A role for the auxin precursor anthranilic acid in root gravitropism via regulation of PIN-FORMED protein polarity and relocalization in Arabidopsis

analogues and transformed lines to over-produce AA while inhibiting its conversion to downstream IAA precursors.  root rescue root affecting Treatments or deficiency defects in PIN and gravistimulus-induced PIN in root cells.  Our results reveal a previously unknown role for AA in the regulation of PIN subcellular localization and dynamics involved in root gravitropism, which is independent of its better-known role in a we we we show that this role of its role in IAA biosynthesis. treatment medium incubated for 5 h without any seedlings. For long-term treatments with 5 µM ES8 compounds, Col-0 and wei2wei7 seedlings were grown for 9 d on ES8, ES8.7 and ES8.7-Trp-supplemented solid medium before harvesting in liquid nitrogen along with samples of solid treatment medium incubated for 9 d without any seedlings. Samples from two biological replicates were harvested and divided into two technical replicates each. The medium samples were diluted 1:100 with 30% v/v acetonitrile and 10 µl was injected onto a Kinetex 1.7 μm C18 100A reversed-phase column 50 x 2.1 mm (Phenomenex, Torrance, CA, USA) followed by analysis by LC-MS/MS. The seedling samples (around 10 mg fresh weight) were extracted in 0.4 ml acetonitrile using a MixerMill MM 301 bead mill (Retsch GmbH) with 2 mm ceria-stabilized zirconium oxide beads at a frequency of 27 Hz for 3 min. The plant tissue extracts were then incubated at 4 °C with continuous shaking for 30 min, centrifuged in a Beckman Coulter μl triple After injection of 10 µl, the purified samples eluted using an 11 min gradient comprised of 0.1% acetic acid methanol (A) and 0.1% acetic acid in water (B) at a flow rate of 0.5 ml/min and column 40 °C. The binary linear gradient of 0 min 2:98 A:B, 11 min 95:5 A:B used, after with 100% methanol for 1 min and re-equilibrated to initial conditions for 2 min. The effluent was introduced into the MS system with the optimal settings: source/desolvation temperature 150/600 cone/desolvation gas flow 150/1000 l h -1 , capillary cone voltage 25-40 15-30 and collision gas flow Quantification and confirmation of the ES8 compounds were obtained by the multiple reaction monitoring mode using the following mass transitions: 274>111/274>230, 457>378/457>413 and for ES8, ES8.7 and ES8.7-Trp, respectively. The non-AA and non-Trp parts of the ES8 compounds were monitored by high resolution mass spectrometry (HRMS) using a Synapt G2-Si hybrid Q-TOF tandem mass spectrometer (Waters Corporation) equipped with electrospray ionization


