Auxin steers root cell expansion via apoplastic pHregulation in Arabidopsis thalianaElke Barbeza,b,1, Kai Dünserb, Angelika Gaidoraa, Thomas Lendla, and Wolfgang Buscha,c,1

aGregor Mendel Institute, Austrian Academy of Sciences, Vienna Biocenter, 1030 Vienna, Austria; bDepartment of Applied Genetics and Cell Biology,University of Natural Resources and Life Sciences, 1190 Vienna, Austria; and cPlant Biology Laboratory, Salk Institute for Biological Studies, La Jolla,CA 92037

Edited by Mark Estelle, University of California at San Diego, La Jolla, CA, and approved May 8, 2017 (received for review August 12, 2016)

Plant cells are embedded within cell walls, which provide structuralintegrity, but also spatially constrain cells, and must therefore bemodified to allow cellular expansion. The long-standing acid growththeory postulates that auxin triggers apoplast acidification, therebyactivating cell wall-loosening enzymes that enable cell expansion inshoots. Interestingly, this model remains heavily debated in roots,because of both the complex role of auxin in plant development aswell as technical limitations in investigating apoplastic pH at cellularresolution. Here, we introduce 8-hydroxypyrene-1,3,6-trisulfonic acidtrisodium salt (HPTS) as a suitable fluorescent pH indicator forassessing apoplastic pH, and thus acid growth, at a cellular resolutionin Arabidopsis thaliana roots. Using HPTS, we demonstrate that cellwall acidification triggers cellular expansion, which is correlated witha preceding increase of auxin signaling. Reduction in auxin levels,perception, or signaling abolishes both the extracellular acidificationand cellular expansion. These findings jointly suggest that endogenousauxin controls apoplastic acidification and the onset of cellularelongation in roots. In contrast, an endogenous or exogenous in-crease in auxin levels induces a transient alkalinization of the ex-tracellular matrix, reducing cellular elongation. The receptor-likekinase FERONIA is required for this physiological process, whichaffects cellular root expansion during the gravitropic response.These findings pinpoint a complex, presumably concentration-dependent role for auxin in apoplastic pH regulation, steering therate of root cell expansion and gravitropic response.

apoplastic pH | auxin | cellular expansion | root growth | root gravitropism

Plant cells are surrounded by a rigid cell wall, which providesform and stability, enabling plants to grow to extreme heights

despite the absence of a skeleton. However, these advantagescome with the price that plant cells are encased within the stiffcell wall matrix, which must be remodeled to allow for cellularelongation. How cell walls are modified to enable cellular expan-sion has been of scientific interest since the 1930s, as insight intothis physiological process would provide a wealth of knowledge onhow plants grow (1). In the early 1970s, a physiological mechanismexplaining cell expansion, the acid growth theory, was proposed(2–4). This theory postulates that the plant hormone auxin triggersthe activation of plasma membrane (PM)-localized H+-ATPases(proton pumps), resulting in acidification of the intercellular space(apoplast). The reduction in apoplastic pH activates cell wall-loosening enzymes, which, in concert with turgor pressure, en-ables cellular expansion (1). Auxin was the first plant hormoneshown to be involved in processes important for plant growth anddevelopment, including tissue growth, apical dominance, woundresponse, flowering, and tropisms, such as the gravitropic response(5). Auxin is known to play a complex role in plant growth regu-lation, as it can both stimulate and inhibit tissue expansion,depending on the tissue and its concentration (6–8). A positiveeffect of auxin on growth was hypothesized by the acid growththeory (1). Subsequent literature provided significant insight intothe molecular mechanisms of auxin-triggered acid growth inshoots (9–13). However, in roots, the acid growth theory remainsthe subject of debate. On one hand, several studies report the

stimulating effect of apoplast acidification on cell expansion inroots, as well as the requirement of functional PM H+-ATPases forroot growth (14–16). On the other hand, high auxin concentrationsare known to inhibit root cell expansion and overall root growth (8,17). Moreover, exogenous auxin application has been described totrigger apoplast alkalization in roots, which is the opposite effect asin shoots (18–20). Notably, a recent study provides substantialtranscriptomic insight into auxin-triggered cell wall modificationand cell expansion in Brachipodium distachion roots (21). However,the authors also observed that medium acidification does not cor-relate with root cell elongation (21). Notably, most of the afore-mentioned studies indirectly investigated apoplast acidification bymeasuring pH alterations in the medium, thereby failing to directlyassess the apoplastic pH at cellular resolution. The discrepancies inthe current literature point to a complex role for auxin in apoplasticpH homeostasis and highlight the need to reassess the acid growththeory at the cellular level.Here, we introduce 8-hydroxypyrene-1,3,6-trisulfonic acid tri-

sodium salt (HPTS) as a suitable fluorescent pH indicator forassessing apoplastic pH at a cellular resolution. Using HPTS, wedissected the apoplastic pH dynamics in A. thaliana roots andshow that root cell expansion correlates with its acidification andincreased nuclear auxin signaling. In agreement, interference withendogenous auxin levels or signaling abolishes acidification andelongation. However, we also find that exogenous and endogenousincreases in cellular auxin accumulation lead to a transient alka-lization of the apoplast, correlating with the inhibition of root cellexpansion. A significant proportion of this transient alkalization

