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Keywords:

  • auxin;
  • calcium;
  • pH;
  • root;
  • gravitropism;
  • Arabidopsis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plants adapt to a changing environment by entraining their growth and development to prevailing conditions. Such ‘plastic’ development requires a highly dynamic integration of growth phenomena with signal perception and transduction systems, such as occurs during tropic growth. The plant hormone auxin has been shown to play a key role in regulating these directional growth responses of plant organs to environmental cues. However, we are still lacking a cellular and molecular understanding of how auxin-dependent signaling cascades link stimulus perception to the rapid modulation of growth patterns. Here, we report that in root gravitropism of Arabidopsis thaliana, auxin regulates root curvature and associated apoplastic, growth-related pH changes through a Ca2+-dependent signaling pathway. Using an approach that integrates confocal microscopy and automated computer vision-based image analysis, we demonstrate highly dynamic root surface pH patterns during vertical growth and after gravistimulation. These pH dynamics are shown to be dependent on auxin, and specifically on auxin transport mediated by the auxin influx carrier AUX1 in cells of the lateral root cap and root epidermis. Our results further indicate that these pH responses require auxin-dependent changes in cytosolic Ca2+ levels that operate independently of the TIR1 auxin perception system. These results demonstrate a methodology that can be used to visualize vectorial auxin responses in a manner that can be integrated with the rapid plant growth responses to environmental stimuli.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The ability of plants to adapt to the environment by altering their growth and development has long fascinated plant physiologists. As early as 1880, Charles Darwin predicted the existence of a plant growth factor (or ‘influence’) that was vital to plant directional, or tropic, movements towards or away from environmental cues (Darwin and Darwin, 1880). This growth factor was later identified as indole-3-acetic acid (IAA)/auxin (Arteca, 1996). Auxin has since been shown to regulate plant responses ranging from tropisms to embryonic patterning, and in many of these signaling pathways it operates through the now well-understood TIR1/AFB-AUX/IAA co-receptor pathway of protein degradation (Leyser, 2006; Mockaitis and Estelle, 2008). However, some plant growth responses to environmental changes are initiated very quickly, and it is unclear whether transcriptional regulation can be reconciled with such rapid response kinetics. Alternate auxin signaling pathways involving, for instance, the regulation of plasma membrane ion fluxes have been postulated (Barbier-Brygoo et al., 1989; Napier and Venis, 1995; Shishova and Lindberg, 2010), but to date there is little evidence for a functional role of these pathways in a physiological context. For example, although changes in calcium have been suggested to form part of a rapid auxin signaling system, it is unclear whether these changes are linked to auxin-dependent modulation of growth (Tretyn et al., 1991; Ayling et al., 1994; Shishova and Lindberg, 1999, 2004, 2010; Plieth and Trewavas, 2002).

To understand how auxin signals are transduced at a cellular level to elicit short-term growth responses to environmental inputs, we focused our investigation on root gravitropism, as this response has been characterized in considerable detail. There is a clear spatial separation between the site of graviperception (root cap) and site of response (elongation zone), and the requirement for basipetal/shootward auxin transport to transmit information between these two sites is well established (Ottenschlager et al., 2003; Swarup et al., 2005; Muday and Rahman, 2008; Masson et al., 2009). Gravitropic curvature is initiated in the apical elongation zone within a few minutes of stimulus perception (Selker and Sievers, 1987; Mullen et al., 2000), and ionic events such as proton fluxes related to the gravitropic response occur even earlier (Zieschang et al., 1993; Monshausen and Sievers, 2002). Rapid changes in cytosolic Ca2+ and pH have been proposed as components of the gravisignaling machinery, although to date Ca2+ changes clearly associated with gravistimulation of the root have not been detected (Gehring et al., 1990b; Legue et al., 1997; Plieth and Trewavas, 2002; Toyota et al., 2008a,b). Fluxes of Ca2+ and H+ have also been linked to the gravitropic growth response. For example, Ca2+ fluxes have been monitored using either ion-selective electrodes or as redistribution of radioactive 45Ca2+ from agar blocks applied to the top or bottom of tropically responding maize and pea roots (Lee et al., 1983a, 1984; Evans, 1986; Bjorkman and Cleland, 1991). Gravistimulation leads to a flux across the root tip, from top to bottom at 10 min (Bjorkman and Cleland, 1991), or between 45 and 90 min (Lee et al., 1983a, 1984). These measurements were made in the apoplast and, as such, could not distinguish between the contributions of apoplastic and symplastic transport to the tropic growth response. A role for these Ca2+ changes in modulating auxin transport has been proposed (Evans et al., 1992), which would be consistent with current ideas about the likely Ca2+-dependency of the function of pin-formed auxin efflux transporter (PIN) inferred from the calmodulin-related regulation of the pinoid protein kinase that plays a role in PIN targeting (Robert and Offringa, 2008).