Introduction
Auxin distribution in controlled concentration gradients within certain tissues plays an important role in regulating the dynamically plastic growth and development of plants (Vanneste & Friml, 2009). An intense research effort has revealed many of the complex mechanisms by which plasma membrane-localized auxin carrier proteins are polarly distributed in order to direct the flow of auxin in plant tissues and maintain these gradients (reviewed by Luschnig & Vert, 2014;and Naramoto, 2017). These proteins, including the well-studied PIN-FORMED (PIN) auxin efflux carriers, are remarkably dynamic in that they rapidly relocalize within the cell in response to signals, resulting in changes in their polarity. This dynamic responsiveness, which is facilitated by vesicular cycling and complex endomembrane trafficking pathways, is essential for altering the direction and strength of cell-to-cell auxin flow and redistributing auxin in response to external cues, thereby regulating cell and tissue growth and plasticity.
Root development in Arabidopsis thaliana has received particular attention as a model system demonstrating the importance of auxin gradients for plant development (Clark et al., 2014). Mutations affecting auxin transporters often disturb root gravitropism and specific PIN proteins within the root tip have been shown to relocalize in response to changes in the gravity vector, leading to altered auxin flow and consequently, organ growth adjustment (reviewed by Geisler et al., 2014). In the root columella, the cellular relocalization of PIN3 and PIN7 plays an important role in root gravitropic growth responses. While these proteins are generally apolar in columella cells, they redistribute toward the downward-facing plasma membranes upon horizontal reorientation of the root (Friml et al., 2002b;Kleine-Vehn et al., 2010), which is presumed to redirect the flow of auxin within the columella, thus contributing to auxin accumulation at the lower root side. PIN2 is also involved the root gravitropic response. Displaying shootward (apical) polarity within root epidermal cells (Müller et al., 1998), PIN2 transports auxin upward, balancing the correct auxin maximum required in the root apical meristem for root development (Adamowski & Friml, 2015). However, in the case of a horizontal reorientation of the root, PIN2 is rapidly redistributed from the plasma membranes to the vacuoles within epidermal cells at the upper organ side (Abas et al., 2006;Kleine-Vehn et al., 2008).
This results in accumulation of auxin and consequent inhibition of cell elongation at the lower root side, contributing toward the root tip bending downward.
In our previous work, we employed a chemical biology approach, whereby we isolated and characterized small synthetic molecules selectively altering the polarity of specific PIN proteins, to dissect the trafficking pathways involved in regulating their localization (Doyle et al., 2015a). This approach led us to identify a potential role for the endogenous compound anthranilic acid (AA) in PIN polarity regulation, which we investigated in the current study. AA is an important early precursor of the main natural plant auxin indole-3-acetic acid (IAA) (Maeda & Dudareva, 2012) and as auxin itself has been shown to regulate PIN polarity in a feedback mechanism to control its own flow (Paciorek et al., 2005), we hypothesized that AA may play a similar regulatory role. Herein, using Arabidopsis root gravitropism as a model system for auxin-regulated plastic growth, we provide strong evidence in favor of this hypothesis. Ultimately, we reveal a previously unknown role for AA in the regulation of PIN polarity and relocalization required for root gravitropic responses and furthermore, we show that this role of AA is distinct from its well-known role in IAA biosynthesis.
We were interested in the strong agravitropic and short phenotypes of wei2wei7 roots compared to Col-0 seedlings (Fig. 1b,c), considering that ES8 treatment reduces both gravitropic root growth and root length in Col-0 (Doyle et al., 2015a). To investigate AA-mediated rescue of these root phenotypes in wei2wei7, we performed long-term AA treatments by growing WT and mutant seedlings on medium supplemented with a range of AA concentrations. In Col-0, none of the tested concentrations affected root gravitropic growth, while concentrations of 10 µM or more decreased root length in a dose-dependent manner (Fig. S2a), possibly due to increased IAA biosynthesis. As expected, in wei2wei7 both root gravitropic growth and length were rescued by AA (Fig. 1c,d), however we observed a striking difference between the AA rescue patterns of these two root phenotypes. While root gravitropic growth in the mutant was almost fully rescued to the WT level at all AA concentrations applied, root length rescue was only partial and was concentrationdependent, with maximal rescue at 5 µM (Fig. 1d). We hypothesized that these different rescue patterns in wei2wei7 might reflect two different roles of AA, one known role in auxin biosynthesis and a distinct, as yet unknown role in regulating auxin distribution, considering that ES8 disturbs auxin distribution patterns in the root (Doyle et al., 2015a).
We next investigated whether ES8, as an analog of AA, could rescue either root gravitropic growth or length in wei2wei7. While long-term treatments with ES8 decreased both root gravitropic growth and length in a dose-dependent manner in Col-0 ( Fig. S2b), only the highest ES8 concentrations (15 and 20 µM) decreased root gravitropic and length in wei2wei7 (Fig. 1e). Moreover, while root length was not rescued in the mutant at any ES8 concentration, 5 µM ES8 partially rescued the root gravitropic phenotype of the mutant (Fig. 1c,e). The partial root gravitropic rescue of wei2wei7 by ES8 without any effect on root length suggest, considering that ES8 is known to affect auxin distribution in the root (Doyle et al., 2015a), that the root gravitropic rescue of wei2wei7 by AA may occur via a previously unknown role of AA in auxin distibution.
To further test our hypothesis, we used another analog of both ES8 and AA -ES8 analog no. 7 (ES8.7; Fig. S3a) and its analog ES8.7-Trp, in which the AA was exchanged for a Trp (Fig. S3b). In Col-0, long-term ES8.7 treatment revealed a similar but weaker effect than ES8 on dose-dependent reduction of root gravitropic growth and length (Fig. S3c). ES8.7 rescued root gravitropic growth in wei2wei7 at a range of concentrations from 1 to 15 µM, with almost no effects on root length (Fig. S3a,d). Moreover, ES8.7-Trp did not rescue root gravitropic growth or length at any concentration in either  or wei2wei7 (Fig. S3b,f), strongly suggesting that it is the AA part of ES8 and ES8.7 that rescues gravitropic growth of wei2wei7 roots. Together, these results suggest that a potential role for AA in auxin distribution may regulate root gravitropic growth, while the well-known role of AA in auxin biosynthesis may be more important for root length regulation.
To investigate any possible degradation or metabolism of the ES8 compounds to release AA or Trp, we performed both short-term and long-term treatments of Col-0 and wei2wei7 seedlings with the ES8 compounds, followed by compound analysis (Fig. S4). We measured the concentrations of the relevant ES8 compound, AA or Trp and the non-AA or non-Trp part of the ES8 compound in planta as well as in ES8 compound-supplemented treatment medium to which no seedlings were added. After short-term treatment (5 h incubation in liquid treatment medium) with 5 µM ES8, high levels of ES8 were detectable in the seedlings and the seedling-free treatment medium remained at about 5 µM ES8 (Fig. S4a). Importantly, these findings confirm that ES8 readily enters plant tissues during short-term treatment. After long-term treatment (9 d growth on solid treatment medium), the concentrations of ES8 in the seedlings and the seedling-free treatment medium had lowered considerably, suggesting degradation of ES8 over time, and/or slower uptake from solid than liquid treatment medium. Compared to ES8, much lower levels of ES8.7 and ES8.7-Trp were present in the seedlings after short-term treatment (Fig. S4b,c), suggesting that ES8 may be more efficiently taken up into seedling tissues or ES8.7 and ES8.7-Trp may be degraded or metabolized during the short-term treatment. Degradation of ES8.7-Trp was supported by our measurements of its concentration in the seedlingfree treatment medium, which had already lowered to 3.3 µM after short-term incubation and to 0.5 µM after long-term incubation (Fig. S4c). Moreover, the levels of ES8.7-Trp were considerably lower in the seedlings after long-term compared to short-term treatment. While these results suggest that ES8 and ES8.7-Trp are likely degraded over time, the levels of AA and Trp in the seedlings after ES8 compound treatment were not different to the levels after mock treatment and neither AA nor Trp were detected in the treatment medium samples (Fig. S4d,e). Furthermore, we did not detect non-AA or non-Trp parts of the ES8 compounds at any time point, neither in the seedlings nor in the seedling-free treatment medium (Fig. S4f). Therefore, the observed activities of the ES8 compounds are not due to the release of AA or Trp leading to increased IAA biosynthesis.

AA and ES8 can rescue root gravitropic growth in wei2wei7 without rescuing IAA level
As AA is a precursor of IAA, we investigated the possibility that the rescue of root gravitropic growth by ES8 and AA might indirectly result from increased IAA biosynthesis. First, we measured IAA concentrations after long-term treatments with AA. In Col-0, only 10 µM AA significantly increased the IAA level ( Fig. 2a), which likely explains why treatments of Col-0 with 10 µM and higher AA resulted in significantly shorter roots (Fig. S2a). While treatment of wei2wei7 with 1 or 10 µM AA rescued the IAA level to that of mock-treated Col-0, treatment with 0.5 µM AA had no effect on IAA content (Fig. 2a), despite this concentration having almost fully rescued root gravitropic growth and partially rescued root length in wei2wei7 seedlings (Fig. 1d). Next, we measured IAA content in seedlings treated long-term with 5 µM ES8, ES8.7 or ES8.7-Trp. While the IAA level was slightly but significantly reduced in mock-treated wei2wei7 compared to Col-0, none of the ES8 compounds significantly affected IAA content compared to mock treatment in either genotype (Fig. 2b). As our IAA analysis was performed on whole seedlings, we cannot rule out small, local changes in IAA levels in specific regions of the root.
However, taken together, our results suggest that ES8 and ES8.7 rescue wei2wei7 root gravitropic growth without affecting general IAA content, thereby supporting the hypothesis that AA plays a role in the regulation of root gravitropic growth independently from its function in IAA biosynthesis and potentially via a previously unknown role in auxin distribution.