Significance

Cellular growth in plants is constrained by cell walls; hence,loosening these structures is required for growth. The long-standing acid growth theory links auxin signaling, apoplasticpH homeostasis, and cellular expansion, providing a conceptualframework for cell expansion in plant shoots. Intriguingly, thismodel remains heavily debated for roots. Here, we present afluorescent dye that allows for the correlation of cell size andapoplastic pH at a cellular resolution in Arabidopsis thaliana.This enabled us to elucidate a complex involvement of auxin inroot apoplastic pH homeostasis, which is important for root cellexpansion and gravitropic response. These findings shed lighton the poorly understood acid growth mechanism in roots.

Author contributions: E.B. and W.B. designed research; E.B., K.D., and A.G. performedresearch; T.L. contributed a new analytic tool; E.B., K.D., and A.G. analyzed data; and E.B.and W.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The raw data files of the presented experiments have been uploaded tothe Dryad database, datadryad.org (doi: 10.5061/dryad.sq7s3).1To whom correspondence may be addressed. Email: wbusch@salk.edu or elkebarbez@gmail.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1613499114/-/DCSupplemental.

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is dependent on the receptor-like kinase FERONIA. Takentogether, our data suggest a complex role of auxin in apoplasticpH regulation, which is important for root organ growth andgravitropic response.

ResultsHPTS Enables the Assessment of Apoplastic pH at a Cellular Resolution.To efficiently dissect acid growth in A. thaliana roots, we aimed toidentify a fluorescent dye that would enable the assessment ofapoplastic pH with a cellular resolution. We screened the literaturefor nontoxic, fluorescent, pH-sensitive dyes that are also watersoluble so they would easily penetrate the root apoplast, but notenter the root cells (22). Our search identified HPTS as a suitablecandidate to assess apoplastic pH in A. thaliana roots. HPTS is awater-soluble fluorescent dye displaying pH-dependent spectralcharacteristics (23). This fluorescent pH indicator has been pre-viously described to be suitable for the pH assessment of neuronalorganelles, as well as liposomes designed for drug delivery (24–26).In plants, HPTS has been used to define the pH of extractedapoplastic fluid from plant tissues (27, 28).To test whether HPTS can be used to directly assess apoplastic

pH with a cellular resolution in planta, we immersed 4-d-old seed-lings in HPTS containing liquid growth medium for 30 min andimaged roots, hypocotyls, and cotyledons, using confocal microscopy(Fig. S1A). The protonated and deprotonated forms of HPTS werevisualized in 2 different channels with excitation wavelengths of405 and 458 nm, respectively. The signal was restricted to the apo-plastic area, confirming that the cells do not take up HPTS (Fig.S1A). This is crucial for the specific assessment of pH in the cell wall.Next, we tested whether HPTS can reliably record pH changes.

We therefore adjusted the apoplastic pH of 4-d-old seedlings byincubating them for 30 min in HPTS containing liquid growthmedium with different pH levels, and imaged them as describedearlier. Dividing the signal intensity of the 458-nm channel by the405-nm channel for each pixel resulted in a ratiometric image fromwhich the intensity values correlate with the apoplastic pH (Fig.S1B). We designed a custom-made Fiji (29) script to enable fast andconsistent ratiometric image conversion with an easy color code-based read out (Dataset S1). Indeed, increases in the pH of themedia led to an increase in the apoplastic pH, as indicated by the458/405 ratio (Fig. S1B). This indicates that HPTS is suitable todetect exogenously imposed apoplastic pH changes.Next, we tested whether HPTS allows for the detection of

physiological changes in apoplastic pH. To accomplish this, wepharmacologically modulated the activity of the PM-localizedH+-ATPases and analyzed the 458/405 ratio after HPTS staining andimaging. Fusicoccin treatment, which activates the PM H+-ATPases(30), resulted in decreased 458/405 ratio values, suggesting a decreasein apoplastic pH (Fig. 1A). To test whether we can also measure anincrease in apoplastic pH, we used N,N′-dicyclohexylcarbodiimide(DCCD), which is a chemical known to inhibit the proton-conductingactivity of H+-ATPases, including the PMH+-ATPases (31). DCCDtreatment gave rise to higher 458/405 values, indicating a higherapoplastic pH (Fig. 1B). Finally, we used HPTS to measure apo-plastic pH on genetic perturbation by analyzing OPEN STOMATA2 (ost2-2) mutant seedlings. The ost2-2 mutant expresses the con-stitutively active PM H+-ATPase AHA1 (32). Compared with WTseedlings, ost2-2 mutants displayed lower 458/405 values (Fig. 1C),confirming the apoplastic acidification in ost2-2mutants (29). Thesedata show that HPTS is able to report on physiological changes inapoplastic pH.To obtain a reliable measure of the apoplastic pH in Arabidopsis

roots, we performed an in vivo calibration of the HPTS dye. Forthis, we stained 4-d-old seedlings for 30 min in HPTS and rinsedthe roots with medium adjusted to pH values between 4.6 and 6.4.The 458/405 ratios were used to plot a calibration curve fromwhich a best-fitting regression curve was calculated (Fig. S2). Al-though we cannot exclude the possibility that a potential buffering

capacity of the root (33) affects our measurements, these short-term treatments allowed us to approximate an absolute pH valueof 5.4 in the cell wall of meristematic cells (Fig. 1 E and F). Ac-cordingly, we conclude that HPTS is a suitable dye for assessingapoplastic pH in Arabidopsis roots at a cellular resolution.

