Abstract: The rate, polarity, and symmetry of the flow of the plant hormone auxin are determined by the polar cellular localization of PIN-FORMED (PIN) auxin efflux carriers. Flavonoids, a class of secondary plant metabolites, have been suspected to modulate auxin transport and tropic responses. Nevertheless, the identity of specific flavonoid compounds involved and their molecular function and targets in vivo are essentially unknown. Here we show that the root elongation zone of agravitropic pin2/eir1/wav6/agr1 has an altered pattern and amount of flavonol glycosides. Application of nanomolar concentrations of flavonols to pin2 roots is sufficient to partially restore root gravitropism. By employing a quantitative cell biological approach, we demonstrate that flavonoids partially restore the formation of lateral auxin gradients in the absence of PIN2. Chemical complementation by flavonoids correlates with an asymmetric distribution of the PIN1 protein. pin2 complementation probably does not result from inhibition of auxin efflux, as supply of the auxin transport inhibitor N-1-naphthylphthalamic acid failed to restore pin2 gravitropism. We propose that flavonoids promote asymmetric PIN shifts during gravity stimulation, thus redirecting basipetal auxin streams necessary for root bending. The rate, polarity, and symmetry of the flow of the plant hormone auxin are determined by the polar cellular localization of PIN-FORMED (PIN) auxin efflux carriers. Flavonoids, a class of secondary plant metabolites, have been suspected to modulate auxin transport and tropic responses. Nevertheless, the identity of specific flavonoid compounds involved and their molecular function and targets in vivo are essentially unknown. Here we show that the root elongation zone of agravitropic pin2/eir1/wav6/agr1 has an altered pattern and amount of flavonol glycosides. Application of nanomolar concentrations of flavonols to pin2 roots is sufficient to partially restore root gravitropism. By employing a quantitative cell biological approach, we demonstrate that flavonoids partially restore the formation of lateral auxin gradients in the absence of PIN2. Chemical complementation by flavonoids correlates with an asymmetric distribution of the PIN1 protein. pin2 complementation probably does not result from inhibition of auxin efflux, as supply of the auxin transport inhibitor N-1-naphthylphthalamic acid failed to restore pin2 gravitropism. We propose that flavonoids promote asymmetric PIN shifts during gravity stimulation, thus redirecting basipetal auxin streams necessary for root bending. The plant hormone auxin (3-indolyl acetic acid, IAA) 5The abbreviations used are: IAA, 3-indolyl acetic acid; PAT, polar auxin transport; NPA, N-1-naphthylphthalamic acid; EZ, elongation zone; RT, root tip; HPLC, high performance liquid chromatography; DPBA, diphenylboric acid 2-aminoethyl ester; MS, mass spectrometry; GFP, green fluorescent protein. 5The abbreviations used are: IAA, 3-indolyl acetic acid; PAT, polar auxin transport; NPA, N-1-naphthylphthalamic acid; EZ, elongation zone; RT, root tip; HPLC, high performance liquid chromatography; DPBA, diphenylboric acid 2-aminoethyl ester; MS, mass spectrometry; GFP, green fluorescent protein. controls virtually all plant developmental and physiological processes. In roots, the differential growth response associated with gravity stimulation (gravitropism) occurs in the elongation zone (1Muday G.K. J. Plant Growth Regul. 2001; 20: 226-243Crossref PubMed Scopus (144) Google Scholar, 2Chen R. Hilson P. Sedbrook J. Rosen E. Caspar T. Masson P.