IAA precursors is repressed
To test our hypothesis that AA may regulate root gravitropic growth via a role independent of IAA biosynthesis, we generated transformed Arabidopsis lines in which the gene encoding ASA1 (WEI2) (Niyogi & Fink, 1992) is constitutively overexpressed and that encoding PHOSPHORIBOSYLANTHRANILATE TRANSFERASE 1 (PAT1), which converts AA to the next downstream IAA precursor (Rose et al., 1992), is subject to estradiol-induced silencing. Of several homozygously transformed 35S::ASA1 (35S::WEI2) and XVE::amiRNA-PAT1 lines, in which the promoters are known to be widely expressed (Odell et al., 1985;Zuo et al., 2000), we used qPCR analysis of ASA1 (WEI2) and PAT1 expression in whole seedlings ( Fig. S5a,b) to select two lines for each construct displaying reproducible and strong constitutive ASA1 induction (35S::ASA1 lines 3B6 and 3B7) or inducible PAT1 silencing (XVE::amiRNA-PAT1 lines 2D4 and 4B10). We then crossed the selected lines and analyzed ASA1 and PAT1 expression in the progeny that were homozygous for both transformations, which we named AxP (ASA1 x PAT1) lines ( Fig. S5c,d). While ASA1 was overexpressed in all AxP lines, there was a tendency for increased PAT1 expression in non-estradiol-induced conditions in those lines with highest ASA1 expression, suggesting positive regulation between ASA1 and PAT1 genes. We selected two AxP lines for further experiments; AxP1 (3B6 x 2D4 line no. 4) in which ASA1 was five-fold overexpressed compared to the non-treated WT without affecting non-induced PAT1 expression and AxP2 (3B7 x 2D4 line no. 21), in which ASA1 was 10-fold overexpressed, resulting in three-fold overexpression of PAT1 in non-induced conditions (Fig. S5c,d). Additionally, an estradiol-inducible five-and three-fold reduction in PAT1 expression compared to non-treated Col-0 was shown for AxP1 and AxP2, respectively (Fig. S5d).
We analyzed the levels of IAA and several IAA precursors/conjugates/catabolites in WT and AxP lines treated long-term with estradiol (grown on supplemented medium) (Fig. S6a). Importantly, AA levels were significantly higher in both AxP lines compared to the WT, while the IAA content was not affected. The levels of Trp, IAN, IAM and oxIAA were also not significantly affected in the lines, while the levels of the IAA conjugates IAA-Aspartate (IAAsp) and IAA-Glutathione (IAGlu) showed rather variable results (Fig. S6a). These analyses suggest that simultaneous overexpression of ASA1 and silencing of PAT1 result in significantly increased AA levels, but do not alter IAA levels.
We next investigated the root phenotypes of the AxP lines. In control conditions, both lines displayed similar root gravitropic growth to, but slightly shorter roots than, the WT (Fig. S6b,c). After long-term estradiol treatment, the gravitropic growth of WT and AxP roots was slightly reduced, to a similar extent (Fig. S6b), while the root length of all genotypes was reduced, but more severely in AxP lines than the WT (Fig. S6c). To analyze root gravitropic responses in the AxP lines, we turned the seedlings 90° and subsequently measured the gravistimulated root bending All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint . http://dx.doi.org/10.1101/422733 doi: bioRxiv preprint first posted online Sep. 21, 2018; angles (Fig. S6d). We divided the total number of roots, by percentage, into several categories of bending angles (Fig. 3). In control conditions, Col-0 and both AxP lines responded to the gravistimulus with a very similar range of root bending angles, with the majority of roots bending 75-105° (Fig. 3). Estradiol treatment inhibited the gravitropic response of Col-0 roots, reducing their bending angles, resulting in a significant reduction in the proportion of total roots bending 75-105° and a significant increase in the proportion bending <75° (Fig. 3a,b). The AxP lines, however, responded differently to estradiol than the WT. As for the WT, estradiol treatment resulted in both a significant reduction in the proportion of AxP roots bending 75-105° and, in the case of AxP1, a significant increase in the proportion bending <75°, but additionally resulted in a significant increase in the proportion bending >105° in both AxP lines ( Fig. 3c-f). Therefore, while estradiol treatment specifically reduces root bending in the WT, this treatment results in both under-and over-bending in AxP1 roots and over-bending in AxP2 roots, in response to a gravistimulus. This suggests that increased AA levels in these lines interferes with root gravitropic responses, although we cannot rule out potential effects of changes in other IAA metabolite levels in root gravitropic responses.