Pharmacological treatments thought to affect Ca2+ changes alter tropic growth (Lee et al., 1983b; Bjorkman and Cleland, 1991), but again it remains unclear whether these treatments affect signaling or response events, and whether they specifically target gravitropic response elements such as auxin-dependent signals or more generally disrupt growth. Interestingly, treatment of maize and pea roots with auxin transport inhibitors also blocked the movement of Ca2+ (Lee et al., 1984), suggesting the potential for complex feedback between the Ca2+ and auxin signaling and response systems.

Although changes in cytosolic pH may play a role in the gravity-sensing machinery (Scott and Allen, 1999; Fasano et al., 2001; Johannes et al., 2001), the proton fluxes elicited around the root elongation zone in response to gravitropic stimulation are consistent with a role in acid growth (Mulkey and Evans, 1981; Mulkey et al., 1982; Pilet et al., 1983; Versel and Pilet, 1986; Zieschang et al., 1993; Monshausen et al., 1996; Taylor et al., 1996), with steady-state acidification occurring on the more rapidly growing flank of the root. These changes are thought to be triggered by auxin redistribution, and to occur, at least in part, through the regulation of plasma membrane proton pump activity. Precisely how these proton fluxes are linked to auxin action and redistribution in response to gravistimulation at the molecular level remains unknown.

These well-defined initial phases of root gravitropism coupled to the extensive background implicating ionic signaling in the earliest phases of the graviresponse provided us with a clear temporal and spatial framework in which to try to position H+ and Ca2+ relative to auxin signaling events. We report that roots show a spatially and temporally highly dynamic H+ flux profile during vertical growth that is modified during the graviresponse, consistent with the process of acid growth. Auxin can modulate these changes in a Ca2+-dependent fashion, and auxin export from the root cap is associated with elevations in cytosolic Ca2+ levels that in gravitropically-stimulated plants reflects a wave of Ca2+-dependent auxin response migrating shootwards along the root.

Results and Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Arabidopsis roots are characterized by auxin transport-dependent surface pH dynamics

In order to characterize the spatial and temporal characteristics of growth-related proton fluxes, we developed a measurement approach integrating confocal microscopy and automated computer vision-based image analysis. Vertical-stage confocal imaging of roots immersed in pH-sensitive fluorescein-dextran enabled us to capture the dynamics of extracellular pH around an entire growing root tip during vertical orientation and after gravistimulation. The high temporal and spatial resolution of such image acquisition was preserved during subsequent rounds of automated image processing and analysis, which provided numerical pH information for each pixel along a defined contour line edging the root surface (Figure 1a). Thus, a detailed map of extracellular pH dynamics over time was generated (Figure 1). Using this approach, we were able to visualize the global reshaping of the pH pattern around the entire root tip upon gravistimulation. Within 3 min of reorienting the root by 70–80°, i.e. well before the initiation of gravitropic curvature (Figure S1; Fasano et al., 2001; Mullen et al., 1998), the upper flank of the root began to acidify (2.8 ± 0.4 min after stimulation at 250 μm from the tip; mean ± SD of n = 6), whereas the surface pH on the lower flank increased (3.0 ± 0.4 min after stimulation; mean ± SD of n = 6). The resulting pH gradient across the root extended from the elongation zone down to the root cap (Figure 1b,c; Video Clip S1; see also Fasano et al., 2001; Monshausen and Sievers, 2002; Zieschang et al., 1993). The speed of these pH changes suggest that they may function in the earliest phase of gravitropic signal transmission, as well as in the subsequent activation of cell wall loosening required for differential cell expansion (Ishikawa et al., 1991; Zieschang and Sievers, 1991).

image

Figure 1.  Surface pH of Arabidopsis roots during vertical growth and after gravistimulation on a vertical-stage confocal microscope. (a) The extracellular pH of an Arabidopsis root was monitored using the pH indicator fluorescein(-dextran). Automated computer vision-based analysis of the surface pH was accomplished by: (i) finding the root tip; (ii) performing ratio analysis of pixel intensities along the left (red) and right (yellow) contour lines of the root; (iii) color-coding pH-dependent ratios; and (iv) repeating the process for each image in a series. (b) Gravistimulation of an Arabidopsis root. At time 0, the seedling was reoriented to 75° (see also Video Clip S1). Scale bar: 100 μm. (c) Gravity-induced surface pH changes along the root shown in (b). The extracellular pH at every position along both sides of the root over time is displayed by color code. *Panels shown in (b). Note that the pH asymmetry induced by gravistimulation is reversed when the root is returned to the vertical orientation (0°). (d) Loss of gravitropic pH response in the agravitropic aux1 mutant. (e) Surface pH dynamics of a vertically growing wild-type root (see also Video Clip S2). (f) Heat plot of surface pH changes along the left side of the root shown in (e). *Panels shown in (e). (g) Altered surface pH pattern along a vertically growing root of the auxin transport mutant aux1. The pH in all plots is color coded according to the scale bar. All panels show representative examples of five or more independent experiments.

