Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging

Noninvasive pH Sensors Are Anchored to the PM.

To investigate proton homeostasis in intact roots, we utilized ratiometric pHluorin. This is the best-described genetically encoded pH sensor with little sensitivity to other parameters such as redox status, potassium, sodium, and calcium ions (15, 22, 23). To record pH values close to the PM, we fused pHluorin to a transmembrane domain such that the fusion protein would be anchored in the PM (24) with the pH-sensing moiety facing the apoplast. As represented in Fig. 1A, this apoplastic membrane pH sensor, PM-Apo, has its fluorochrome positioned a few nanometers away from the PM, which is nearly 1,000 times closer to the cell surface than what can be achieved using classical pH electrodes. When transiently expressed in tobacco leaf epidermis, PM-Apo fluorescence was evenly distributed at the cell periphery, as expected for PM labeling (Fig. 1B). This is in contrast to the labeling pattern obtained with Apo, a secreted soluble pHluorin that can diffuse away from the membrane in the apoplast (Fig. 1A). As shown in Fig. 1C, Apo fluorescence labeling was scarce, weak, and restricted to occasional “pockets” that are likely due to acidic quenching of the pHluorin in most of the apoplast (Fig. S1A).

To quantify the PM delta pH, we also designed a sensor anchored in the inner face of the PM. For this, we fused pHluorin to the farnesylation sequence of Ras (25) and named it PM-Cyto (Fig. 1A). As shown in Fig. 1D, the labeling of tobacco epidermal cells expressing PM-Cyto is restricted to the cell periphery (Fig. 1D) and does not include the nucleus, in contrast to Cyto labeling (Fig. 1E) obtained with a cytoplasmic soluble pHluorin (Fig. 1A). The membrane localization of PM-Apo and PM-Cyto was further assayed by the plasmolysis experiment and microsomal fractionation (Fig. S1 B and C, respectively).

Next, the fluorescence ratio was assessed from tobacco epidermal cells expressing the different sensors, and the corresponding pH values were calculated using an in situ calibration with a recombinant pHluorin (Fig. S2). An average pH value of 6.5 was detected at the outer face of the PM in the tobacco epidermis (PM-Apo, Fig. 1F). The different labeling patterns obtained for the two apoplastic sensors (Fig. 1 B and C) and the acidic quenching of Apo (Fig. S1A) suggest that the extracellular environment has a heterogenous pH. In contrast, the PM–cytosol interface and the entire cytosol (PM-Cyto and Cyto, respectively; Fig. 1F) both displayed a pH of 7.5. Due to the pH value obtained at the outer face of the PM, two altered versions of PM-Apo were generated with modified linkers between the sequences of the transmembrane domain and the pHluorin—PM-Apo(bis) and PM-Apo(ter)—and their PM localization was further assayed by plasmolysis (Fig. S3A). The three PM-Apo sensors are predicted to expose the pHluorin at various distances from the PM (Fig. S3B). The pH values obtained in tobacco epidermal cells with the two additional membrane-anchored sensors were slightly but significantly more alkaline than PM-Apo (Fig. S3C, P value <0.05). Interestingly, in tobacco protoplasts, this difference is abolished (Fig. S3D). Altogether, this suggests that pH measurement in close vicinity to PM is little impacted by the linker structure but is affected by the cell wall. Lastly, the membrane orientation of the PM-associated pH sensors was assayed by two different approaches (Fig. S4). A proteinase K digest on living cells (26) was performed on tobacco protoplasts, which led to a clear decrease of PM-Apo fluorescence as compared to PM-Cyto (Fig. S4A). Similarly, a proteinase K digest on Arabidopsis microsomes led to opposite responses of two PM sensors (Fig. S4B), a result that is also in accordance with pHluorin being exposed outside of the cell for PM-Apo and protected from digestion in PM-Cyto configuration. Altogether, our results with membrane-anchored sensors revealed an apoplastic pH of 6.5 to 6.7 close to the PM, and a cytoplasmic pH of 7.5 in tobacco epidermal cells.

Arabidopsis Roots Present Radial Variation in pH at the Apoplastic Interface of the PM, Leading to a Maximal PM Delta pH in Stele Cells.