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15112-15117Crossref PubMed Scopus (367) Google Scholar) and is a result of the asymmetric distribution of auxin to the lower side of epidermal cells (3Moore I. Curr. Biol. 2002; 12: R452-R454Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In these tissues accumulating auxin, cell elongation is inhibited and the root tip bends downwards. This cell-to-cell or polar auxin transport (PAT) is determined by the asymmetric cellular localization of auxin in- and efflux components of the ABCB/PGP/MDR, AUX1/LAX, and PIN-FORMED (PIN) family (4Vieten A. Sauer M. Brewer P.B. Friml J. Trends Plant Sci. 2007; 12: 160-168Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 5Wisniewska J. Xu J. Seifertova D. Brewer P.B. Ruzicka K. Blilou I. Rouquie D. Benkova E. Scheres B. Friml J. Science. 2006; 312: 883Crossref PubMed Scopus (662) Google Scholar, 6Geisler M. Murphy A.S. FEBS Lett. 2006; 580: 1094-1102Crossref PubMed Scopus (293) Google Scholar, 7Kerr I.D. Bennett M.J. Biochem. J. 2007; 401: 613-622Crossref PubMed Scopus (63) Google Scholar). Although ABCBs are apparently involved in long-range auxin transport and movements of auxin out of apical regions (8Blakeslee J.J. Bandyopadhyay A. Lee O.R. Mravec J. Titapiwatanakun B. Sauer M. Makam S.N. Cheng Y. Bouchard R. Adamec J. Geisler M. Nagashima A. Sakai T. Martinoia E. Friml J. Peer W.A. Murphy A.S. Plant Cell. 2007; 19: 131-147Crossref PubMed Scopus (326) Google Scholar, 9Lewis D.R. Miller N.D. Splitt B.L. Wu G. Spalding E.P. Plant Cell. 2007; 19: 1838-1850Crossref PubMed Scopus (151) Google Scholar, 10Wu G. Lewis D.R. Spalding E.P. Plant Cell. 2007; 19: 1826-1837Crossref PubMed Scopus (129) Google Scholar), AUX1 and PIN2/EIR1/WAV6AGR1 have been demonstrated to channel auxin from the lateral root cap basipetally to the expanding epidermal cells (11Swarup R. Friml J. Marchant A. Ljung K. Sandberg G. Palme K. Bennett M. Genes Dev. 2001; 15: 2648-2653Crossref PubMed Scopus (469) Google Scholar, 12Marchant A. Kargul J. May S.T. Muller P. Delbarre A. Perrot-Rechenmann C. Bennett M.J. EMBO J. 1999; 18: 2066-2073Crossref PubMed Scopus (454) Google Scholar, 13Abas L. Benjamins R. Malenica N. Paciorek T. Wisniewska J. Moulinier-Anzola J.C. Sieberer T. Friml J. Luschnig C. Nat. Cell Biol. 2006; 8: 249-256Crossref PubMed Scopus (455) Google Scholar). The regulation of auxin transport during root gravitropic responses is still largely unclear. Among various possible mechanisms, the localized synthesis and directed transport of flavonoids, plant-specific phenylpropanoid compounds, have been shown to modulate the rate of the gravity response (14Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (312) Google Scholar, 15Buer C.S. Muday G.K. Djordjevic M.A. Plant Physiol. 2007; 145: 478-490Crossref PubMed Scopus (192) Google Scholar). A number of lines of experimentation have suggested that flavonoids may act as non-essential auxin transport inhibitors (16Brown D.E. Rashotte A.M. Murphy A.S. Normanly J. Tague B.W. Peer W.A. Taiz L. Muday G.K. Plant Physiol. 2001; 126: 524-535Crossref PubMed Scopus (555) Google Scholar, 17Murphy A. Peer W.A. Taiz L. Planta. 2000; 211: 315-324Crossref PubMed Scopus (282) Google Scholar, 18Peer W.A. Bandyopadhyay A. Blakeslee J.J. Makam S.I. Chen R.J. Masson P.H. Murphy A.S. Plant Cell. 2004; 16: 1898-1911Crossref PubMed Scopus (305) Google Scholar, 19Peer W.A. Murphy A.S. Grotewold E. The Science of Flavonoids. Springer, Berlin2006: 239-268Crossref Scopus (81) Google Scholar, 20Peer W.A. Murphy A.S. Trends Plant Sci. 2007; 12: 556-563Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). This is mainly based on the finding that flavonoids displace binding of synthetic auxin transport inhibitors, like N-1-naphthylphthalamic acid (NPA), a herbicide (Naptalam®), in vitro (31Jacobs M. Rubery P.H. Science. 1988; 241: 346-349Crossref PubMed Scopus (490) Google Scholar, 50Morris D.A. Plant Growth Regul. 2000; 32: 161-172Crossref PubMed Scopus (69) Google Scholar, 51Lomax T.L. Muday G.K. Rubery P.H. Davies P.J. Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer, Dordrecht, Netherlands1995: 509-530Crossref Google Scholar, 52Luschnig C. Trends Plant Sci. 2002; 7: 329-332Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Moreover, roots of transparent testa (tt) Arabidopsis mutant with manipulated flavonoid levels exhibit altered gravitropic curvature and auxin transport, which are restored to the wild-type level by exogenous application of flavonoids (16Brown D.E. Rashotte A.M. Murphy A.S. Normanly J. Tague B.W. Peer W.A. Taiz L. Muday G.K. Plant Physiol. 2001; 126: 524-535Crossref PubMed Scopus (555) Google Scholar, 21Taylor L.P. Grotewold E. Curr. Opin. Plant Biol. 2005; 8: 317-323Crossref PubMed Scopus (446) Google Scholar). Nonetheless, the identity of the specific flavonoid compounds involved, their molecular targets as well as their mode of action in vivo are essentially unknown. Several lines of evidence suggest that ABCBs are directly (8Blakeslee J.J. Bandyopadhyay A. Lee O.R. Mravec J. Titapiwatanakun B. Sauer M. Makam S.N. Cheng Y. Bouchard R. Adamec J. Geisler M. Nagashima A. Sakai T. Martinoia E. Friml J. Peer W.A. Murphy A.S. Plant Cell. 2007; 19: 131-147Crossref PubMed Scopus (326) Google Scholar, 9Lewis D.R. Miller N.D. Splitt B.L. Wu G. Spalding E.P. Plant Cell. 2007; 19: 1838-1850Crossref PubMed Scopus (151) Google Scholar, 10Wu G. Lewis D.R. Spalding E.P. Plant Cell. 2007; 19: 1826-1837Crossref PubMed Scopus (129) Google Scholar, 22Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 23Geisler M. Blakeslee J.J. Bouchard R. Lee O.R. Vincenzetti V. Bandyopadhyay A. Titapiwatanakun B. Peer W.A. Bailly A. Richards E.L. Ejendal K.F.K. Smith A.P. Baroux C. Grossniklaus U. Muller A. Hrycyna C.A. Dudler R. Murphy A.S. Martinoia E. Plant J. 2005; 44: 179-194Crossref PubMed Scopus (421) Google Scholar) or indirectly (24Bailly A. Sovero V. Vincenzetti V. Santelia D. Bartnik D. Koenig B.W. Mancuso S. Martinoia E. Geisler M. J. Biol. Chem. 2008; 283: 21817-21826Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) regulated by aglycone flavonols. High NPA concentrations cause inhibition of auxin efflux catalyzed by ABCB1/PGP1, ABCB19/PGP19/MDR1 (22Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. 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In contrast, the expression and subcellular location of PIN auxin efflux carriers is thought to be a consequence of flavonoid-mediated alteration of auxin concentrations (18Peer W.A. Bandyopadhyay A. Blakeslee J.J. Makam S.I. Chen R.J. Masson P.H. Murphy A.S. Plant Cell. 2004; 16: 1898-1911Crossref PubMed Scopus (305) Google Scholar, 19Peer W.A. Murphy A.S. Grotewold E. The Science of Flavonoids. Springer, Berlin2006: 239-268Crossref Scopus (81) Google Scholar). In a pioneer study evidence that flavonoids are functioning as endocrine effectors that specifically determine individual PIN gene expression and protein localization was provided (18Peer W.A. Bandyopadhyay A. Blakeslee J.J. Makam S.I. Chen R.J. Masson P.H. Murphy A.S. Plant Cell. 2004; 16: 1898-1911Crossref PubMed Scopus (305) Google Scholar). Here, we report that agravitropic loss-of-function mutant pin2/eir1/wav6/agr1 has impaired patterns of flavonol glycosides. We found that nanomolar concentrations of exogenous flavonols, which have apparently only a mild inhibitory effect on root elongation and gravitropic response in wild-type plants, can partially rescue the agravitropic phenotype of pin2 roots by promoting asymmetric PIN1 shifts, re-establishing polar auxin fluxes. Chemicals—The following substances were obtained as indicated MeCN (HPLC Supra grade, Scharlau, E-Barcelona), HCOOH (Fluka, Puriss, Switzerland), methanol (MEOH, Fisher Scientific, UK), HPLC-grade acetonitrile (Fisher Scientific, UK), and H3PO4 (Applichem, Germany). Water was purified with a MilliQ Gradient apparatus (<5 ppb, Millipore, Milford, MA). Diphenylboric acid 2-aminoethyl ester (DPBA, Sigma, Germany), NPA (Fluka, Germany), kaempferol (Calbiochem, La Jolla, CA), and quercetin (Fluka, Germany) were dissolved in 100% dimethyl sulfoxide. Growth