PIN polarity in the stele is altered in wei2wei7 and partially rescued by ES8
ES8 has been shown to disturb auxin distribution patterns in the root by altering PIN polarity (Doyle et al., 2015a). Considering that IAA itself can influence its own transport by regulating PIN abundance at the plasma membrane (Paciorek et al., 2005;Robert et al., 2010), we reasoned that AA, as a precursor of IAA, might also play such a role. To investigate this possibility, we first studied the effects of long-term ES8 and AA treatments on the expression pattern of the auxin-responsive promoter DR5 in the root. To observe the effects of ES8 more easily, we used treatment at a high concentration of 15 µM, which led to a strong decrease in green fluorescent protein (GFP) signal in the stele of DR5::GFP WT roots (Fig. S7a), in agreement with previously published work (Doyle et al., 2015a). Furthermore, DR5::GFP crossed into the wei2wei7 background showed a similarly low GFP signal in the stele in control conditions, which was reduced even further by 15 µM ES8 treatment (Fig.   S7a). While 10 µM AA treatment did not noticeably affect the GFP signal in the stele of the WT, the signal in the wei2wei7 stele was rescued by this treatment (Fig. S7a).
These results suggest that AA may play a role in auxin distribution in the stele.
Next, we focused on the GFP signal in the root tip, particularly around the quiescent center (QC) and in the columella (Fig. S7b). We used both 5 and 15 µM ES8 treatment concentrations, which, in agreement with previously published work (Doyle et al., 2015a), led to an accumulation of GFP signal in cell file initials surrounding the QC in DR5::GFP WT, which were not labeled in control conditions ( Fig. S7b). This accumulation of signal was rather striking, extending into lateral columella and root cap cells, at the higher ES8 treatment concentration of 15 µM. As found for the stele, DR5::GFP crossed into the wei2wei7 background showed a similar GFP signal pattern in the root tip in control conditions as that induced by ES8 in the WT, with an accumulation of signal in the file initials surrounding the QC (Fig.   S7b). This signal pattern was also apparent in the wei2wei7 background after 5 µM ES8 treatment and was enhanced after 15 µM ES8 treatment. While 0.5 µM and 10 µM AA treatment did not noticeably affect the GFP signal in the root tip of Col-0, the signal in the wei2wei7 root tip was slightly increased by 0.5 µM AA treatment and rescued to that of the control WT by 10 µM AA treatment (Fig. S7b). Therefore, we observed a negative correlation between the DR5::GFP signal strength in the root stele and tip. We suggest that a balanced AA level is important for proper auxin distribution in both the root stele and tip, as both addition of exogenous AA/ES8 and deficiency in endogenous AA levels disturbed DR5::GFP signal patterns. Together, these results indicate that AA may indeed play a role in auxin distribution in the root stele and tip, which likely affects gravitropic growth. However, it is important to note that we cannot rule out possible effects of AA/ES8 on local auxin biosynthesis within specific groups of cells.
Our observations of DR5::GFP signal in the stele prompted us to investigate the rootward-to-lateral plasma membrane fluorescence ratio (hereafter referred to as rootward polarity) of PIN1, PIN3 and PIN7 in the provascular cells of Col-0 and wei2wei7 root tips. We treated seedlings short-term (2 h) with 15 µM ES8 or 10 µM AA, performed immunolabeling to observe endogenous PIN1 and PIN7 and used the PIN3::PIN3-GFP line crossed into the wei2wei7 background due to poor labeling of antibodies against PIN3. The fluorescence signals for these PIN proteins were consistently weaker in the mutant than in the WT (Fig. 4a-c), suggesting decreased abundance at the plasma membranes. As previously reported by Doyle et al. (2015a), short-term ES8 treatment significantly, albeit slightly, reduced immunolocalized PIN1 rootward polarity in Col-0 and importantly, AA treatment produced a similar result All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint . http://dx.doi.org/10.1101/422733 doi: bioRxiv preprint first posted online Sep. 21, 2018;(Fig. 4d). In contrast, PIN1 rootward polarity was significantly increased by about 20% in untreated wei2wei7 compared to Col-0, while ES8 treatment appeared to rescue this hyper-polarity of PIN1 in the mutant back to almost that of the WT (Fig.   4d). Although PIN3-GFP rootward polarity was not affected by ES8 or AA treatments in either the Col-0 or wei2wei7 backgrounds, it was increased by over 20% in the mutant compared to the WT (Fig. 4e). Finally, although PIN7 rootward polarity was not affected by ES8 or AA treatment in Col-0, it was strongly increased in the mutant compared to the WT, and like PIN1, was rescued in the mutant back to the level of the WT by ES8 treatment (Fig. 4f). These results suggest that AA may play a role in maintenance of PIN polarity in root provascular cells. One possible speculation on why treatment with AA, in contrast to ES8, did not rescue PIN1 or PIN7 polarity in the mutant may be a rapid conversion of AA to downstream IAA precursors within the seedlings.
As AA is a precursor of auxin, which is known to affect transcription of PIN genes (Vieten et al., 2005;Paponov et al., 2008), we investigated gene expression levels for all the plasma membrane-localized PIN proteins (PIN1, PIN2, PIN3, PIN4 and PIN7) in WT and mutant seedlings at 9 d old, the age at which we performed our root gravitropic growth and length studies. The expression levels of PIN1, PIN2 and PIN4 were strongly decreased in wei2wei7 compared to Col-0, while PIN3 and PIN7 expression levels were somewhat decreased, but not significantly (Fig. S8a). We next investigated the expression levels of PIN1, PIN3 and PIN7 in the same conditions used for our PIN polarity studies in root provascular cells (five-d-old seedlings treated with ES8 and AA for 2 h). At this stage, expression levels of PIN1, PIN3 and PIN7 were somewhat decreased in the mutant compared to the WT, but not significantly ( Fig. S8b). Furthermore, treatment with ES8 and AA did not significantly affect the expression of these genes (Fig. S8b). These results imply that while transcription of PIN genes is decreased in wei2wei7, the effects of ES8 and AA on PIN polarity are not due to PIN gene transcriptional changes. Overall, our data suggest that endogenous AA may play a role in regulating the polarity of PIN1, PIN3 and PIN7 in root provascular cells via a mechanism unrelated to PIN gene expression levels. We previously determined that ES8 targets a secretory pathway delivering newly produced PIN1 toward the rootward plasma membranes of root provascular cells (Doyle et al., 2015a) and it is tempting to speculate that AA might play regulatory roles in similar PIN trafficking routes in order to guide auxin distribution in the root.