Transgenic Arabidopsis plants expressing one of the two pHluorin-based sensors anchored on either side of the PM (PM-Apo and PM-Cyto) were generated. These plants did not present any visible growth phenotype compared with wild-type plants (Fig. S5A) and were able to acidify their growth media (Fig. S5B and ref. 27). A detailed analysis of pH by confocal macroscope on intact Arabidopsis plantlets revealed two acidic root zones (Fig. S6A), in agreement with previous reports using other approaches (11, 28–30). Next, the ability of PM-associated sensors to detect transmembrane transport mechanisms in mature root was confirmed using orthovanadate, an inhibitor of P-ATPase (Fig. S6B), and nigericin, a proton ionophore (Fig. S6C). In contrast, applying PM depolarization treatments either by increasing potassium concentration in the media or by adding the K⁺ ionophore valinomycin (31) did not affect PM-Apo pH (Fig. S7 B or C, respectively). To confirm the impact of the different treatment on membrane potential, we used the voltage-sensitive fluorescent dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] which exhibits an enhanced fluorescence when bound to depolarized membranes (refs. 32 and 33 and Fig. S7A). This suggests that fluorescence from PM-Apo was not affected by the electron motive force at the PM.

Subsequently, we analyzed PM-associated pH on the portion of roots with expanded hairs located roughly 2 cm from the apex and a fully differentiated Casparian strip, as confirmed by staining with propidium iodide (Fig. S8A, purple). The fluorescent signals of the PM-Cyto (Fig. 2A) and PM-Apo (Fig. 2B) sensors were limited to the periphery of the root cells and were detected all the way down to the stele using confocal microscopy on intact plantlets, as revealed in xy optical sections and xz projections (Fig. 2 A and B). In comparison, no signal was detected from nontransformed plants (Fig. S9A).

Using xz projections of transformed mature roots, cell layer-specific fluorescence data were collected at the medium–epidermis interface or between two isotypic cells (Fig. S8B), and the pH was calculated using in situ calibration curves (Fig. S2B). As shown in Fig. 2C (Bottom curve), the apoplastic pH observed close to the PM (i.e., pH_apo-PM) was stable at pH 6.4, from the medium–epidermis interface up to the cortex. Several approaches were performed to investigate the apoplastic pH further away from the PM, including the transformation of Arabidopsis plants with the Apo construct. Unfortunately, as previously described for the pH sensor apopHusion (16), most of the fluorescence signal was retained in the endoplasmic reticulum (ER), especially in the mature part of the root (Fig. S10A, arrows), making accurate pH measurements extremely difficult. We then used the fluorescent probe 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) (Fig. S10C) and estimated the overall apoplastic pH as 5.7 (Fig. S10H). This is likely an overestimation of the apoplastic space pH, since the 450-nm excitation peak of HPTS was missing at pH 5. Lastly, the pH dye Oregon Green dextran was used as previously described (34), except for application of an additional gentle vacuum (Fig. S10B); this permitted us to estimate the overall apoplastic pH to be close to 4 (Fig. S10H). Altogether, these results indicate that in mature roots, the apoplastic pH close to the PM is more alkaline than the average pH of the apoplastic space.

Deeper in the root tissue (e.g., the endodermis and the stele), pH_apo-PM was significantly more acidic than in more peripheral cell layers (Fig. 2C and Fig. S11). This difference in pH_apo-PM along the radial axis in the mature root was named radial delta pH_apo-PM (Fig. 2C). This positive radial delta pH value is in opposition to a simple model of proton diffusion between the medium and the entire root apoplastic space and shows a tight regulation of PM-associated pH in the mature root (including the outermost cell layer).

Similarly, cell layer-specific cytoplasmic pH values were measured from roots expressing PM-Cyto (i.e., pH_cyto) (Fig. 2C, Top curve). Importantly, the two sets of collected pH values allowed us to calculate the transmembrane proton gradient (PM delta pH = pH_cyto − pH_apo-PM) for each cell layer in a noninvasive manner. This PM delta pH measurement is directly linked to the PMF of the PM, which increases from the medium–epidermis interface to the internal cell layers where it reaches an optimum of 1.7 pH units in the endodermis and the stele (Fig. 2D).

The Radial Variation of pH_apo-PM Is Highly Robust in Mature Roots.