Conditions and Plant Material—Seeds were surface sterilized for 5 h in a chamber containing vaporous HCl and sodium hypochlorite and stratified in a 0.1% agar solution for 2 days at 4 °C. Subsequently, the seeds were plated on sterile half-strength MS medium at pH 5.7 containing 2% sucrose solidified with 0.6% phytagel (Sigma), and vertically grown at 22 °C with a 16-h/8-h light/dark cycle. The mutant alleles used in this study were pin2-1 (29Muller A. Guan C.H. Galweiler L. Tanzler P. Huijser P. Marchant A. Parry G. Bennett M. Wisman E. Palme K. EMBO J. 1998; 17: 6903-6911Crossref PubMed Scopus (663) Google Scholar) and eir1-4 (30Luschnig C. Gaxiola R.A. Grisafi P. Fink G.R. Genes Dev. 1998; 12: 2175-2187Crossref PubMed Scopus (641) Google Scholar). Flavonoid Fluorescence Staining—Flavonoid compound locations were visualized in vivo by the fluorescence of flavonoid-conjugated DPBA compounds after excitation with blue light. Plants were grown for 5 days before staining. Fluorescent staining of whole seedlings was performed according to Buer and Muday (14Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (312) Google Scholar). Fluorescence was achieved by excitation with fluorescein isothiocyanate filters (450-490 nm, suppression long pass 515 nm) on a Leica DMR fluorescence microscope and ×10 or 20 objectives. Digital images were captured with a Leica DC300 F charge coupled device camera. Extraction of Phenolic Compounds and HPLC Analysis—Excised roots were incubated overnight in the dark at 4 °C in 0.5 ml of 80% (v/v) methanol (MeOH), extracted, and centrifuged at 18,000 × g for 10 min. The supernatant was concentrated to dryness and resuspended in 0.1 ml of 80% MeOH. Aliquots (50 μl) were analyzed by a reverse-phase HPLC (Gynkotek, Germany). Absorbance spectra were recorded with a UVD340S diode array detector (Dionex, Switzerland). Data integration analysis was conducted using the Chromeoleon software (version 6.4, Dionex, Switzerland). The peak height was quantified at 330 nm. A calibration curve for kaempferol was used as reference for single peak quantification. All analyses were performed with at least three independent replicates, each representing 100 roots. Chromatographic conditions were Nucleosil 100-5 C18 column (5 μm, 2 × 250 mm, Macherey-Nagel, Düren, Germany); flow rate 1.00 ml min-1, gradient (step, time, %B over A) 1, 25 min, 10-25%; 2, 10 min, 25-70%). Solvent A was H2O, 0.1% (v/v) H3PO4 and solvent B was MeCN. Structural Elucidation: HPLC-ESI-MS/MS Analysis—HPLC-MS analyses were performed on an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA) fitted with a HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland), an Agilent 1100 binary pump, and an Agilent 1100 photodiode array detector. Chromatographic conditions were Nucleosil 100-3 C18 column (3 μm, 2 × 250 mm, Macherey-Nagel, Hoerdt, France); flow rate 0.170 ml min-1, gradient (step, time, %B over A) 1, 25 min, 10-25%; 2, 10 min, 25-70%). Solvent A was H2O, 0.1% (v/v) HCOOH and solvent B was MeCN, 0.1% (v/v) HCOOH. The HPLC was connected to a Bruker ESQUIRE-LC quadrupole ion trap instrument (Bruker Daltonik GmbH, Bremen, Germany), equipped with a combined Hewlett-Packard Atmospheric Pressure Ion source (Hewlett-Packard Co., Palo Alto, CA). The HPLC output was directly interfaced to the ESI ion source. The MS conditions were: nebulizer gas (N2) 40 p.s.i., dry gas (N2) 9 liters/min, dry temperature 300 °C, HV capillary 4000 V, HV EndPlate offset -500 V, capillary exit -100 V, skimmer1-28.9 V, and trap drive 53.4. The MS acquisitions were performed in the negative electrospray ionization mode, at normal resolution (0.6 unit at half-peak height), under ion charge control conditions (10,000) in the mass range from m/z 100 to 1000. The MS2 acquisitions were obtained in the auto-MS/MS mode. The isolation width was 4 units, the fragmentation cut-off set by “fast calc,” and the fragmentation