AA regulates root gravitropism via repolarization of PIN3 and PIN7 in the columella
Our observations of DR5::GFP signal in the columella (Fig. S7b) indicate that AA may also play a role in auxin distribution specifically in this particular root tissue.
Additionally, previous studies of the expression patterns of ASA1 (WEI2) and ASB1 (WEI7) promoter-GUS fusions in dark-grown Arabidopsis roots revealed strong expression in the root meristem and columella (Stepanova et al., 2005). Plasma membrane-localized PIN3 and PIN7 in the columella are thought to act in redistribution of auxin in response to gravistimulus (Friml et al., 2002b;Kleine-Vehn et al., 2010), potentially redundantly with PIN4, which is also localized in columella cells (Friml et al., 2002a;Vieten et al., 2005). We therefore reasoned that high expression of anthranilate synthase genes in the columella may reflect a role of AA in regulating gravity-responsive polarity of these PIN proteins. First, to investigate the expression patterns of the ASA1 and ASB1 promoters in light-grown roots, we performed GUS staining of ASA1::GUS (WEI2::GUS) and ASB1::GUS (WEI7::GUS) seedlings. We observed strong expression of the ASB1 promoter, but not the ASA1 promoter, in the stele of the upper root, while neither ABA1 nor ASB1 promoter expression was detected in the lower part of the root excluding the root tip ( Fig.   S9a,b). We observed strong ASA1 and ASB1 promoter expression in the tip of the root meristem and in the columella, with ASA1::GUS expressed throughout the columella, while ASB1::GUS expression was limited to the innermost columella cells (Fig. S9c).
Next, we investigated the localization of endogenous PIN3, PIN4 and PIN7 in the columella of Col-0 and wei2wei7. Interestingly, the fluorescence intensity of these proteins was consistently increased in the innermost cells of the columella in wei2wei7 compared to Col-0 ( Fig. S9d-f), suggesting that the abundance and/or localization of these proteins are altered in the mutant columella. The antibodies against these PIN proteins did not label the outermost columella cells, in agreement with previous studies using PIN3 and PIN4 antibodies (Friml et al., 2002a;Friml et al., 2002b). We therefore continued our studies using PIN3::PIN3-GFP and PIN7::PIN7-GFP lines crossed into the wei2wei7 background ( Fig. 5a,b). We performed long-term treatments of these lines with ES8 and AA and investigated the shootward-plus-rootward to lateral-plus-lateral fluorescence ratio (hereafter referred to as shootward-rootward polarity) of the GFP-labeled PIN proteins. While the All rights reserved. No reuse allowed without permission.
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We next investigated gravity-induced relocalization of PIN3-GFP and PIN7-GFP in the columella. After a 90° gravistimulus for 30 min, about 15% more PIN3-GFP and PIN7-GFP were present on the now downward-facing (formerly lateralfacing) plasma membranes of the columella cells in WT seedlings ( To further investigate a potential role for PIN proteins in AA-regulated root gravitropism, we analyzed root gravitropic growth in a range of pin mutants and their crosses with wei2wei7. Interestingly, while the ethylene insensitive root1-4 (eir1-4, a pin2 allele) mutant showed intermediate root gravitropic growth between wei2wei7 and Col-0, crossing these mutants caused an additive effect, with wei2wei7eir1-4 being more severely agravitropic than wei2wei7 (Fig. S11a). Of the tested pin3, pin4 and pin7 alleles, none of the single mutants were affected in root gravitropic growth compared to the WT and introduction of the pin3-4 or pin7-2 mutations into the All rights reserved. No reuse allowed without permission.
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While pin1-501 showed increased root gravitropic growth compared to the WT, this mutation also partially rescued the root gravitropic growth of wei2wei7 (Fig. S11a).
We next tested the effects of long-term treatments with high concentrations of AA on root gravitropic growth in the mutants. Similarly to the WT, none of the pin mutants tested showed any sensitivity to AA in terms of changes in root gravitropic growth (Fig. S11b). While the introduction of pin3-4 or pin7-2 to wei2wei7 did not alter its sensitivity to AA in terms of increase in gravitropic index, crossing eir1-4 into wei2wei7 significantly increased its sensitivity to AA (Fig. S11b). In contrast, introduction of pin3-5, pin4-3 or pin1-501 to wei2wei7 reduced its sensitivity to AA, resulting in decreased rescue of root gravitropic growth (Fig. S11b).
The recovery of, as well as the reduction in AA-induced rescue of, wei2wei7 root gravitropic growth by introducing pin1, pin3 or pin4 mutations, provide further evidence for the involvement of PIN1 and PIN3, as well as implicating involvement of PIN4, in AA-regulated root gravitropism. It is unclear why pin3-5 but not pin3-4 partially rescues wei2wei7 gravitropism and suppresses rescue of wei2wei7 gravitropism by AA, but these different effects may be due to potential secondary mutations in one or both of the mutants, and/or the different positions of the T-DNA insertions. These insertions occur in an exon in pin3-4 and in the UTR preceding the start codon in pin3-5. The well-known important role of PIN2 in root gravitropism (Abas et al., 2006;Kleine-Vehn et al., 2008), however, is most likely not related to AA-regulated root gravitropism, considering the strong additive effect of eir1-4 and wei2wei7 mutations in reducing root gravitropic growth and increasing sensitivity to AA.
Taken together, our results strongly support a new role for endogenous AA in root gravitropism via regulation of selective PIN protein polarity and dynamics and thereby auxin distribution in both the stele and columella and that this role of AA is independent of its well-known function in IAA biosynthesis.