Apoplastic pH Determines Cellular Expansion in A. thaliana Roots. Hav-ing established an accurate method for assessing apoplastic pH, ournext step in dissecting acid growth in A. thaliana roots was to de-termine the relationship between apoplastic pH and root cell ex-pansion. Several studies have reported a stimulating effect ofapoplast acidification on root cell expansion in maize, pea, and bean(14, 15). To assess apoplastic pH dynamics in A. thaliana roots, wemeasured the epidermal cell length of root cells, beginning at theheight of the quiescent center, and recorded their correspondingapoplastic pH (Fig. 1 D–G). Under our growth and experimentalconditions, cells in the meristematic zone harbor an approximateapoplastic pH of 5.4. During cellular elongation, we observed thatthe apoplastic pH decreases below pH 5 (Fig. 1 D–G). When rootcells reach their final length in the differentiation zone, the apo-plastic pH increases again, suggesting apoplast acidification onlyoccurs in elongating cells (Fig. 1F and Fig. S1C).This correlation led us to test whether apoplast acidification

induces root cell elongation. Therefore, we transferred 4-d-oldseedlings (grown on standard medium with pH 5.8) to growthmedium with a pH of 4.6 for 2.5 h. These seedlings displayedlonger epidermal cells in the distal (late) meristem (the last4 cells of the meristem) compared with seedlings transferred tocontrol medium with a pH of 5.8 (Fig. 1I). These data indicatethat exogenously imposed apoplast acidification is sufficient totrigger root cell expansion. Seedlings transferred to pH 4.6 me-dium also display longer epidermal cells in the proximal (early)meristem and differentiation zone compared with control seed-lings, indicating that these cells are also responsive to exoge-nously imposed apoplast acidification (Fig. S3 A and B). We nexttested the opposite response: whether apoplast alkalizationcan inhibit cell elongation. Indeed, seedlings transferred topH 6.4 medium for 2.5 h displayed shorter epidermal cells in thedistal meristem compared with seedlings transferred to controlmedium (Fig. 1I). These data indicate that apoplast acidificationcorrelates with and affects root cell expansion in A. thaliana. Wenote that, in this study, we focus on epidermal root cells in thestage just before elongation. From here on, the term root epi-dermal cells comprises the root epidermal cells in the distalmeristem (including the last four cells of the meristem).After having established that our dye faithfully reports pH

changes, we studied the relationship between cell elongation andapoplastic pH. To test whether root cell elongation is affected by aphysiological change in apoplastic pH, we treated seedlings withfusicoccin for 19 h and observed longer epidermal root cells com-pared with in mock-treated seedlings (Fig. 1J). In agreement withthis, ost2-2 mutant seedlings, which had a lower apoplastic pHcompared with WT seedlings (Fig. 1C), also displayed longer rootepidermal cells compared with WT seedlings (Fig. 1K). Eventhough DCCD treatment significantly increased the root apoplasticpH (Fig. 1B), we did not observe differences in cell length under ourgrowth and experimental conditions (Fig. S3D). However, previousliterature reports an inhibiting effect of DCCD on root cell elon-gation in A. thaliana roots (34). Altogether, our data show thatapoplast acidification and alkalization promotes and inhibits epi-dermal root cell expansion, respectively.

Auxin Signaling Affects Apoplastic pH Regulation. The phytohor-mone auxin has been shown to be indispensable for plant growthand development because of its involvement in a broad spectrumof plant developmental processes (35). In plant aerial tissues,auxin is known to trigger the activation of PM-localized pro-ton pumps (PM H+-ATPases), resulting in acidification of the