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Gravity-induced asymmetric redistribution of auxin in the root tip is well established as a key event in gravitropic signaling (Ottenschlager et al., 2003; Swarup et al., 2005). However, because of the limited temporal resolution of currently available assays for auxin relocation (Peer and Murphy, 2007) or localized auxin action (e.g. DR5:GFP; Ottenschlager et al., 2003; Peer and Murphy, 2007; Sukumar et al., 2009), it is unclear whether such tropically-induced auxin redistribution occurs sufficiently fast to be functionally linked to the gravity-induced pH changes described above. To explore the relationship between root surface pH changes and auxin redistribution, we therefore first monitored the effect of gravistimulation on the root pH pattern of aux1, a mutant in which a lesion in the auxin influx carrier AUX1 impairs mobilization of auxin from the root tip, and renders the roots agravitropic (Swarup et al., 2001; Marchant et al., 2002). This disruption in AUX1 function completely abolished the development of pH asymmetry upon gravistimulation (Figure 1d).

Intriguingly, aux1 also showed altered root surface pH in the absence of gravistimulation, compared with wild type. During vertical growth, the extracellular pH of wild-type roots was seen to be unexpectedly dynamic, and fluctuated in a complex pattern between periods of alkalinization and acidification, with bursts of alkalinity migrating along the root longitudinal axis or alternating between opposing flanks (Figures 1e,f, S2; Video Clip S2). Thus, the expectation of steady-state wall acidification in regions of the root showing rapid cell expansion predicted from the acid growth theory was not precisely followed. Indeed, the pH at the surface of the root increased above the pH of the medium, suggesting a net influx of protons to the root. There was significant variability among individual roots in the frequency of fluctuations (2–11 identifiable pH peaks in 50 min), with the highest frequency for a single root typically found in the central elongation zone.

The extracellular pH of aux1 roots, on the other hand, showed little of these dynamic fluctuations, being almost uniformly acidic, with only a small alkaline shift close to the root apex (Figure 1g). However, both the pH dynamics and the development of pH gradients after gravistimulation were restored (along with normal growth; Swarup et al., 2005) when AUX1 was selectively expressed in the lateral root cap and epidermis of aux1 using a transactivation approach (aux1-J0951≫AUX1; Swarup et al., 2005), demonstrating that localized perturbation of auxin transport in these tissues severely impacts processes involved in growth-related pH regulation (Video Clip S3a,b).

Auxin triggers a rapid increase in root surface pH

To further define the role for auxin in these root growth-related events, we treated Arabidopsis seedlings with auxin while directly measuring pH along the root surface. Exogenous treatment with auxin has previously been shown to alter extracellular pH in growing plant organs, causing acidification in plant shoots and alkalinization of the root environment within 6–15 min (Luthen and Bottger, 1988; Luthen et al., 1990; Felle et al., 1991; Spiro et al., 2002). However, such slow response times are inconsistent with the rapid pH dynamics described above. By improving the spatiotemporal resolution of such measurements using our confocal imaging technique, we found that 0.1–1 μm IAA triggered a virtually instantaneous (within 15 sec) extracellular alkalinization along the elongation zone, quickly followed by the meristematic region (Figures 2a, S3). Treatment with IAA also elicited a small but significant decrease in cytosolic pH (Figure S4), suggesting that auxin activated proton fluxes across the plasma membrane. IAA-induced alkalinization was also observed in the aux1 mutant, although the extent of the alkalinization was attenuated (Figure 2b), indicating that defects in auxin influx do not abolish auxin responsiveness. Importantly, statistically significant increases in root surface pH were also elicited by 1–10 nm IAA (Figure S3), i.e. a response occurred in the range of hormone levels that might be expected when auxin fluxes are re-directed to the lower side of a root during gravistimulation (Tanimoto, 2005; Petersson et al., 2009).

image

Figure 2.  pH response of Arabidopsis roots to auxin. (a) Surface pH changes along a wild-type Arabidopsis root triggered by the global application of 100 nm indole-3-acetic acid (IAA). (b) Surface pH changes along the aux1 root triggered by the global application of 100 nm IAA. (c) Protocol for localized application of auxin to the root tip. The tip of an Arabidopsis root embedded in agar (containing fluorescein-dextran) was cut free. An agar block containing 1 μm IAA was then placed in the path of the growing root. A gap of 140–220 μm between the two agar surfaces prevented diffusion of IAA from the agar block to the agar medium surrounding the root. Once the root tip made contact with the agar block, IAA could be taken up by the root cap and transported shootwards along the root length (Peer and Murphy, 2007). (d) Surface pH changes along a wild-type Arabidopsis root triggered by the tip-localized application of 1 μm IAA. Note the progression of extracellular alkalinization along the root length; also see Video Clip S4. (e) Surface pH along the Arabidopsis aux1 root after the tip-localized application of 1 μm IAA. The surface pH in all plots is color coded according to the scale bars. All panels show representative examples of six or more similar measurements.