Discussion
We provide evidence in favor of a role for AA in root gravitropic growth through regulation of the subcellular localization of auxin transporter proteins, which likely All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/422733 doi: bioRxiv preprint first posted online Sep. 21, 2018; influences the flow of auxin within the organ. Following their synthesis, most plasma membrane-targeted proteins are sorted and packaged into selective secretory trafficking routes (Gendre et al., 2014). It has been shown, for instance, that the auxin importer AUXIN-RESISTANT1 (AUX1) and exporter PIN1, when targeted to shootward or rootward plasma membranes of root tip cells respectively, are transported in distinct endosomes, subject to control by different regulatory proteins (Kleine-Vehn et al., 2006). The trafficking routes of such proteins may be distinct even if targeted to the same plasma membrane, as is the case, for example, for AUX1 and PIN3 in epidermal hypocotyl cells of the apical hook (Boutté et al., 2013). Such a remarkably complex system of endomembrane trafficking pathways is thought to allow for a high level of control, suggesting the likely existence of an array of selective endogenous compounds and/or signals regulating these trafficking routes.
Once polar plasma membrane-targeted auxin carriers have reached their destination, they remain remarkably dynamic, being subject to constant vesicular cycling (Geldner et al., 2001) to enable rapid retargeting in response to external stimuli (reviewed by Luschnig & Vert, 2014;and Naramoto, 2017). Auxin itself promotes its own flow by inhibiting clathrin-mediated endocytosis of PIN transporters, thus enhancing their presence at the plasma membrane (Paciorek et al., 2005). Our results suggest that AA, an important early precursor in IAA biosynthesis, may also act on PIN plasma membrane localization to regulate the flow of auxin, through currently unknown mechanisms.
The use of pharmacological inhibitors, identified through chemical biology approaches, has proven a powerful strategy that has greatly assisted in unravelling the details of auxin transporter trafficking mechanisms (reviewed by Hayashi & Overvoorde, 2014;and Doyle et al., 2015b). We previously employed such a strategy, revealing that the AA analog ES8 selectively inhibits an early endoplasmic reticulum (ER)-to-Golgi secretory pathway, regulated by the adenosine diphosphate (ADP) ribosylation factor guanine nucleotide exchange factors (ARF-GEFs) GNOM and GNOM-LIKE 1 (GNL1), involved in rootward targeting of PIN1 without affecting the polarity of shootward plasma membrane proteins (Doyle et al., 2015a). We suggest that AA itself is likely to act endogenously on PIN trafficking regulation in a similar way to ES8, but detailed studies on AA mechanisms may prove difficult due to the potential conversion of AA to other IAA precursors in plant tissues. As ES8 appears to mimic the effects of AA on PIN localization and root gravitropic growth without All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint . http://dx.doi.org/10.1101/422733 doi: bioRxiv preprint first posted online Sep. 21, 2018; releasing AA through degradation and without affecting IAA levels, this synthetic compound provides great potential for understanding the mechanisms of AA on PIN localization in more detail, having already been extremely useful for distinguishing this newly discovered role of AA from its better-known role in auxin biosynthesis.
Any pharmacological treatments of biological tissues raise the question of compound uptake efficiency. Based on our analysis of the ES8 compounds inside plant tissues, we can conclude that uptake, either passive or active, of all these compounds occurs, with ES8 being taken up about 10 times faster than ES8.7 or ES8.7-Trp during short-term treatments. Although we currently do not know how these compounds enter plant tissues, one may speculate that AA and Trp transporters are likely to exist in planta, which might also transport ES8/ES8.7 and ES8.7-Trp, respectively. It will be of great interest in future studies to investigate the distribution and dynamics of compound uptake, which could potentially be observed by their labeling, fluorescently or otherwise.
Interestingly, the amino acid para-aminobenzoic acid, which has a similar structure to AA and is produced from the same precursor, has also recently been shown to play a role in root gravitropism, distinct from its better known role in folate biosynthesis (Nziengui et al., 2018). However, unlike AA, para-aminobenzoic acid promotes gravitropic root growth in WT plants as well as promoting gravistimulated root bending by enhancing the asymmetric auxin response between the two root sides (Nziengui et al., 2018). Another study linking AA, or more specifically Trp, with root bending, revealed that a mutation in the ASA1 gene confers a more compressed wavy root phenotype than the WT when seedlings are grown on agar surfaces tilted 30° from the vertical (Rutherford et al., 1998). As the mutant roots respond normally to gravistimuli when grown in non-waving conditions (within agar), it could be concluded that the root waving phenotype is not caused by agravitropism.
Furthermore, the phenotype is rescued in the mutant by supplementing with AA or Trp but not IAA. Mutations in the TRYPTOPHAN SYNTHASE α and β1 genes confer similar phenotypes, suggesting that a Trp deficiency is responsible. While these results suggest that Trp may be involved in regulating non-agravitropic root waving, potentially related to thigmotropism, independently of IAA, our study did not use tilted plates and moreover, in our conditions (vertical plates), single mutants in ASA1 and ASB1, namely wei2-2 and wei7-1, did not show differences in root growth compared to the WT (Fig. S12). We would therefore argue that the reduced All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint . http://dx.doi.org/10.1101/422733 doi: bioRxiv preprint first posted online Sep. 21, 2018; gravitropic index of wei2wei7 roots is indeed due to a gravitropic defect, caused by AA deficiency. It is however very interesting that Trp, another IAA precursor, appears to regulate root directional growth in response to another stimulus. Indeed, the existence of several complex root growth regulatory mechanisms is hardly surprising considering the remarkable plasticity of this organ, the growth of which must respond to a wide array of internal signals and external stimuli, including gravity, touch, light, temperature, humidity and various chemical substances.
The agravitropic growth of wei2wei7 roots may be due to a combination of decreased auxin content caused by reduced AA levels and the AA deficiency itself, as both auxin and AA affect the localization of PIN proteins. As was shown previously for ES8 (Doyle et al., 2015a), AA appears to act selectively depending on the PIN protein and the root tissue. PIN1, PIN3 and PIN7 all display increased rootward polarity in provascular cells of wei2wei7 compared to Col-0, suggesting increased flow of auxin toward the root tip in the mutant. Correspondingly, we found decreased expression of the auxin-responsive promoter DR5 in the root stele and increased expression around the root tip QC in the mutant, a pattern that was also observed in WT roots upon ES8 treatment. PIN7, but not PIN3, is also abnormally polarized in columella cells of wei2wei7, while both these proteins appear to be completely unresponsive to gravistimulus in the mutant columella. Furthermore, the high expression of ASA1 (WEI2) and ASB1 (WEI7) in the root columella of the WT suggests the importance of AA in this tissue in particular, which our results suggest is due to a role for this compound in gravity-regulated PIN distribution amongst the plasma membranes. The particular importance of PIN1 and PIN3 in AA-regulated root gravitropism was further supported by the rescue, as well as the reduction in AAinduced rescue, of wei2wei7 root gravitropic growth by introduction of pin1 or pin3 mutations. Taken together, our results suggest that the endogenous compound AA plays a role in root gravitropism by regulating the polarity and gravity-induced relocalization of specific PIN proteins in the provascular and columella cells.
Furthermore, this role of AA is distinct from its well-known function in auxin biosynthesis, which we suggest is more important for root elongation than gravitropic growth.
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The copyright holder for this preprint . http://dx.doi.org/10.1101/422733 doi: bioRxiv preprint first posted online Sep. 21, 2018; standard error of the mean of the biological replicates. Values in square brackets indicate treatment concentrations in µM. Tissue was sampled from a mixture of 20 ground seedlings per sample per each of five (a) or four (b) biological replicates.  (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Supporting Information
Additional supporting information may be found in the online version of this article: Methods S1 Selection of homozygous crossed mutants, generation of 35S::ASA1 (35S::WEI2) and XVE::amiRNA-PAT1 lines and root growth measurements.

Methods S2
Chemical synthesis and IAA metabolite analysis.

Methods S3 Compound degradation analysis.
Methods S4 qPCR, GUS staining and generation of PIN7 antibody.

Methods S5 Microscopy image quantifications.
Table S1 Arabidopsis thaliana mutants and transformed lines used in this study.

Table S2
Genotyping, cloning and qPCR primers used in this study.

Fig. S1
AA and other IAA precursors are deficient in wei2wei7.
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Fig. S2
Effects of AA and ES8 on root gravitropic growth and length in the wild type.

SI References
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. The following Supporting Information is available for this article: Methods S1 Selection of homozygous crossed mutants, generation of 35S::ASA1 (35S::WEI2) and XVE::amiRNA-PAT1 lines and root growth measurements.

Methods S2
Chemical synthesis and IAA metabolite analysis.

Methods S3 Compound degradation analysis.
Methods S4 qPCR, GUS staining and generation of PIN7 antibody.

Table S1
Arabidopsis thaliana mutants and transformed lines used in this study.

Table S2
Genotyping, cloning and qPCR primers used in this study.