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Fig. 1. Apoplastic pH and epidermal cell length correlation in A. thaliana roots. (A and B) The effect of 5 μM fusicoccin (FC) treatment for 60 min and 5 μMDCCD treatment for 17 h on the apoplastic pH in A. thaliana roots, visualized using HPTS staining. y-axis: mean 458/405 values of epidermal cells in rootmeristems of the pharmacologically treated seedlings relative to mock-treated seedlings. (C) Comparison of the apoplastic pH in WT and ost2-2 mutantseedlings, visualized by HPTS staining. y-axis: the mean 458/405 values of the ost2-2 mutant roots relative to the WT. (A–C) Error bars represent SEM (n ≥13 roots per line/condition). Student t test P values: *P < 0.05, **P < 0.01, ***P < 0.001. (D, E, and G) Epidermal cell length with the corresponding absoluteapoplastic pH, as visualized using HPTS staining. Error bars represent SEM (n = 11 roots). (F) Apoplastic pH of the epidermal apoplast in the root meristem,elongation, and differentiation zones (Fig. S1C). Error bars represent SEM. Statistical significance (0.05%) was tested using a one-way ANOVA test with aTukey-Kramer post hoc test (n ≥ 14 roots). (H and I) Epidermal cell length in the distal meristem of seedlings grown on pH 5.8 growth medium and transferredfor 2.5 h on pH 4.6 or 6.4 growth medium. (J) The effect of 19 h of 10 μM FC treatment on the root epidermal cell length. (K) Root epidermal cell length of WTand ost2-2 seedlings. (H–K) Error bars represent SEM. (n ≥ 13 roots). Student t test P values: *P < 0.05, **P < 0.01, ***P < 0.001. (A–C and G) Color code (blackto white) depicts (low to high) 458/405 intensity, and thus, pH values. (Scale bars: 10 μm.)

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apoplast (1). Our data suggest apoplast acidification is also im-portant for root cell elongation in A. thaliana. To investigate therole of auxin in controlling apoplast acidification, and therebyroot cell expansion, we used the auxin response reporter linesDR5v2:n3GFP and R2D2. DR5v2:n3GFP expresses a GFP re-porter with a nuclear localization signal under a synthetic auxininducible promoter (36). R2D2 seedlings express a Venus-taggedauxin degradable reporter protein (DII:n3xVenus) under controlof an RPS5A promoter along with an RFP-tagged undegradableprotein (mDII:ntdTomato), allowing for the quantitative assess-ment of auxin responses (36). In root epidermal cells, DR5v2:n3GFP expression is very low in the meristem, but gradually in-creases toward the elongation zone (Fig. 2 A, C, and D). DII-Venussignal intensity, normalized by the mDII signal, is strong in epider-mal, meristematic root cells and gradually decreases toward theelongation zone (Fig. 2 B, C, and E). The increased DR5v2:n3GFPexpression along with the decreased DII/mDII signal intensity ratio,both indicating increased auxin signaling, suggest an increase in auxinsignaling in epidermal root cells at the onset of elongation (Fig. 2 A–E). This raises the question of whether auxin could be involved in theapoplast acidification required for root cell expansion.To test this hypothesis, we analyzed apoplastic pH as well as root

epidermal cell length in mutants that are compromised in auxincontent and signaling. Proteins from the GRETCHEN HAGEN3 family are conjugating enzymes, of which some, includingGH3.5 and GH3.6, conjugate auxin to amino acids (37), and thuslower the levels of cellular free auxin. To transiently decrease freeauxin levels, we induced GH3.6 expression in an estradiol-inducible

GH3.6 line and analyzed the effects on apoplastic pH and cell size.On 6 h of induction, we observed higher apoplastic pH in GH3.6-induced seedlings compared with control (empty vector expressing)seedlings (Fig. 3 A and B). At that point, we did not observe asignificant effect of GH3.6 expression on cell length. However, 19 hof GH3.6 expression significantly decreased the epidermal root cellsize compared with control seedlings (Fig. 3C). These data suggestdecreased endogenous auxin levels give rise to a higher apoplasticpH and subsequent inhibition of cell elongation.Next, we genetically interfered with the auxin signaling pathway.

Transcriptional auxin responses are known to be mediated by thenuclear TIR/AFB auxin signaling pathway. Auxin binding to mem-bers of the TIR1/AFB F-box family receptors triggers the protea-somal degradation of AUX/IAA repressors. This degradation causesthe subsequent release of AUXIN RESPONSE FACTOR (ARF)transcription factors, which then execute auxin responsive gene ex-pression (38). Auxin receptor triple mutant seedlings, tir1afb2afb3,display a higher apoplastic pH along with smaller epidermal cellscompared with WT seedlings (Fig. 3 D–F). This suggests TIR/AFB-dependent auxin signaling is required for normal apoplast acidifi-cation with a concomitant effect on root cell elongation.We also aimed to test the effect of transiently compromised

auxin signaling. bodenlos (bdl) is a mutant allele of IAA12 thatproduces a nondegradable form of the AUX/IAA repressorIAA12 (39). Hence, induction of bdl expression inhibits auxinsignaling (40). We analyzed the apoplastic pH and root cell sizeof seedlings in which bdl expression was induced. On 6 h in-duction, bdl-expressing seedlings displayed a higher apoplastic