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Taken together, these results demonstrate an unexpectedly dynamic pH regulatory system, with surface pH likely to reflect the dynamics of AUX1-dependent auxin transport and redistribution. To directly address this hypothesis, we locally applied auxin to the tip of the root cap by placing an agar block containing 1 μm IAA in the path of a vertically growing root (Figure 2c), and monitored how uptake and transport of this auxin affected the surface pH. Within 49 ± 7 sec (mean ± SD of n = 9) of making contact with the agar block, an increase in pH was detectable along the root surface 200 μm from the tip. This pH change rapidly migrated towards the more mature regions of the root (i.e. basipetally/shootwards; detectable after 58 ± 9 sec at 300 μm, 81 ± 10 sec at 400 μm and 117 ± 14 sec at 500 μm; Figure 2d, Video Clip S4). The progression of the alkalinization thus occurred at a velocity (150–600 μm min−1 depending on position along the root length) similar to the reported auxin transport rates (5–20 mm h−1; Naqvi and Gordon, 1966; Lomax et al., 1995). The gap in the gel necessary to isolate the auxin-containing block from the rest of the growth medium in order to block diffusion of auxin to the root prevented us from making pH measurements closer to the root tip than 200 μm. To ascertain that the wave of alkalinization was indeed caused by auxin transport rather than diffusion of auxin along the root surface, we performed control experiments using the aux1 mutant, which showed only a subtle alkalinization at the meristem/apical elongation zone upon touching the auxin–agar block, consistent with the impaired long-distance transport of auxin away from the root cap in the mutant (Figures 2e, S5).

Auxin regulates root surface pH changes through a Ca2+-dependent signaling pathway

The results described above support the idea that auxin plays a central and immediate role in regulating the shape of the root pH pattern, both during vertical and tropic growth. However, even though this signaling pathway is likely to be important for the dynamic control of root growth, the auxin perception and response mechanisms underlying such auxin-induced alkalinization in roots remain unknown. We observed that the pH change in response to exogenously applied auxin is extremely rapid, and occurs in both the tir1-1 and tir1-1 afb2-3 afb3-4 (Parry et al., 2009) backgrounds (Figure S6), suggesting the involvement of a post-transcriptional signaling pathway that is independent of the well-characterized TIR1/AFB-AUX/IAA co-receptor pathway of protein degradation (Leyser, 2006; Mockaitis and Estelle, 2008).

Indole-3-acetic acid (IAA) is known to modulate ion fluxes across the plasma membrane (Napier and Venis, 1995), and there are isolated reports of changes in cytosolic Ca2+ levels in guard cells, shoot protoplasts, root hairs and whole seedlings in response to a wide range of auxin concentrations (Gehring et al., 1990a; Tretyn et al., 1991; Irving et al., 1992; Ayling et al., 1994; Plieth and Trewavas, 2002; Shishova and Lindberg, 2010). This suggested to us a potential relationship between auxin and Ca2+ signaling that might underlie the dynamic pH changes we have characterized. Such a link would also be consistent with our previous observation that rapid extracellular alkalinization can be elicited by increases in cytosolic Ca2+ levels related to mechanical stimulation, and during root hair elongation (Monshausen et al., 2007, 2008, 2009).

In order to explore the hypothesis that in roots, Ca2+ functions as a second messenger to translate local auxin signals into appropriate physiological responses, we monitored cytosolic Ca2+ levels using confocal imaging of Arabidopsis roots expressing the FRET-based Ca2+ sensor yellow cameleon YC3.6 (Nagai et al., 2004; Monshausen et al., 2008). In tissues of the growing root tip, global treatment with auxin triggered a rise in cytosolic Ca2+ within 7–14 sec (Figure 3a; Video Clip S5). Furthermore, the application of auxin localized to the root tip via the agar block elicited a wave of Ca2+ advancing shootwards along the root axis (detectable after 45 ± 8 sec at 200 μm, 58 ± 11 sec at 300 μm, 87 ± 22 sec at 400 μm and 127 ± 37 sec at 500 μm, mean ± SD of n = 5–7 roots; Figure 3b,c; Video Clip S6). Control experiments using agar blocks containing 1 μm benzoic acid did not result in detectable Ca2+ elevation (Figure S7), suggesting that this Ca2+ response was not an artifact of mechanical stimulation upon contacting the agar block, or of the action of weak acids in general. Exposure to auxin thus elicited Ca2+ changes similar in both time and space to the pH changes monitored previously. In addition, pre-treatment with the Ca2+ channel blocker La3+ completely inhibited not only auxin-induced Ca2+ transients but also auxin-induced pH changes (Figure 4), supporting the idea that Ca2+ acts downstream of exogenous auxin to mediate auxin-induced alkalinization, and that auxin rapidly triggers Ca2+ influx to the cytoplasm across the plasma membrane.