amplitude set at 0.9 V in the “SmartFrag” mode. The total amounts of flavonoid compounds were calculated as the sum of the areas (×106 arbitrary unit) of the mass signals identified during HPLC-ESI-MS analysis. Each extraction consists of a pull of 100 different roots. Quantification of RT-EZ flavonoid compounds is the result of two independent extractions in which each time 150 5-mm long root apices from 12 different agar plates were pulled together. Gravitropic Assays—4-Day light-grown seedlings were transferred from control plates to plates containing nutrient media optionally supplemented with quercetin or kaempferol (100 or 200 nm) or NPA (100 nm to 5 μm). After 24 h of adaptation and growth in the new media, plates were turned 90°. In one type of gravitropic assay, after 24 h of growth under gravistimulation, seedlings were scanned using Epson Perfection photo 2450 and angles of gravitropic curvature were measured from digital pictures using the tool “Image Manager” of the Leica IM1000 software (Leica, Heerbrug, CH). Each gravity stimulated root was assigned to one of 12, 30° sectors; the length of each bar represents the percentage of seedlings showing the same direction of root tip growth. To enable direct comparison of root bending, percentual occurrence of 60 and 90° bending (sum of 60 and 90° sectors), the dominant bending sectors of wild-type roots under control conditions (98.4%), was defined as relative root bending. Short pulses of gravity stimulation were achieved by turning the plates 90° for 2 h, which corresponds to the peak of gravity-induced flavonoid accumulation (14Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (312) Google Scholar). After 2 h of gravity stimulation, roots were excised and flavonoids extracted as described, or expression of DR5rev-GFP reporter protein analyzed on a Leica TCS SP2 CLSM. Kinetics of root bending were performed and analyzed as described in Ref. 31Jacobs M. Rubery P.H. Science. 1988; 241: 346-349Crossref PubMed Scopus (490) Google Scholar. All gravitropic assays were performed in the dark to prevent phototropic responses. In some cases (Table 2 and supplemental Figs. S1 and S2), angles of gravitropic curvature were measured in a blind assay, to reduce possible unbiased calculations.TABLE 2The majority of quercetin-treated pin2 roots form IAA gradients upon gravity stimulation Immunocytochemistry—PIN1 immunolocalization was performed as previously described (32Friml J. Benkova E. Mayer U. Palme K. Muster G. Plant J. 2003; 34: 115-124Crossref PubMed Scopus (117) Google Scholar) with PIN1 specific antibody (33Paciorek T. Zazimalova E. Ruthardt N. Petrasek J. Stierhof Y.D. Kleine-Vehn J. Morris D.A. Emans N. Jurgens G. Geldner N. Friml J. Nature. 2005; 435: 1251-1256Crossref PubMed Scopus (615) Google Scholar) at 1:1000 dilution and anti-rabbit Cy3-conjugated secondary antibodies. Confocal imaging of Cy3 and DR5rev::GFP was carried out on a Leica SP2 AOBS microscope. In some cases (Fig. 4C and supplemental Fig. S3), symmetries of DR5-GFP gradients and PIN1 distribution were measured in a blind assay, to reduce possible unbiased calculations. Data Analyses—Statistical analysis was performed using SPSS 11.0 (SPSS Inc., Chicago, IL). pin2 Roots Have an Altered Flavonoid Pattern—pin2/eir1/wav6/agr1 Arabidopsis mutant (referred to as pin2 hereafter), one of the best characterized auxin transport mutants, exhibits reduced basipetal auxin transport and agravitropic root growth (2Chen R. Hilson P. Sedbrook J. Rosen E. Caspar T. Masson P.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15112-15117Crossref PubMed Scopus (367) Google Scholar, 29Muller A. Guan C.H. Galweiler L. Tanzler P. Huijser P. Marchant A. Parry G. Bennett M. Wisman E. Palme K. EMBO J. 1998; 17: 6903-6911Crossref PubMed Scopus (663) Google Scholar, 30Luschnig C. Gaxiola R.A. Grisafi P. Fink G.R. Genes Dev. 1998; 12: 2175-2187Crossref PubMed Scopus (641) Google Scholar, 34Utsuno K. Shikanai T. Yamada Y. Hashimoto T. Plant Cell Physiol. 