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pH compared with control seedlings containing an empty vector(Fig. 3 G and H). Similar to GH3.6 expression, we only observedan inhibitory effect on root cell elongation on 19 h of bdl induction(Fig. 3I), again suggesting pH regulation precedes growth.Next, we assessed the apoplastic pH and cell length of seedlings

defective in ARF10 and ARF16. ARFs are transcription factorsthat act in the nuclear auxin signaling pathway that activate theexpression of auxin responsive genes in the presence of auxin (41).ARF10 and ARF16 are readily expressed in root epidermal cells,and arf10 arf16mutant roots are impaired in auxin responses (42).Similar to the auxin receptor triple mutant, we observed a higherapoplastic pH and smaller cells in arf10arf16 mutant seedlingscompared with WT (Fig. 3 J–L). Finally, we induced the expres-sion of the auxin responsive gene SMALL AUXIN UP RNA 19(SAUR19) fused to a GREEN FLUORESCENT PROTEIN(GFP) in an estradiol-inducible GFP-SAUR19 line. SAUR19 wasshown to acidify the apoplast through PM H+-ATPase activation(12). Moreover, GFP-SAUR19 overexpression increases the size

of hypocotyls in A. thaliana, as well as in tomato (43, 44). Becauseof the emission of the GFP fluorophore, which overlaps with theHPTS spectrum, we were not able to directly confirm the GFP-SAUR19 effect on the root apoplastic pH. However, we observedlonger epidermal root cells in the GFP-SAUR19-induced linecompared with estradiol-treated control seedlings (expressing anempty vector) (Fig. S3C). These data show that the ectopic ex-pression of the auxin response gene GFP-SAUR19 is sufficient totrigger epidermal cell elongation in A. thaliana roots. Notably, ithas been shown that the stabilizing effect of the GFP tag is re-quired for the GFP-SAUR19-dependent phenotypes (43). It hasto be seen whether the ectopic overexpression of an untaggedSAUR19 is able to trigger cell expansion in A. thaliana roots.Our data suggest auxin signaling is important for apoplast

acidification and cell elongation in A. thaliana roots. This impliesendogenous auxin levels could trigger apoplast acidification, andthus stimulate cell expansion in the distal meristematic rootepidermal cells (Fig. S4).

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Fig. 3. Apoplastic pH homeostasis in auxin signaling mutants. (A–C) Apoplastic pH and corresponding epidermal cell length in estradiol-induced GH3.6 andcontrol (empty vector) seedlings, as visualized by HPTS staining. (D–F) Apoplastic pH and corresponding root epidermal cell length of WT and tir1afb2afb3mutant seedlings, as visualized by HPTS staining. (G–I) Apoplastic pH and corresponding root epidermal cell length in estradiol-induced bdl and control(empty vector) seedlings, as visualized by HPTS staining. (J–L) Apoplastic pH and corresponding root epidermal cell length of WT and arf10arf16 mutantseedlings, as visualized by HPTS staining. (A, D, G, J) Color code (black to white) depicts (low to high) 458/405 intensity, and thus, pH values. Error barsrepresent SEM (n ≥ 19). Student t test P values: *P < 0.05, **P < 0.01, ***P < 0.001. (Scale bars: 10 μm.)

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Exogenous Auxin Causes a Biphasic pH Response in the Apoplast. Ourdata suggest low endogenous auxin levels in the root meristem andelongation zone are required for apoplast acidification and sub-sequent root cell elongation. This is quite surprising, however, asincreased auxin levels are assumed to inhibit root cell elongation,rather than stimulate it (8). A possible explanation could come fromthe fact that auxin is also known to affect growth in a concentration-dependent manner, stimulating or repressing tissue expansion inlow or high concentrations, respectively, depending on the tissue inquestion (6, 7).Because high auxin concentrations are known to inhibit root

tissue expansion (8), we aimed to address how inhibitory levels ofauxin affect root apoplastic pH and cell expansion. For this, wetransferred seedlings to growth medium supplemented with250 nM IAA. In contrast to our observations in untreated roots,and in agreement with previous reports, we observed a rapidauxin-induced alkalization of the apoplast (Fig. 4 A and B (20,45). Notably, this alkalizing effect of auxin was transient. After2 h of auxin treatment, the apoplastic pH returned to levelscomparable to mock-treated seedlings (Fig. 4 A and B). More-over, prolonged auxin treatments of up to 8 h decreased theapoplastic pH relative to mock-treated seedlings (Fig. 4 A and

B). We conclude that the application of high auxin concentra-tions affects apoplastic pH in a biphasic manner. Accordingly,the contradictions in the current literature may be explained bythe varying experimental conditions of previous studies, such astimes. Importantly, the auxin-induced inhibition of cell expan-sion occurs after the alkalization phase (2 h) and precedes thelater apoplast acidification (Fig. 4C).To test whether this behavior is also observable using endoge-

nous elevation of auxin levels, we induced the expression of theauxin biosynthesis gene YUCCA6. Similar to exogenous auxintreatment, YUCCA6 induction initially resulted in increasedapoplastic pH, whereas long-term YUCCA6 induction of 19 hgave rise to a lower apoplastic pH compared with control seedlingsexpressing an empty vector (Fig. S5 A and B). Accordingly, in-duction of auxin biosynthesis also inhibited epidermal root cellelongation (Fig. S5C). Overall, these data show that external auxinapplication in an inhibitory concentration range, as well as in-creased endogenous auxin levels, gives rise to a biphasic apoplasticpH response resulting in the inhibition of root cell expansion.Accordingly, we conclude that the initial apoplast alkalizationcould be the causal factor for repressing cellular elongation.