SI References
Methods S1 Selection of homozygous crossed mutants, generation of 35S::ASA1 (35S::WEI2) and XVE::amiRNA-PAT1 lines and root growth measurements. For selection of homozygous lines at the F2 generation, seedlings displaying a wei2wei7 phenotype were initially selected and then genotyped for the third mutation for triple mutants (see Table S2 for primers used), or observed on a fluorescence stereomicroscope for GFP marker line crosses. Finally, selected lines were confirmed for homozygous wei2-1 and wei7-1 mutations by genotyping (see Table S2 for primers used). The heterozygous line pin1-501 (we added 01 to the name of this mutant to distinguish from another pin1-5 allele) and its cross with wei2wei7 were transferred to soil after imaging on treatment plates and only those seedlings that later formed pin-like inflorescences were included in root measurements in the initial images.
For generation of 35S::ASA1 and XVE::amiRNA-PAT1 lines, see Table S2 for primers used. For constitutive overexpression of ASA1 (AT5G05730), the coding region of the gene was amplified from Arabidopsis thaliana Col-0 cDNA. Primers for artificial microRNA (amiRNA) to knock down the PAT1 (AT5G17990) gene were designed using the Web MicroRNA Designer tool (http://wmd3.weigelworld.org). The amiRNA was obtained using the pRS300 vector as a PCR template, as described previously (Ossowski et al., 2008). The fragments were introduced into the Gateway pENTR/D-TOPO cloning vector (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and verified by sequencing. The ASA1 coding sequence was then cloned into the DL-phosphinothricinresistant vector pFAST-R02 (Shimada et al., 2010), while the amiRNA was cloned into the hygromycinresistant vector pMDC7b containing the estradiol-inducible XVE system (Curtis & Grossniklaus, 2003),

using the LR reaction (Invitrogen). Agrobacterium-mediated transformation of the constructs into
Arabidopsis thaliana Col-0 was achieved by floral dipping (Clough & Bent, 1998). Transformed plants were selected via antibiotic resistance on agar plates supplied with the respective antibiotics, 50 µg ml -1 hygromycin B or 25 µg ml -1 DL-phosphinothricin. Four and six independent homozygous 35S::ASA1 and XVE::amiRNA-PAT1 lines were analyzed, respectively. Two lines per construct were then selected that displayed strong and reproducible constitutive ASA1 induction or induced PAT1 silencing, as determined by performing qPCR (Methods S4) using RNA extracted from 1-wk-old whole seedlings ( Fig. S6a,b). To induce silencing in the XVE::amiRNA-PAT1 seedlings, the seedlings were germinated and grown on agar plates supplemented with 20 µM estradiol, with DMSO used as a mock treatment control. Each selected 35S::ASA1 line was crossed with each selected XVE::amiRNA-PAT1 line and homozygous F2 generation offspring, named AxP (ASA1 x PAT1) lines, were selected as before via antibiotic resistance. The homozygous lines were then tested for gene expression as before via qPCR (12 independent lines were analyzed) (Fig. S6c,d). Finally, two lines displaying reproducible simultaneous constitutive ASA1 induction and induced PAT1 silencing were selected for use in further experiments.
All root length and gravitropic index measurements were performed on 9-d-old seedlings, while microscopy studies were performed on 5-d-old seedlings due to cell collapse in a proportion of the tips of older wei2wei7 roots. ImageJ software (https://imagej.nih.gov/ij/) was used to measure root length and vertical gravitropic index, which was calculated for each root as a ratio of L y : L, where L y is the vertical distance from root base to tip, or the real depth of root tip penetration, and L is the root length, as described in Grabov et al. (2005). For gravistimulated root bending experiments, seedlings were grown vertically for 5 d in treatment-free conditions, then transferred to mock or estradiol-supplemented medium for 24 h, and then gravistimulated by turning the plates 90° for 24 h before measuring the root bending angles. The angles were measured in the direction of root bending between two lines intersecting at the former root tip position before gravistimulus, one being horizontal and the other originating from the current root tip position (Fig. S6d).