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Figure 3.  Ca2+ response of Arabidopsis roots to auxin and gravistimulation measured using the FRET-based Ca2+ sensor YC3.6. (a) Cytosolic Ca2+ levels in response to global application of 100 nm indole-3-acetic acid (IAA). Ca2+ levels are color coded according to the scale bar. Green corresponds to a ratio value of approximately 1.3; red corresponds to ratios ≥ 2. IAA was applied at time 0. Also see Video Clip S5. (b) Cytosolic Ca2+ levels along the root length after the tip-localized application of 1 μm IAA (see Figure 2c). Note the basipetal progression of Ca2+ elevation indicated by the arrowheads. The root tip made contact with the agar block containing IAA at time 0. Also see Video Clip S6. (c) Increases in cytosolic Ca2+ levels upon tip-localized application of 1 μm IAA measured in four regions of interest along the left flank of the root shown in (b). Black, 200 μm; red, 300 μm; green, 400 μm; purple, 500 μm from the extreme root tip. (d) Protocol for monitoring cytosolic Ca2+ after gravistimulation. Seedlings were pre-stimulated by placing the cuvette holding the seedling horizontally for 7 min. The cuvette was then turned by 180° and placed on the microscope stage, resulting in a reversal of the graviresponse (cf. Figure 1c). The physically upper side of the root epidermis in the apical elongation zone was monitored using an upright Zeiss 510 LSM; the lower side was monitored using an inverted Zeiss 510 LSM. (e) Cytosolic Ca2+ levels in epidermal cells of the apical elongation zone on the upper side (left) and lower side (right) of the gravistimulated root. The white arrow indicates the position of the root tip. Also see Video Clip S7. (f, g) Numeric analysis of cytosolic Ca2+ levels in four consecutive cells of the epidermal cell files shown in (e). (f) Upper flank and (g) lower flank of the gravistimulated root. Black, most apical (i.e. closest to root tip); red, second; green, third; purple, fourth cell in the epidermal cell file. See Figure S8 for the outlines of the analyzed regions of interest. Representative measurements of n ≥ 6 experiments.

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image

Figure 4.  LaCl3 inhibition of auxin-induced (a) cytosolic Ca2+ and (b) extracellular pH changes. Black, Ca2+ and pH response of Arabidopsis roots treated with 100 nm indole-3-acetic acid (IAA). Grey, roots were incubated with 1 mm of the Ca2+ channel blocker La3+ for ca. 3 min prior to the addition of 100 nm IAA.

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To determine if the cascade linking auxin to pH-dependent growth regulation via Ca2+ is of physiological significance during endogenous auxin signaling, we investigated a potential role for Ca2+ in gravitropism, where the involvement of differential auxin accumulation (Swarup et al., 2001; Ottenschlager et al., 2003) and surface pH changes (Mulkey and Evans, 1981; Versel and Pilet, 1986; Zieschang et al., 1993; Monshausen and Sievers, 2002) is established. Research over three decades has amassed a substantial body of circumstantial evidence linking Ca2+ to gravitropism in roots and aerial organs (Sinclair and Trewavas, 1997; Fasano et al., 2002). Many of these studies have utilized pharmacological approaches to demonstrate that Ca2+ is required for a normal graviresponse, i.e. the development of gravitropic curvature. However, actual measurements of gravity-related changes in cytosolic Ca2+ have remained complex to interpret, in that rapid Ca2+ elevations upon gravistimulation are often difficult to distinguish from mechanical signaling associated with reorienting the plant (Gehring et al., 1990b; Plieth and Trewavas, 2002; Toyota et al., 2008b).

