additional role that enhances tropic responses. Auxinaccumulation can also produce flavonoid accumulation.Gravistimulated roots exhibiting localized auxin accumu-lation demonstrate increased flavonoid accumulation [46].Mutations in PGP auxin transport genes result in ectopicflavonoid accumulation [35] that can be mimicked withexogenous auxin application [4,46]. One way that flavo-noids might modulate tropic responses is through scaven-ging ROS because an oxidative burst seems to be an earlysignal to stimulate gravitropism in the root cap and colu-mella cells [44,62], and the flavonol quercetin, an excellentROS scavenger, increases in the columella response toauxin accumulation [4]. After the ROS signal is initiated,flavonoids might quench this signal so that it does notcontinue to stimulate a response. Oxidative catabolism oflaterally accumulated IAA trapped in the elongation zone[28,35] also seems to generate ROS [27,67,68]. Flavonoid

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quenching of ROS generated in this manner would preventcellular oxidative damage [25].

Phosphorylation might also have a role in gravi-responses [69]. In addition to modulating the phosphoryl-ation of PGPs, flavonoids might also mediate the serine/threonine kinases PINOID and WAG. PIN2 reorientationis observed in the PID overexpressor mutants [41].Although PIN2 localization was not affected by proteinphosphatase inhibitors [61] or in tt4 [4], PIN2-mediatedgravitropism is regulated by protein phosphatases that arebiochemically similar to RCN1 [61].

The role of flavonoids in gravitropism is most probably acombination of factors: modulation of PGP activity, ROSquenching and retardation of phosphorylation events. Fla-vonoid modulation of physiological responses is tissue andprocess specific, involving mechanisms that evolved overtime. Systems biology approaches utilizing mutants andprecise studies of auxin movement out of the lateral rootcap (such as those shown in Box 2) are required to develop amore complete model.

Evolution of flavonoid modulation of auxin transportFlavonoid synthesis is observed in extant members of thefirst land plants but not in algae [70]. Unlike angiosperms,gymnosperms and pteridophytes, bryophytes only accumu-late flavonoids synthesized by the biosynthetic genes earlyin the pathway (Figure 1), and the moss (Physcomitrellapatens) genome does not seem to contain the genesrequired for the later steps in flavonoid synthesis, despitethe presence of 19 CHS-like genes [71,72]. Bryophytesexhibit polar growth and have different auxin transportmechanisms within each bryophyte division, ranging fromNPA-insensitive diffusion to NPA-sensitive polar auxintransport [73,74]. Poli et al. [73] conclude that auxin trans-port developed independently in each extant bryophytelineage. Because polar IAA transport in some bryophytesis NPA sensitive, one hypothesis is that polar auxin trans-port and auxin transporters, such as the PINs and, to alesser extent, PGPs, evolved in the presence of flavonolsbecause all bryophytes examined accumulate flavonols.

The activity of PGPs is inhibited by flavonoids; however,plant PGP activity is less sensitive to flavonoids than aremammalian transporters because flavonoids are endogen-ous in plants [28,29,75]. PINs evolved more recently thanPGPs [76,77] and exhibit less apparent sensitivity to fla-vonoids, perhaps as a result of undergoing much of theirevolution into auxin transporters in the presence of flavo-noids. By contrast, PGPs are part of the ancient ABCtransporter lineage [77] and flavonoids inhibit their essen-tial ATPase activity [28,29]. Therefore, it is unsurprisingthat PGPs are more sensitive to flavonoids than are PINs.Thus, we might regard auxin transport proteins and theirregulators as cellular components that have been adaptedto function with greater efficiency in a cellular environ-ment that includes flavonoid compounds, rather than asspecific targets of flavonoid regulation.

Concluding remarks and future perspectivesFlavonoids modulate auxin transport through direct andindirect interactions with cellular transport and regulat-ory mechanisms. Developmental regulation of flavonoid

562 Review TRENDS in Plant Science Vol.12 No.12

biosynthesis also determines where and when theseinteractions take place. This makes it difficult to ascertainwhen flavonoids are functioning primarily as ROS scaven-gers, defense compounds, regulators of phosphorylation,enzyme inhibitors or modulators of membrane fluidity. Anevolutionary approach using natural variation among thebryophytes could provide a context for assessing the speci-ficity of flavonoid regulation. It seems that as the complex-ity of flavonoid biosynthesis increased during plantevolution, flavonoid function also expanded. A systemsbiology approach comparing bryophytes and tracheophytescould be used to identify which flavonoid functions are themost fundamental and which are the most divergent.When compared with the evolution of auxin transportmechanisms, those processes that evolved in the presenceof flavonoids could then be differentiated from those thatare more specifically regulated by these compounds.

AcknowledgementsThis work was supported by the US National Science Foundation and theUK Biotechnology and Biological Sciences Research Council (BBSRC).We thank Simon Gilroy and John Courage for suggesting the Box 2experiment.

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