1998; 39: 1111-1118Crossref PubMed Scopus (160) Google Scholar). As a starting point of this work, we investigated whether defects in basipetal auxin transport in pin2, which result in agravitropic responses (35Rashotte A.M. Brady S.R. Reed R.C. Ante S.J. Muday G.K. Plant Physiol. 2000; 122: 481-490Crossref PubMed Scopus (268) Google Scholar), are linked to an altered accumulation of specific endogenous flavonoids and whether flavonoids could be directly implicated in the control of the gravitropic responses. DPBA, a fluorescent dye that specifically interacts with flavonoids, allows in situ flavonoid staining and localization in Arabidopsis seedlings (18Peer W.A. Bandyopadhyay A. Blakeslee J.J. Makam S.I. Chen R.J. Masson P.H. Murphy A.S. Plant Cell. 2004; 16: 1898-1911Crossref PubMed Scopus (305) Google Scholar, 20Peer W.A. Murphy A.S. Trends Plant Sci. 2007; 12: 556-563Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 36Peer W.A. Brown D.E. Tague B.W. Muday G.K. Taiz L. Murphy A.S. Plant Physiol. 2001; 126: 536-548Crossref PubMed Scopus (269) Google Scholar). In wild-type seedlings, flavonoid DPBA staining is restricted to the shoot apex and cotyledons, the root-shoot junction, along the primary root, and most intensely to the root elongation zone (Fig. 1A and supplemental Fig. S1, A-C) (36Peer W.A. Brown D.E. Tague B.W. Muday G.K. Taiz L. Murphy A.S. Plant Physiol. 2001; 126: 536-548Crossref PubMed Scopus (269) Google Scholar). In contrast, flavonoid-DPBA fluorescence in the pin2 mutant was clearly lower at the root tip-elongation zone (RT-EZ) (Fig. 1B and supplemental Fig. S1, E-G). Manipulation of endogenous auxin levels by addition of 100 nm IAA increased DPBA fluorescence in the wild type (14Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (312) Google Scholar) and, although to a lesser extend, also in the pin2 RT-EZ (supplemental Fig. S1, D and H), suggesting that auxin and flavonoid levels in planta are interconnected (18Peer W.A. Bandyopadhyay A. Blakeslee J.J. Makam S.I. Chen R.J. Masson P.H. Murphy A.S. Plant Cell. 2004; 16: 1898-1911Crossref PubMed Scopus (305) Google Scholar, 36Peer W.A. Brown D.E. Tague B.W. Muday G.K. Taiz L. Murphy A.S. Plant Physiol. 2001; 126: 536-548Crossref PubMed Scopus (269) Google Scholar). To determine how flavonoid distribution was affected by PAT alterations, we qualitatively and quantitatively investigated endogenous flavonoid derivatives present in wild-type and pin2 RT-EZ and entire roots using HPLC-UV-(-)-ESI-MS and HPLC-ESI-MS/MS, respectively (Table 1). Consistent with DPBA staining profiles (Figs. 1, A and B and supplemental S1), we found that the total amount of flavonoids was significantly reduced in the RT-EZ of pin2 mutant (Fig. 1, E and G), whereas no significant difference was observed over the entire root (Figs. 1E and supplemental S2). pin2 roots showed altered accumulation of specific flavonol glycosides both in the RT-EZ and in the entire root (Table 1 and arrows in the extraction ion chromatograms of the masses of interest in Figs. 1G and supplemental S2). In pin2 entire roots and RT-EZ, a shift from di- and triglycosylated flavonols to monoglycosylated flavonols, such as K-G-3 (compound 18), was observed (Table 1), which suggests that auxin levels may have an effect on the expression or activity of corresponding glycosyltransferases. This is supported by in silico (www.genevestigator.ethz.ch) expression analysis of two glycosyltransferase genes that are involved in flavonoid biosynthesis in Arabidopsis (37Tohge T. Nishiyama Y. Hirai M.Y. Yano M. Nakajima J. Awazuhara M. Inoue E. Takahashi H. Goodenowe D.B. Kitayama M. Noji M. Yamazaki M. Saito K. Plant J. 2005; 42: 218-235Crossref PubMed Scopus (743) Google Scholar). At5g17050, which encodes for a flavonoid 3-O-glucosyltransferase, is induced by 1-naphtylacetic acid and 2,4-dichlorophenoxyacetic acid treatment, whereas At4g14090, anthocyanin 5-O-glucosyltransferase, is down-regulated by auxin transport inhibitor treatments. Conversely, those peaks, whose accumulation is affected in pin2 roots, may be functionally important for the regulation of auxin transport during root gravitropism.TABLE 1Flavonoid derivatives detected in the MeOH extracts of entire root and RT-EZ of wild type and pin2 As previously reported (14Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (312) Google Scholar, 38Buer C.S. Sukumar P. Muday G.K. Plant Physiol. 