The root apoplastic space is in direct contact with the bulk media, shown by diffusion of propidium iodide in the root (Fig. S8A). As a consequence, the apoplastic liquid should be highly sensitive to any fluctuation in proton concentration in the medium. The impact of modifying the pH of half-strength Murashige and Skoog (MS/2) medium on steady-state apoplastic PM-associated pH was therefore investigated in mature roots and was found to have no effect at all on either the endodermis or the stele pH_apo-PM (Fig. 3A). Only small changes were detected in the outer cell layers up to the cortex, where pH_apo-PM was observed to increase even when a pH value of 5 was applied, although further analysis of the data revealed no statistical difference between pH 5 and pH 6 or between pH 7 and pH 8 for the epidermis and the cortex (Fig. 3A). As shown in Fig. 3B, the measured pH_apo-PM was mostly preserved, even at the interface between the root and the medium, as changing the bulk proton concentration by up to 3 orders of magnitude resulted in only a variation of 0.7 pH units at most. No effect on the apoplastic pH was observed in the stele (Fig. 3B). We observed that the radial delta pH_apo-PM was not abolished by acidifying the external medium, and it was even amplified in more alkaline conditions (Fig. 3C). A similar mild effect was observed with stronger buffering capacities of the medium (Fig. S12 A and B).

These results demonstrate that the pH of the apoplast in close proximity to the PM is very stable in MS/2 medium and that it is mostly unaffected by external conditions. This indicates that a radial PM-associated apoplastic delta pH value of at least 0.4 pH units was preserved throughout the root tissue, with the pH in the stele strictly maintained at pH 6.1 in MS/2 medium.

The Calcium and Proton Variation in Minimal Medium Influences the PM-Associated pH Observed in Mature Roots.

To further investigate the effect of ions on root pH, we replaced the complete medium with minimal ionic medium composed of 5 mM KCl to which we added various Ca²⁺ concentrations below and above that of MS/2 medium. At equilibrium, the calcium concentration was observed to have a major effect on the radial delta pH_apo-PM, which appears to follow a saturating mechanism, with an optimum of 0.5 pH units for 5 mM calcium and above (Fig. 3D). The best fit was found using an exponential relation for radial delta pH_apo-PM and calcium concentration (R² = 0.97, K_d = 1.9 mM). Moreover, the value previously obtained under MS/2 conditions (2 mM Ca²⁺ and 0.75 mM Mg²⁺) fits perfectly with this curve (Fig. 3D, square).

For the following experiments, we chose a concentration of 10 mM CaCl₂, which provides a CaCl₂/KCl ratio of 2 and, under this condition, the potassium concentration associated with the cell wall is predicted to be marginal (18). Using these conditions, the pH_apo-PM values obtained in minimal medium were compared with those previously obtained in complete medium (MS/2 medium) at the same pH level (pH 6), and no differences were detected (Fig. S12C). Next, pH_apo-PM robustness was challenged with various external proton concentrations. As shown in Fig. 3E, the pH_apo-PM values followed the bulk pH in the most external cell layers, but not in the endodermis or the stele. Consequently, the steady-state radial delta pH_apo-PM increased in a linear manner together with the alkalinization of the minimal medium, which is in contrast to what was observed in complete medium (see Fig. 3F vs. Fig. 3C).

For further insight into pH regulation across the PM, we monitored proton concentration at the inner face of the PM in mature roots (Fig. S12D) and calculated the PM delta pH (Fig. S12E). At any of the three pH levels tested, the PM delta pH increased from the epidermis to the stele where it reached an optimum of 2 pH units (Fig. S12E). For the endodermis, we observed an alkalinization of pH_cyto at pH 7 (Fig. S12D, blue), suggesting that the plant can also compensate for environmental constraints by modulating cytoplasmic PM-associated pH by cell layer-specific mechanisms. As a result of decreasing the minimal medium pH to 5, the radial delta pH_apo-PM is close to zero (Fig. 3E). This result strongly differs from those obtained in complete medium (Fig. 3A, red).

Altogether, these results demonstrate that K⁺, Cl⁻, and Ca²⁺ are less efficient at maintaining proton homeostasis in the external cell layer than the complex set of ions provided by complete medium (MS/2). It also demonstrates that Ca²⁺ has a significant impact on the radial delta pH_apo-PM in mature roots equilibrated with a minimal KCl medium.

In Minimal Medium, the Anion Species Has Little Impact on the PM-Associated pH Observed in Mature Roots.