Based on our analysis of gravity-induced changes in root surface pH, we surmised that cytosolic Ca2+ was likely to play a role in gravitropic signal transmission during asymmetric basipetal auxin transport along the root length. To obtain a clear graviresponse unmasked by endogenous fluctuations likely to occur during vertical growth (cf. Figure 1e), we pre-stimulated roots for 7 min horizontally (−90°), and then turned them by 180° (to +90°) when placing them onto the microscope stage (Figure 3d). pH measurements had demonstrated a strong reversal of gravi-induced surface pH asymmetry under similar conditions, which therefore seemed optimal for evoking a detectable Ca2+ response (see Figure 1c). By monitoring Ca2+ levels in the epidermis of the upper or lower side of the gravistimulated root (Figure 3d), we found that gravitropic signal transmission is associated with asymmetric changes in cytosolic Ca2+ levels. From 2 to 4 min (3.2 ± 0.4 min; n = 6) after gravistimulation, Ca2+ levels on the upper flank began to decrease (Figure 3e,f). Conversely, on the lower flank, the Ca2+ concentration increased at 3–6 min after reorientation (4.3 ± 1.0 min; n = 12; i.e. well after Ca2+ signals are elicited by mechanical stimulation of the root; Monshausen et al., 2009), and subsequently fluctuated for the duration of the experiment (Figure 3e,g; Video Clip S7). The timing of these Ca2+ changes is consistent with the pH changes found after reorientation to vertical (0°), which were slightly slower than during the initial gravistimulation (acidification after ca. 3 min and alkalinization after 2–6 min; cf. Figure 1c). Although cytosolic Ca2+ changes have been reported upon reorientation of Arabidopsis (Plieth and Trewavas, 2002; Toyota et al., 2008a; Toyota et al., 2008b), these rapid changes have been proposed to reflect sensory events linked to as yet to be defined downstream signaling systems. The temporal and spatial characteristics of these Ca2+ transients are dissimilar to those we have observed, in that they occur rapidly, within seconds of reorientation, and it is unclear if they arise in roots (Legue et al., 1997; Toyota et al., 2008b). The changes we have observed are more consistent with the propagation of auxin efflux from the root cap eliciting a wave of Ca2+ response that travels back to the elongating cells of the root, where it regulates proton fluxes related to the modulation of growth through increasing apoplastic pH. Ca2+ increases to the apoplast of the lower side of the gravistimulated root have been previously reported (Lee et al., 1983a, 1984; Evans, 1986; Bjorkman and Cleland, 1991), and these changes can be extremely large (e.g. 3 mm difference from top to bottom of the root apex; Bjorkman and Cleland, 1991). These apoplastic fluxes may be driven by asymmetrical active Ca2+ transport through the symplast, or possibly via the electrical gradient formed, for example, by the proton pumping associated with graviresponse (Weisenseel and Meyer, 1997).

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The data presented here support and extend the widely accepted model where asymmetrical auxin efflux from the root cap elicited by gravitropic stimulation leads to pH-dependent acid growth related to tropic curvature. However, our data now suggest that this signal reflects the propagation of a Ca2+-dependent auxin response from the root apex to the elongating cells that coordinates wall alkalinization, and so modulates growth. In addition, our observations highlight a role for extracellular pH as an important signaling element that could itself feed back to modulate auxin signaling, e.g. by altering the chemisosmotic proton gradient that drives lipophilic and AUX1 symport-mediated auxin uptake, as well as carrier-mediated cellular auxin efflux (Figure 5). Ca2+-induced alkalinization has also been linked to growth control , e.g. in response to touch stimulation or during tip growth (Monshausen et al., 2008, 2009). Thus, a response cassette revolving around the Ca2+-dependent regulation of wall pH may have been recruited during plant evolution to serve in very disparate signaling pathways ranging from immediate responses to abiotic and biotic stresses (Brault et al., 2004; van Loon et al., 2008; Monshausen et al., 2009), to post-translational regulation of tip growth (Monshausen et al., 2007, 2008) and, as shown here, the early phases of hormone-dependent modulation of cellular expansion.

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Figure 5.  Model of gravitropic signal transmission in roots. Gravistimulation results in a redistribution of auxin fluxes in the root cap, leading to auxin accumulation on the lower flank of the root. These higher auxin levels are then conveyed shootward via the concerted action of auxin transporters. Among these, ABCB4 and polarly localized PIN2 (1) mediate auxin (IAA) efflux into the apoplastic space where, depending on cell wall pH, IAA may become protonated. The uptake of auxin across the plasma membrane can occur passively by diffusion (IAAH) or actively as IAA/H+ symport mediated by AUX1 (2) (Blakeslee et al., 2005). In each cell, the increase in auxin levels activates an unidentified receptor localized close to/at the cell periphery (3); whether auxin perception occurs apoplastically or in the cytosol remains to be defined. Activation of the receptor triggers the rapid activation of a plasma membrane Ca2+-permeable channel (4). The resulting increase in cytosolic Ca2+ concentration activates a plasma membrane H+/OH conductance (5), leading to apoplastic alkalinization (6). The rise in extracellular pH may modulate cell wall extensibility and thereby cellular expansion (Tanimoto, 2005), and will shift the IAA/IAAH equilibrium towards IAA. This reduces the level of auxin that can passively diffuse across the plasma membrane. By triggering an elevation of cytosolic Ca2+ and an increase in apoplastic pH, auxin is likely to affect not only root growth but also modulates its own transport by enhancing the contribution of AUX1 to auxin uptake, and potentially by altering PIN2 localization (1) (Robert and Offringa, 2008).