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Fig. 4. Biphasic effect of exogenous auxin on apoplastic Ph. (A–C) Effect of 250 nM exogenous IAA on apoplastic pH and root epidermal cell length, as visualizedby HPTS staining. (B) The mean 458/405 intensities of seedlings treated with 250 nM IAA over time relative to mock-treated seedlings. (C) Epidermal cell length inthe root of seedlings treated with 250 nM IAA for 2 h and 8 h compared with mock-treated seedlings. (D–F) Effect of 250 nM IAA treatment on apoplastic pH andepidermal cell length in the root of WT and mutant seedlings, as visualized by HPTS staining. (E) Mean 458/405 intensities of WT and fer-4 seedlings treated with250 nM IAA for 25 min relative to mock-treated seedlings. (F) Root epidermal cell length of WT and fer-4 seedlings treated with 250 nM IAA for 8 h. Color code(black to white) depicts (low to high) 458/405 intensity, and thus pH values. (B, C, E, and F) Error bars represent SEM (n ≥ 13 roots). Statistical significance wastested using a one-way ANOVA test with a Tukey-Kramer post hoc test (different letters depict statistically significant differences; P < 0.05) (B) or a two-wayANOVA test with Bonferroni posttests (treatment factor P values: *P < 0.05, **P < 0.01, ***P < 0.001) (C, E, and F). (Scale bars: 10 μm.)

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FERONIA Impairs Auxin-Dependent Apoplast Alkalization and CellularElongation. To further assess the importance of auxin-mediatedcell wall alkalization for cellular expansion, we wanted to study theeffects of suppressed cell wall alkalization. Apoplast alkalizationhas been previously reported to be mediated by the PM-localizedreceptor-like kinase FERONIA on the binding of its ligand rapidalkalization factor 1 (RALF1) (46). RALF-FERONIA bindingtriggers the phosphorylation of the PM-localized proton pumps,thereby inhibiting their proton transport capacity and resulting inapoplast alkalization (46). To test whether auxin-triggered apo-plast alkalization is affected by FERONIA, we tested the effect ofauxin on apoplastic pH and cell size in the fer-4 loss-of-functionmutant. Indeed, fer-4 mutant seedlings displayed a substantial

resistance to the apoplast alkalization, as well as an eventual in-hibition of cell expansion triggered by 250 nM IAA (Fig. 4 D–F).This suggests that a significant portion of the auxin-triggeredapoplast alkalization requires FERONIA. Moreover, this experi-ment demonstrated that auxin-induced apoplast alkalization isimportant for the inhibition of root cell expansion.

Auxin Induces Transient Apoplastic Alkalization for Gravitropic RootGrowth. Root gravitropism, the process in which root growth isaligned with the field of gravity, is one of the most prominentprocesses in which auxin-dependent growth responses immedi-ately affect cellular elongation in the root. On gravistimulation,an auxin-signaling maximum is formed at the lower side of the

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Fig. 5. Effect of gravistimulation on apoplastic pH in WT and fer4 seedlings. (A–F) Mean 458/405 signal intensities in the epidermal cell layer at the outer and innerside of the root of WT and fer-4 seedlings after 45 min gravistimulation (90°) compared with nonstimulated seedlings, visualized using HPTS staining. Error barsrepresent SEM (n = 17 roots per line). Statistical significance was tested using a two-way ANOVA test with Bonferroni posttests. Outer/inner factor P values: *P < 0.05,**P < 0.01, ***P < 0.001. (Scale bars: 10 μm.) (G) Gravitropic index (50) of 7-d-old WT and fer-4 seedlings. (H) Gravitropic response of 4-d-old seedlings gravistimulatedfor 6 h (90°). y-axis shows the mean angle of the root tip with respect to the horizontal (base of the root), as depicted in the schematic. (I) Root elongation of 4-d-oldseedlings gravistimulated for 6 h (90°). (G–I) Error bars represent SEM (n ≥ 23 roots per line). Student t test P values: *P < 0.05, **P < 0.01, ***P < 0.001.

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root, resulting in local inhibition of tissue expansion and, as aconsequence, root bending (47). To assess the biological rele-vance of our findings, we tested the effect of gravistimulation onapoplastic pH and its requirement for gravitropic root response.In agreement with previous reports (19, 48), 45 min root grav-istimulation resulted in an increased apoplastic pH at the lowerside of the root (Fig. 5 A–C), which corresponds to the site ofauxin accumulation (49) (Fig. S4).To address whether auxin-induced apoplast alkalization plays a

role in this physiological process, we tested the effect of grav-istimulation on apoplastic pH in the fer-4 mutant. Compared withWT seedlings, fer-4 mutants display a reduced increase in apo-plastic pH at the lower side of the root on gravistimulation (Fig. 5D–F). In agreement with this compromised cellular response, fer-4seedling’s root growth was less aligned with the gravity vector,displaying a lower gravitropic index (50) compared with WTseedlings (Fig. 5G). Moreover, 6 h of gravistimulation revealed areduced gravitropic response in fer-4 mutants compared with WTseedlings (Fig. 5H). In addition, we tested another feronia loss-of-function allele, fer-2, for its responsiveness to a gravitropic stim-ulus. Similar to fer-4 seedlings, fer-2 seedlings displayed a reducedgravitropic response compared with WT seedlings on 6 h ofgravistimulation (Fig. S6A). Importantly, the overall root growthrate is not impaired in fer-4 or fer-2 (Fig. 5I and Fig. S6B), sug-gesting a specific delay in asymmetric growth responses.These data strongly suggest that auxin-triggered transient

apoplast alkalization and the consequent repression of cellularexpansion are required during the initial stages of the gravitropicresponse (Fig. S4).