After determining that minimal medium conditions conferred less root pH robustness to environmental constraints than complete medium, we next tested the impact of anion species. We chose two major anions that are expected to affect root pH: SO₄²⁻, which is less absorbed than Cl⁻; and NO₃⁻, which is more absorbed than Cl⁻ (35). Moreover, both SO₄²⁻ and NO₃⁻ require proton cotransport for their uptake: Sulfate is believed to consume three protons and nitrate is believed to consume two protons (36, 37). The pH on either side of the PM was measured in mature roots of plants expressing PM-Apo and PM-Cyto, after equilibration in either KNO₃/CaCl₂ or K₂SO₄/CaCl₂ conditions at pH 5, 6, or 7 (Fig. S13 A, B, D, and E). Changing the nature of the anion had no effect on the radial delta pH_apo-PM (Fig. S13C). The calculated PM delta pH of each cell layer for the NO₃⁻ (Fig. S13F) and SO₄²⁻ (Fig. S13G) conditions was unexpectedly stable in comparison with Cl⁻ (Fig. S12E). These results could be explained by the fact that a majority of the anion channels/transporters characterized so far transport both Cl⁻ and NO₃⁻ (38). Altogether, our results suggest that PM-associated pH is barely affected by the nature of the anion, suggesting the presence of potential compensatory mechanisms.

Homeostatic Regulation of PM-Associated Apoplastic pH in Arabidopsis Roots.

Arabidopsis displays a conserved gradient of pH_apo-PM in the mature root, wherein the stele is on average 0.4 pH units more acidic than the surface. When looking more closely at the distribution of pH at the level of each cell layer, it is clear that the proton concentration in the apoplastic interface of the PM does not gradually vary along a radial axis. Instead, the root is divided into two zones: an “alkaline” zone including the surface of the root, the epidermis, and the cortex; and an “acidic” zone containing the endodermis and the stele (Fig. S11). Because we performed our measurements in a fully developed part of the root with a mature Casparian strip, the pH “break” in the root apoplast can be observed to coincide with this structure in the endodermis. The Casparian strip is the main barrier for apoplastic diffusion of solutes and plays a central role in current models of plant nutrient uptake by roots. Indeed, when this barrier is deficient, as in the schengen3 mutant, a major defect in maintaining sufficient levels of macronutrients such as potassium can be observed (39). Therefore, it is possible that the Casparian strip contributes to the control of PM-associated apoplastic pH in plants.

Surprisingly, even the peripheral cell layers that are in direct contact with the medium appeared to “resist” against pH variation within the medium. These observations suggest the potential existence of strong compensatory mechanisms that appear more efficient in complete medium (Fig. S13H). The two major P-type ATPases in Arabidopsis roots, AHA1 and AHA2, are known to participate in media acidification and are necessary for plant growth in alkaline conditions (40–43). Interestingly, AHA2 activity is regulated by PKS5 kinase, which is in turn regulated by apoplastic pH (44). One in vitro study has elegantly demonstrated that a PM delta pH value higher than 2 acts directly on AHA2 activity via allosteric regulation (45). This illustrates the tight link between apoplastic pH and the activity of P-type ATPase, and might explain why in our in vivo measurement the PM delta pH saturates at 2 pH units. We could not exclude that the channel and transporter, in addition to the P-type ATPase regulation, participate in pH homeostasis. To test this hypothesis, we modified the anion species, but this resulted in little effect, suggesting that chloride, nitrate, or sulfate alone cannot restore the apoplastic pH robustness observed in complete medium. We also examined the role of calcium and determined that it acts on the radial difference of pH_PM-Apo between the two sides of the endodermal cell layer. Cell wall appears to be the biggest pool of calcium ion in plant tissues (46), with a several-millimolar concentration in horse bean and yellow lupine root (18) and 6 mM in soybean roots (47). Calcium is known as a crucial regulator of growth and development in plants (48). Modification of cell wall extensibility and cell growth are essential for the processes leading to shape plants. These modifications in stiffness are in part attributed to Ca²⁺ interaction with pectate, more precisely to the free carboxyl groups that are present in pectin, a major component of primary cell wall (49, 50). Thus, calcium depletion could free up the cell wall acidic group and modify the cell wall cation exchange capacity or buffering effect of pectin. Interestingly, radial heterogeneity of calcium association with the cell wall has been described in Brachypodium roots (51).