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Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and growth conditions

Seeds of Arabidopsis thaliana Columbia (wild type, aux1-21, aux1-J0951≫AUX1, Swarup et al., 2005; tir1-1 and tir1-1 afb2-3 afb3-4, Parry et al., 2009) were surface sterilized with 75% (v/v) ethanol and germinated under 24-h light conditions at 22 °C on quarter-strength Murashige and Skoog medium, pH 5.8, supplemented with 1% (w/v) sucrose. Four- to five-day-old seedlings were selected for experiments and transferred to experimental chambers as described by Monshausen et al. (2009).

Fluorescence microscopy

Root surface pH was measured as described by Monshausen et al. (2007, 2009). Briefly, seedlings were embedded in agar containing fluorescein-dextran (10 kDa) and were ratio-imaged using the 458- and 488-nm lines of the argon laser of the Zeiss LSM 510 confocal microscope and a ×10, 0.3 NA Plan-Neofluar objective (Zeiss, http://www.zeiss.com). A 90º objective inverter (LSM Tech, http://www.lsmtech.com) and custom-built vertical stage were used for pH measurements during vertical or gravitropic growth experiments, and during tip-localized auxin application experiments. Calibration was conducted as described previously (Monshausen et al., 2007).

Cytosolic Ca2+ levels of Arabidopsis seedlings expressing the Ca2+ sensor YC3.6 were measured as described by Monshausen et al. (2008, 2009), with the following modifications: to monitor Ca2+ changes during gravitropic stimulation, we used the ×63, 1.4 NA, oil immersion Plan-Apochromat objective of either an upright (to monitor the upper side of the root) or inverted (to visualize the lower side of the root) Zeiss LSM 510 confocal microscope.

Automated analysis of root surface pH

To isolate the root from the background, the images were smoothed with a 6 × 6 pixel-averaging kernel and Otsu’s threshold method was applied to each frame of the image stack. The boundary of the root was traced and curvature was calculated along the boundary curve according to the following formula:

  • image

To remove noise, the signal was first smoothed with a Gaussian kernel before applying finite differences to approximate the derivatives.

The location along the curve with the highest curvature was isolated as the root tip. To obtain the pH values along the left and right flanks, the pixel intensities along the boundary curve starting at the root tip were sampled for each image (488- and 458-nm excitation), and pH values were calculated from the pixel ratios according to the calibration.

For experiments where no root tips were visible, the junction formed by the root and the air interface was tracked using an optical flow technique (Lucas and Kanade, 1981). Otsu’s threshold method was applied to the image and the edge of the binarized image was traced in the basal direction, starting from the tracked junction. The image was then sampled along the edge of the root.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr Malcolm Bennett (University of Nottingham) for the gift of Arabidopsis aux1-21 and aux1-J0951AUX1, and Dr Mark Estelle (UCSD) for sharing seeds of Arabidopsis tir1-1 and tir1-1 afb2-3 afb3-4 lines. We are grateful to Dr Edgar Spalding for his critical reading of the manuscript, and thank Drs Edgar Spalding and Marisa Otegui (University of Wisconsin) for many helpful discussions. We thank Dr Judith Kimble (University of Wisconsin) for the use of the upright Zeiss LSM510. This work was funded by the National Science Foundation (grant MCB 0641288 to GBM and SG, IOS 0820648 to ASM, and DBI-0621702 to Edgar Spalding).

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  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Gravitropic bending response of gravistimulated Arabidopsis roots. Black, wild type; red, aux1 roots. The root tip angle of wild type roots had increased significantly (= 0.05; student t-test) by 8.5 min after turning the roots to 70–80°. Measurements were made on roots also monitored for gravitropic pH responses. Means ± SD of n = 7 (wild type) and n = 5 (aux1) measurements.

Figure S2. Surface pH dynamics of vertically growing Arabidopsis roots. Surface pH on left (black) and right (red) sides of the elongation zone ca. 300–350 μm from the root tip. Note that pH peaks on opposite sides of the root are displaced with respect to each other and occur in an approximately alternating pattern.

Figure S3. Dose--response curve of auxin-induced root surface alkalinization. *Statistically significant increase in surface pH at the < 0.05 level; Student t-test was performed by comparing average pH values measured during the time period 30 s prior to auxin addition to the average pH at specific time points after auxin addition. Means ± SE of n = 6 (ethanol control; 100 nm IAA) and n = 4 (1 nm IAA; 10 nm IAA) measurements. Black, 200 μm; red, 300 μm; green, 400 μm; purple, 500 μm from the extreme root tip.