DiscussionPlant cells are surrounded by a rigid cell wall, which needs toundergo loosening to allow cellular expansion. The long-standingacid growth theory postulates that the plant hormone auxin ac-tivates PM-localized proton pumps, decreasing the pH of theintercellular space. This apoplast acidification activates cell wall-loosening enzymes, enabling cell expansion (2–4). Subsequentstudies confirmed the acid growth theory in shoot tissues ofnumerous species including Avena coleoptiles, pea stem, andmaize coleoptiles, as well as Arabidopsis thaliana hypocotyls (2,9–13). In roots, however, the acid growth theory has been in-tensely debated during the last decades. On one hand, previousstudies demonstrated the stimulating effect of apoplast acidifi-cation on root elongation, as well as the requirement for func-tional PM proton pumps for root growth (14–16). In addition,low auxin concentrations are described to have a positive effecton root growth (6, 7). However, the root growth-promoting ef-fect of low auxin concentrations remains controversial, as only afew laboratories were able to observe this presumably condition-dependent phenomenon. On the other hand, opponents raise theargument that auxin in higher concentrations inhibits, ratherthan stimulates, root cell expansion, as well as overall rootgrowth (8, 17, 51). Moreover, although exogenous auxin appli-cation triggers apoplast acidification in shoots, it was proposed totrigger the opposite in roots (18–20). Interestingly, a recent studyon B. distachion roots provides arguments both for and againstthe acid growth theory (21). Altogether, the contradictory con-clusions obtained from previous studies point to a highly com-plex regulation of apoplastic pH homeostasis in roots. Moreover,the overall role of auxin in this physiological process is currentlyonly poorly understood. A possible reason for this partiallycontradictory information may be the limited number of mo-lecular tools available to tackle this biological question. Apo-plastic pH has been previously investigated using pH-sensitivefluorescent dye combinations (48, 52–55), as well as by thetransgenic expression of pH-sensitive fluorescent proteins facingthe apoplast (20). Even though these approaches substantiallyincreased our physiological insight into apoplastic pH homeo-

stasis, they still face technical limitations in easily assessingapoplastic pH at a cellular resolution and/or assessing a largenumber of genotypes.Here we present HPTS as a versatile pH indicator enabling

apoplastic pH assessment at a cellular resolution. We show, viapharmacologic and genetic modulation of PM-localized protonpump activity, that HPTS reliably reports on subtle changes inapoplastic pH. In A. thaliana roots, we observed a decrease inapoplastic pH around expanding cells. Moreover, we show thatmedia-driven acidification of the apoplast triggered root cellexpansion, whereas alkalization prevented it. These data suggestthat root cell expansion in A. thaliana is driven by apoplastacidification. Interestingly, recent literature reports a negativeeffect of apoplast acidification on cell expansion in Brachipodiumroots (21). The reason for these opposing observations may be aresult of the different duration of pharmacological treatments. Inour study, we decided to assess the short-term effects of apoplastacidification on cell expansion to avoid secondary effects, whichmay complicate the interpretation of the experimental outcome.Previous literature demonstrates that the stimulating effect ofapoplast acidification on cell expansion is transient (56, 57). Inaddition, long-term acidity is known to affect nutrient availabilityand is correlated with a large energy cost to maintain the cellularinflux of necessary cations (58–60). On root gravistimulation, wedemonstrate the biological relevance of a short/transient asym-metrical apoplast alkalization, which enables differential tissuegrowth, and ultimately, root bending.Using HTPS, we show that apoplast acidification is important

for root cell expansion and requires nuclear auxin signaling.Consistent with our data, a recent study on A. thaliana hypocotylsreported that auxin-induced hypocotyl growth correlates with localapoplast acidification and requires nuclear auxin signaling (13).Moreover, another study reported a correlation between reducedauxin influx and altered PM H+-ATPase phosphorylation status,resulting in reduced proton protrusion capacity and impaired rootgrowth (61). These recently published findings support our data insuggesting that endogenous auxin levels may be required forapoplastic pH homeostasis, which is important for sustainable cellelongation during root growth (Fig. S4). In addition, we demon-strate that growth inhibitory concentrations of auxin trigger fasttransient root apoplast alkalization and subsequent acidification.Our data suggest that the initial auxin-triggered apoplast alkali-zation is causal for the inhibition of root cell expansion (Fig. S4). Itremains to be seen whether the effect of inhibitory auxin con-centrations on the root apoplastic pH is indeed the result of al-tered PM H+-ATPase activity or whether other molecularcomponents also contribute to the observed dynamics.To address the biological relevance of high auxin concentrations,