The strong homeostatic behavior of root cell apoplastic pH at the membrane interface is paralleled by the conservation of the delta pH formed through the PM. In MS/2 medium, we found that the PM delta pH gradually increases from the outer cell layer to the stele (from 1 to almost 2 pH units). Numerous transport and channel activities are regulated by PM delta pH. Nitrate transporter 1/peptide transporter family (NPF) proteins like NPF6.3 or NPF2.7 that are expressed in root outer layers are functional in heterologous systems when the PM delta pH is close to 1 pH unit (31, 52, 53). By contrast, NPF7.2 or NFP4.6, which are found in the root inner tissue, require a higher PM delta pH (close to 2 pH units) to generate a substantial transport activity (54–56). The expression pattern of these NPF genes is in agreement with our in vivo observed PM delta pH, in which the outer layers of the root have smaller PM delta pH values than those in the stele. In the case of potassium channels, their regulation by pH at the PM is also well documented (57). For example, the stelar K⁺ outward rectifier channel SKOR that is involved in potassium release into the xylem sap toward the shoot (58) is modulated both by inner and outer pH levels at the PM (59). Interestingly, with pH 7.7 in the cytosol and pH 6 in the apoplasm in the stele (Fig. 2C), SKOR should only be at 50% of its optimal activity, suggesting a full potential to regulate potassium secretion into xylem via pH. The inwardly rectifying potassium channel AKT1 (60) is functional in Sf9 with a PM delta pH of 1 unit (61) and is expressed in the root epidermis and cortex (62). It is therefore functional in the most external cell layers where PM delta pH values are the lowest, which agrees with the attributed function of AKT1 as the main channel for potassium uptake from the soil by roots in high potassium concentration (63).

Apoplastic pH Close to the PM Is More Alkaline than in the Overall Apoplastic Space.

To date, apoplastic pH in plants has primarily been investigated using either electrodes or fluorescent ratiometric components that diffuse into the apoplast. Although these methods allow the rapid measurement of pH, they are limited by the accessibility of the internal cell layers. Even small fluorescent molecules cannot pass through the Casparian strip, leaving the central cell layers inaccessible. This limitation was potentially surmounted by generating genetically encoded pH sensors that can accumulate in the apoplast via secretion. However, such sensors can diffuse out of the tissue and can also accumulate in the ER, which complicates apoplastic pH measurements, especially in turgescent cells such as mature root cells (15, 16). We resolved these problems by anchoring pHluorin to the PM with a transmembrane domain and executing repetitive imaging of all root cell layers up to the central cylinder in Arabidopsis. The pH_apo-PM values that were obtained with our PM-anchored sensor in mature roots grown in rich medium ranged from 6.0 to 6.3, depending on the cell type. This range is close to the value found using a secreted At-pHluorin (pH 6.3) in the lateral roots of 1- to 4-wk-old Arabidopsis plants (15, 16). This is in contrast to the more acidic cell wall measurements obtained from roots perfused with small fluorescent probes, with pH levels detected between 4 and 5.7 in mature roots (this work) and ranging from pH 4.2 to 5.3 in Arabidopsis root cells, from pH 5 to 6 in maize, or close to pH 5.2 in lupine tap roots (64, 65). Altogether, these results indicate the existence of a complex apoplastic environment, with a potential unstirred layer in the vicinity of the PM where pH is more alkaline than in the entire apoplastic liquid and with an acidic Donnan space associated with the cell wall. This raises the question of how such a gradient can be maintained, since the source of apoplast acidity should come from the activity of P-type ATPase localized at the PM (40). One possible explanation for this paradoxical situation is that the net proton extrusion recorded in Fig. S6 B and C is not coming from the root zone observed in our experiment. Indeed, it is generally accepted that the Arabidopsis root tip is acidic and therefore could alone be responsible for the medium acidification. If true, this would suggest that in fully differentiated tissue of Arabidopsis, a net influx of protons is taking place (66). It would be of great interest to further study this complex apoplastic environment, including in regions more than a few nanometers from the lipid bilayer, although this would require sensors that are not quenched below pH 5. The ability of these PM-associated sensors to monitor pH variation independent of membrane potential changes and within minutes after treatment also provides a great tool for the future study of pH on either side of the PM in potentially all cell types (including those not directly accessible), including using caged protons (67).