Methods S2
Chemical synthesis and IAA metabolite analysis. The following procedure was used to synthesize (4-(N-(4-chlorobenzyl) methylsulfonamido)benzoyl)tryptophan (ES8.7-Trp). Methyl sulfonyl chloride (3.1 ml, 45.5 mmol, 1.2 equivalent) and pyridine (3 ml, 37.1 mmol, 1.1 equivalent) were added to a solution of 4-aminoethylbenzoate (5.1 g, 33.7 mmol) in acetonitrile (100 ml) at 0 °C and then stirred overnight at room temperature. The reaction mixture was concentrated and the resulting residue was dissolved in ethyl acetate. The organic layer was washed with HCl (2 N, 100 ml), saturated NaHCO 3 , H 2 O and brine, dried over Na 2 SO 4 , and concentrated to give an off-white solid (7.1 g, 94%), ethyl 4-(methylsulfonamido) benzoic acid, which was used without further purification. Then 4-chlorobenzyl bromide (1.43 ml, 11 mmol, 1.2 equivalent) and K 2 CO 3 (3.8 g, 27.4 mmol, 3 equivalent) were added to a solution of ethyl 4-(methylsulfonamido)benzoate (2.1 g, 9.15 mmol) in dimethylformamide (DMF) (14 ml). The reaction mixture was stirred overnight after which liquid chromatography-mass spectrometry indicated the reaction was complete, yielding 4-(N-(4-chlorobenzyl)methylsulfonamido) ethylbenzoate (2.8 g, 88%). Next, NaOH (2 N, 15 ml) and H 2 O (10 ml) were added to 2 g of 4-(N-(4chlorobenzyl)methylsulfonamido)ethylbenzoate, followed by overnight stirring. The reaction mixture was acidified to pH 5 with concentrated HCl and partitioned between ethyl acetate and H 2 O. The organic layer was washed with brine, dried over Na 2 SO 4 , and concentrated to give 4-(N-(4chlorobenzyl)methylsulfonamido)ethylbenzoate (1.7 g, 92%), which was used without further purification. Then 10 ml thionyl chloride was added to a solution of 1 mmol 4-(N-(4chlorobenzyl)methylsulfonamido)ethylbenzoate and refluxed for 12 h under a nitrogen atmosphere. The thionyl chloride was removed under reduced pressure to give acid chloride. Then 1.2 mmol tryptophan was dissolved in 5 ml DMF and cooled to 0 °C. At this temperature the acid chloride was added slowly by dissolving in 3 ml DMF. The reaction mixture was stirred at room temperature for 24 h. Ice cold water was added to the reaction mixture, which was then extracted with chloroform and washed with brine solution three times to afford the crude mixture. Finally, purification with column chromatography using methanol and chloroform as a solvent system resulted in a light yellow solid, (4-(N-(4chlorobenzyl) methylsulfonamido)benzoyl)tryptophan (ES8.7-Trp) with 68% yield.
For IAA metabolite analysis, frozen samples were homogenized using a MixerMill bead mill (Retsch GmbH, Haan, Germany) and extracted in 1 ml of 50 mM sodium phosphate buffer containing 1% sodium diethyldithiocarbamate and a mixture of 13 C 6 -or deuterium-labeled internal standards. After pH adjustment to 2.7 by 1 M HCl, a solid-phase extraction was performed using Oasis HLB columns PP2A PDF2 (AT1G13320) were analyzed alongside the target genes (see Table S2 for primers used).
Expression levels of the target genes were normalized against the two most stably expressed reference genes, as determined using GeNorm (Biogazelle, Ghent, Belgium) (Vandesompele et al., 2002), using the formula below, where E = efficiency, R = reference gene, Cq = quantification cycle mean, T = target gene. For each target gene, the normalized expression values were scaled relative to that of the WT control.
Normalized expression = √( 1 1 + 2 2 ) ⁄ For GUS staining, seedlings were fixed in 80% acetone at -20 °C for 20 min, washed three times with distilled water and then incubated in 2 mM X-GlcA in GUS buffer (0.1% triton X100, 10 mM EDTA, 0.5 mM potassium ferrocyanide and 0.5 mM potassium ferricyanide in 0.1 M phosphate buffer (Na2HPO4 / NaH2PO4) at pH 7). The samples were then infiltrated for 10 min in a vacuum desiccator before incubation in the dark at 37 °C. The GUS reaction was stopped by replacing the GUS buffer with 70% ethanol. Samples were then mounted in 50% glycerol and observed on a Zeiss (Oberkochen, Germany) Axioplan microscope.
For the generation of anti-PIN7, a region of 882 bp corresponding to the hydrophilic loop of PIN7 was amplified and attB1 and attB2 recombination sites were incorporated (see Table S2 for primer sequences). The amplicon was recombined into the pDONR221 vector (Invitrogen) and the resulting Methods S5 Microscopy image quantifications. For live imaging, seedlings were mounted in their treatment medium for microscopic observations and images were acquired using identical acquisition parameters between mock and chemical treatments and between the WT and mutant in each experiment.
For PIN rootward (basal) polarity index quantification in stele provascular cells, the 'mean gray area' tool in ImageJ was used to measure plasma membrane fluorescence intensity in confocal images and a rootward to lateral fluorescence ratio was calculated for each cell measured. For PIN shootwardrootward polarity index measurements in columella cells, the shootward-plus-rootward to lateral-pluslateral plasma membrane fluorescence ratio was calculated. To monitor gravitropically induced PIN relocalization in columella cells, confocal z-stacks were acquired directly before and after a 30 min gravistimulation, during which the seedlings were rotated 90° to the horizontal position. Fluorescence intensity at the shootward (apical), rootward and lateral plasma membranes of the left-and right-most columella cells was measured on maximal intensity projections of the z-stacks using ImageJ (see Fig.   S10 for examples of the cells measured). The percentage of fluorescence intensity distributed on each of the four plasma membranes of the same cell was calculated before and after gravistimulation.
Relocalization was calculated by comparing the percentage of signal on the right-most lateral plasma membrane of the cell (which became the downward-facing plasma membrane during the gravistimulus) before and after gravistimulation. Table S1: Arabidopsis mutants (a) and transformed lines (b) used in this study. SALK/SAIL T-DNA insertion lines (Alonso et al., 2003) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). Arabidopsis Genome Initiative (AGI) codes are included for all genes. We renamed pin1-5 SALK line 097144 to pin1-501 to distinguish the mutant from another published pin1-5 allele.

a) Mutants
Mutant name ID / mutation type Gene name(s) AGI code(s) Reference(s) Stepanova et al. (2005) b) Transformed lines  Table S2: Genotyping (a), cloning (b) and qPCR (c) primers used in this study. Primers marked SALK were sourced from the SALK Institute Genomic Analysis Laboratory website (http://signal.salk.edu/). Primers with no indicated source have not been published previously (sequences designed in the present study or kindly provided by collaborators). Arabidopsis Genome Initiative (AGI) codes are included (for affected gene AGIs in mutants listed in (a) see Table S1).  All rights reserved. No reuse allowed without permission.

a) Genotyping primers
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Fig. S4
The ES8 compounds are not degraded/metabolized to release AA or Trp. Samples of Arabidopsis thaliana Col-0 and wei2wei7 seedlings after short-term (5-d-old seedlings incubated 5 h in liquid treatment medium) or long-term (9-d-old seedlings grown on solid treatment medium) treatments with ES8 compounds were analyzed for concentrations (Conc) of various compounds. Samples of the short-term and long-term treatment medium, which were incubated for the same length of time but without any seedlings added, were also analyzed. All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint . http://dx.doi.org/10.1101/422733 doi: bioRxiv preprint first posted online Sep. 21, 2018; Normalized expression Line no. and treatment All rights reserved. No reuse allowed without permission.
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Fig. S10
Gravitropic relocalization of PIN3-and PIN7-GFP in root columella cells. Representative images of GFP fluorescence in PIN3::PIN3-GFP (a-b) and PIN7::PIN7-GFP (c-d) in root columella cells of 5-d-old Arabidopsis thaliana Col-0 (a, c) and wei2wei7 (b, d) seedlings grown on anthranilic acid (AA) and Endosidin 8 (ES8)-supplemented medium directly before and after a 90º gravistimulus of 30 min, during which the seedlings were turned clockwise (the left-and right-facing plasma membranes became upward-and downward-facing, respectively). Arrows represent the direction of the gravity vector immediately prior to image acquisition. Scale bars represent 10 µm. Single confocal plane images are displayed for clarity, but maximal intensity projections of confocal z-stacks were used for the plasma membrane fluorescence analyses displayed in Fig. 5c-f. In the example images where asterisks are shown, these indicate the cells for which fluorescence was measured.
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