we assessed the apoplastic pH on root gravitropism. During rootgravistimulation, auxin is known to accumulate at the inner (lower)side of the root (62). We show an asymmetric apoplast alkalizationat the inner side of gravistimulated roots, which presumably inhibitscell elongation and enables root bending (Fig. S4).It has been previously shown that auxin-triggered apoplast acid-

ification in hypocotyls is mediated by the rapid up-regulation ofSAUR19 expression, resulting in the activation of PM H+-ATPases(12). On GFP-SAUR19 induction, we also observed increasedepidermal root cell elongation. Notably, no molecular players havebeen attributed to auxin-triggered apoplast alkalization so far. Herewe show that auxin-triggered apoplast alkalization requires thePM-localized receptor-like kinase FERONIA. FERONIA wasidentified as an important mediator during pollen tube reception,as well as sperm cell release (63–65). Later studies reported thatFERONIA, on binding to its ligand RALF, triggers phosphorylationof Ser899 of the PM H+-ATPase AHA2 (46). This phosphorylationinhibits the proton secretion capacity of the proton pump, resultingin alkalization of the apoplast (46). Intriguingly, auxin-induced roothair elongation is abolished in the absence of FERONIA (66).

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We demonstrate the requirement of FERONIA for auxin-triggeredapoplast alkalization on exogenous auxin treatment, as well asduring root gravistimulation. Moreover, fer-4 loss-of-functionmutants displayed a delayed response to gravitropic stimuluscompared with WT seedlings. Altogether, our data suggest a rolefor auxin-triggered apoplast alkalization in root gravitropicresponse. Intriguingly, it has been previously described that theRALFs, which are ligands of FERONIA, are involved in cellwall integrity maintenance. In particular, RALF4 seems coregu-lated with pectin-modifying enzymes (67), such as pectin methylesterases (PMEs). PMEs play a role in cell wall modificationsthat are associated with growth transitions (68, 69) and displayan alkaline pH optimum. A notable adverse effect of their enzy-matic activity is cell wall acidification, which in turn inhibits PMEactivity. It needs to be seen whether auxin-triggered apoplastalkalization, for example during gravitropism, activates PMEs.The PME activity would subsequently lead to cell wall acidifi-cation, resulting in a PME inhibitory effect that keeps the cellwall modification process transient. Such a process would resultin the local inhibition of root cell elongation, enabling efficientgravitropic response without overbending of the root.

ConclusionIn this work, we have introduced HPTS as a suitable dye forassessing apoplastic pH at a cellular resolution. Our data,obtained by HPTS-based analysis, suggest nuclear auxin signal-ing is required for apoplast acidification important for root cellexpansion. In addition, we show that high auxin levels transientlytrigger root apoplastic alkalization. Finally, we show that auxin-triggered apoplast alkalization requires FERONIA, which isimportant for the root gravitropic response.

Altogether, our data suggest a model in which auxin plays acomplex, concentration-dependent role in apoplastic pH ho-meostasis during A. thaliana growth and development. This hy-pothesis is in line with previous literature reporting that lowauxin concentrations stimulate, whereas high auxin concentra-tions inhibit, root growth (6–8). This view may clarify previouslypublished contradictory results, which either supported or op-posed the acid growth theory in the root. In summary, our workprovides a versatile tool as well as mechanistic insight intoapoplastic pH regulated root growth.

Materials and MethodsWe used A. thaliana of the ecotype Colombia 0. Seeds were sterilized with70% ethanol and stratified at 4 °C for 2 d in the dark on in vitro growthplates. Seedlings were grown vertically on half Murashige and Skoog me-dium [per liter: 2.16 g MS salts (Duchefa), 4.7 mM Mes, 0.8% plant agar].Plants were grown under long-day (16 h light/8 h dark) conditions at 21 °C/18 °C. The following lines have been previously described: ost2-2 (32), DII-Venus (70), tir1-1/afb2-1/afb3-1 (49), arf10-2/arf16-2 (42), feronia-4 (66),feronia-2 (71). pER8 and pER8:YUC6 (72), pER10:bdl (40), pER8:GFP:SAUR19(12), and R2D2 and DR5v2::n3GFP (36). The raw data files of the presentedexperiments have been uploaded to the public database datadryad.org (doi:10.5061/dryad.sq7s3).

More detail can be found in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Herman Höfte, Clara Sanchez Rodriguez,Paul Larsen, Dolf Weijers, Hong-Quan Yang, William Gray, and Mark Estellefor sharing publishedmaterial; J. MatthewWatson for English editing; JuergenKleine-Vehn for expertise, access to scientific equipment, and the kind dona-tion of the pMDC7B(pUBQ10)GH3.6 and the pMDC7B(pUBQ10)empty lines;Youssef Belkhadir for scientific discussion; the W.B. group for critical readingof the manuscript; Pawel Paserbiek, Tobias Müller, Karin Aumayr, and GabrieleStengl from the IMP BioOptics Facility; and the VIBT imaging center for theiraccess and expertise. This work was supported by funds from the AustrianAcademy of Science through the Gregor Mendel Institute (W.B.).

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