Figure S4. Cytosolic acidification of epidermal cells of the root apical elongation zone in response to auxin treatment. (a) Treatment with 1 μm IAA elicits a rapid decrease in cytosolic pH. Representative of n = 7 experiments. In contrast, no change in cytosolic pH was detected in the lower epidermis of gravistimulated roots where auxin is thought to accumulate and trigger a gradual extracellular alkalinization (data not shown). (b) Average cytosolic pH values before and after treatment with 1 μm IAA. For each root, the cytosolic pH of an epidermal cell was averaged over the 30-s period prior to adding IAA and for the 30-s period between 45s and 75s after IAA treatment. ***pH values were significantly different before and after treatment (P < 0.001; paired t-test, n = 7). Cytosolic pH was measured using Arabidopsis roots expressing the pH-sensitive GFP variant H148D. Either the monomeric GFP H148D or – for increased brightness – a tandem version GFP H148D were imaged and calibrated as described (Monshausen et al., 2007).

Figure S5. Basipetal auxin transport in WT and aux1 Arabidopsis roots. Radiotracer assays with 3H-IAA were performed by locally applying 10 nl 3H-IAA to the root surface. Upper panel, the aux1 lesion did not alter net 3H-IAA transport when 3H-IAA was applied to the root surface at the level of the quiescent center. Lower panel, 3H-IAA transport was strongly inhibited in aux1 when 3H-IAA was applied to the root cap at the level of the columella. This reduction was observed both in the presence and absence of ethylene, indicating that when auxin is applied to the root cap, basipetal transport is strongly dependent on AUX1, and ethylene is not directly involved in responses observed in aux1 under such conditions.

Figure S6. Surface pH changes along tir1-1 afb2-3 afb3-4 root triggered by global application of 1 μm IAA. The surface pH in the plot is color-coded according to the scale bar.

Figure S7. Cytosolic Ca2+ levels along Arabidopsis root contacting an agar block containing 1 μm benzoic acid. Note that tip-localized application of benzoic acid did not cause any elevation of cytosolic Ca2+ levels. Black, 200 μm; red, 300 μm; green, 400 μm; purple, 500 μm from the extreme root tip.

Figure S8. Outlines of cells analyzed for Figure 3f, g. Pseudo-colored and fluorescence images of epidermal cell file(s) on (a) upper flank and (b) lower flank of gravistimulated roots. Colors of outlines correspond to colors of curves in Figure 3f, g.

Video Clip S1. Development of a surface pH gradient across a gravistimulated Arabidopsis root growing through agar containing the pH sensor fluorescein conjugated to a 10-kDa dextran. Increasing fluorescence intensity indicates increasing pH. Note that pH asymmetry reverses when the root is returned to vertical orientation. Images were acquired every 5 s, every third image is shown; movie duration = 50 min.

Video Clip S2. Surface pH fluctuations along a vertically oriented Arabidopsis root growing through agar containing the pH sensor fluorescein conjugated to 10-kDa dextran. Increasing fluorescence intensity indicates increasing pH. Images were acquired every 5 s, every third image is shown; movie duration = 50 min.

Video Clip S3a,b. (a) Surface pH fluctuations along a vertically oriented Arabidopsis aux1-J0951>>AUX1 root. (b) Development of a surface pH gradient across a gravistimulated Arabidopsis aux1-J0951>>AUX1 root. Roots were growing through agar containing the pH sensor fluorescein conjugated to 10-kDa dextran. Increasing fluorescence intensity indicates increasing pH. Images were acquired every 10 s; movie duration = 46 min (a), 35 min (b).

Video Clip S4. Effect of root-tip localized application of auxin on the surface pH of an Arabidopsis root. Increasing fluorescence intensity indicates increasing pH. Note the basipetal migration of surface alkalinization once the root tip has made contact with the agar block containing 1 μm IAA. See Figure 2c for experimental set-up. Images were acquired every 10 s; movie duration = 13 min.

Video Clip S5. Auxin-induced increase in root cytosolic Ca2+ levels. At the indicated time-point, 100 nm IAA was added to the medium. See Figure 3a for color-coded scale bar. Images were acquired every 3 s; movie duration = 4 min.

Video Clip S6. Effect of root-tip localized application of auxin on cytosolic Ca2+ levels of an Arabidopsis root. Note the basipetal migration of Ca2+ increase once the root tip has made contact with the agar block containing 1 μm IAA after 10.33 min. See Figure 2c for experimental set-up. Images were acquired every 5 s; movie duration = 16.75 min.

Video Clip S7. Gravity-induced changes in cytosolic Ca2+ levels in the root epidermis. Ca2+ levels were monitored in the apical elongation zone on the lower side of the horizontally oriented root. Image sequence begins 0.5 min after start of gravistimulation. Images were acquired every 3 s; movie duration = 9.6 min.

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