2006; 140: 1384-1396Crossref PubMed Scopus (149) Google Scholar), a 2-h gravity stimulation increased the DBPA fluorescence in wild-type RT-EZ by nearly 2-fold, with a maximum at 1.5 to 2.5 h after stimulation. A smaller but significant increase in DPBA fluorescence was observed also in pin2 mutant (Fig. 1, C-D). Flavonoid quantification by HPLC-UV (Fig. 1F) was consistent with the DPBA staining. Collectively, our results demonstrate that the synthesis and transient accumulation of specific flavonoid glycosides in the root tip-elongation zone, but not over the entire root or in the shoot, are impaired quantitatively and qualitatively in pin2 (Figs. 1, supplemental S1 and S2). Flavonoids Partially Rescue the Agravitropic Response of pin2 Roots—To test whether flavonoid concentrations play a critical role in the response to gravity stimuli, we searched for conditions in which flavonoids could be supplied without negatively affecting root growth and gravitropism most probably by acting as auxin transport inhibitors (16Brown D.E. Rashotte A.M. Murphy A.S. Normanly J. Tague B.W. Peer W.A. Taiz L. Muday G.K. Plant Physiol. 2001; 126: 524-535Crossref PubMed Scopus (555) Google Scholar, 18Peer W.A. Bandyopadhyay A. Blakeslee J.J. Makam S.I. Chen R.J. Masson P.H. Murphy A.S. Plant Cell. 2004; 16: 1898-1911Crossref PubMed Scopus (305) Google Scholar). Concentrations up to 100 nm kaempferol or quercetin did not significantly influence wild-type gravitropic responses after 24 h (95.5 and 99.0% instead of 98.4% (relative root bending (= sum of 90 and 60° sectors, respectively); see “Experimental Procedures” (Fig. 2A)). Moreover, root-bending kinetics demonstrated that treatment with 100 nm quercetin did not significantly alter the bending performance of wild-type roots over 24 h compared with the solvent control (Fig. 2B). Importantly, bending of wild-type roots in the presence and absence of quercetin is virtually identical after 2 and 24 h, time points used in this study), however, small but not significant differences are found between 2 and 24 h. Roots of the eir1-4 mutant, a severe agravitropic allele of pin2 (30Luschnig C. Gaxiola R.A. Grisafi P. Fink G.R. Genes Dev. 1998; 12: 2175-2187Crossref PubMed Scopus (641) Google Scholar), were gravity stimulated for 24 h in the presence of 100 nm flavonoids. Intriguingly, pin2 gravitropic root bending (33.5%) was significantly restored by quercetin (50.0%) and kaempferol (52.1%, Fig. 2, A and B). Restoration of relative root bending by flavonols was roughly 25% and therefore only partial. The same gravitropic assay was performed in the presence of the synthetic inhibitor of polar auxin efflux NPA, which blocks basipetal IAA movement from the root tip (39Casimiro I. Marchant A. Bhalerao R.P. Beeckman T. Dhooge S. Swarup R. Graham N. Inze D. Sandberg G. Casero P.J. Bennett M. Plant Cell. 2001; 13: 843-852Crossref PubMed Scopus (775) Google Scholar). In wild-type plants, treatment with 5 μm NPA, a concentration routinely used, resulted in an agravitropic phenotype (39.4%) (16Brown D.E. Rashotte A.M. Murphy A.S. Normanly J. Tague B.W. Peer W.A. Taiz L. Muday G.K. Plant Physiol. 2001; 126: 524-535Crossref PubMed Scopus (555) Google Scholar, 35Rashotte A.M. Brady S.R. Reed R.C. Ante S.J. Muday G.K. Plant Physiol. 2000; 122: 481-490Crossref PubMed Scopus (268) G