Abstract
The concentration of CO2 in the atmosphere has increased over the past 200 years and is expected to continue rising in the next 50 years at a rate of 3 ppm·year−1. This increase has led to a decrease in seawater pH that has changed inorganic carbon chemical speciation, increasing the dissolved . Posidonia oceanica is a marine angiosperm that uses as an inorganic carbon source for photosynthesis. An important side effect of the direct uptake of is the diminution of cytosolic Cl− (Cl−c) in mesophyll leaf cells due to the efflux through anion channels and, probably, to intracellular compartmentalization. Since anion channels are also permeable to we hypothesize that high , or even CO2, would also promote a decrease of cytosolic (). In this work we have used - and Cl−-selective microelectrodes for the continuous monitoring of the cytosolic concentration of both anions in P. oceanica leaf cells. Under light conditions, mesophyll leaf cells showed a of 5.7 ± 0.2 mM, which rose up to 7.2 ± 0.6 mM after 30 min in the dark. The enrichment of natural seawater (NSW) with 3 mM NaHCO3 caused both a decrease of 1 ± 0.04 mM and a decrease of 3.5 ± 0.1 mM. The saturation of NSW with 1000 ppm CO2 also produced a diminution of the , but lower (0.4 ± 0.07 mM). These results indicate that the rise of dissolved inorganic carbon ( or CO2) in NSW would have an effect on the cytosolic anion homeostasis mechanisms in P. oceanica leaf cells. In the presence of 0.1 mM ethoxyzolamide, the plasma membrane-permeable carbonic anhydrase inhibitor, the CO2-induced cytosolic diminution was much lower (0.1 ± 0.08 mM), pointing to as the inorganic carbon species that causes the cytosolic leak. The incubation of P. oceanica leaf pieces in 3 mM -enriched NSW triggered a short-term external net concentration increase consistent with the leak. As a consequence, the cytosolic diminution induced in high inorganic carbon could result in both the decrease of metabolic N flux and the concomitant biomass N impoverishment in P. oceanica and, probably, in other aquatic plants.
Introduction
Nitrate ion () is the main source of inorganic nitrogen for plants in aerobic conditions. Compared with the concentrations in soils, the concentration of in seawater is persistently very low, particularly in the oligotrophic Mediterranean Sea (; ). Seagrasses are the unique angiosperms that evolved from land plants to live submerged in the sea, forming the basis of the most productive and widespread coastal ecosystems on the planet (). For vascular plants, colonizing the sea implicates losses and gains to effect structural and physiological adaptations to complete the submerged life cycle, achieved by a reverse evolutionary trajectory in the seagrass lineage (). Thus, key land angiosperm innovations were lost in seagrasses including the entire collection of genes involved in stomata differentiation, genes related to the synthesis and sensing of terpenoids and other volatile substances, genes for ultraviolet protection, and phytochromes for far-red sensing (). However, to survive in the conditions of low availability, seagrasses have evolved high affinity uptake systems to capture through their leaves. These systems have been characterized for Zostera marina in which the uptake of and inorganic phosphate (Pi) are driven by the inwardly directed electrochemical gradient for Na+ (; ). In the Mediterranean, Posidonia oceanica is an endemic coastal species of huge ecological importance (). Similar high-affinity and Na+-dependent uptake mechanisms also operate in P. oceanica for both nutrients and some amino acids (). In both cases, the low semi-saturation constants observed (2.3 and 8.7 µM for Z. marina and P. oceanica, respectively: ; ) indicate that those systems are very efficient for uptake at the very low concentrations of in seagrass meadows (; ). Nevertheless, those systems are energetically expensive because seagrass leaf cells have to keep low homeostatic Na+ concentrations in the cytosol to maintain the Na+ motive force (; ). In this scenario, namely low availability of and the energetically expensive mechanism for its high-affinity uptake, the maintenance of inside the cells appears critical. Therefore, as with other vascular plants, seagrasses must maintain intracellular homeostasis to preserve the N metabolic flux.
In a previous work, besides cytosolic H+ and Na+, we also measured cytosolic Cl− (using intracellular ion-selective microelectrodes) in the mesophyll cells of P. oceanica (). In that work, we demonstrated that this seagrass has a direct plasma membrane symporter to uptake driven by the H+ electrochemical potential gradient. A significant increase of photosynthesis in natural seawater supplemented by 3 mM supported the role of uptake in the photosynthetic activity of this species (). Furthermore, the enrichment of seawater with 3 mM also evoked a delayed, but significant, diminution of the cytosolic Cl− concentration (). Similar cytosolic Cl− efflux has been observed in guard cells of the model land angiosperm Arabidopsis thaliana during stomatal closure in response to elevated CO2 (). This Cl− efflux from guard cells is described as taking place through the plasma membrane S-type anion channels, whose activation responded to the cytosolic concentration (). Interestingly, in addition to Cl− these anion channels have a high permeability to (), which is also released from the guard cells during stomatal closure (; ; ).
The release of Cl− from P. oceanica in response to is driven by the outwardly directed electrochemical potential gradient for Cl− (). Considering Cl−-selective microelectrodes are also partially sensitive to () we hypothesize that the increase of inorganic carbon in seawater could also lead to the decrease of cytosolic in P. oceanica leaf cells. Such diminution would affect assimilation () and could partially be responsible for the plant biomass nitrogen impoverishment expected under elevated inorganic carbon in vascular plants with non-saturated photosynthesis ().
Therefore, the aim of this work was to measure the cytosolic concentration in mesophyll leaf cells of P. oceanica to describe the responses to light and dark conditions and to the increase of dissolved inorganic carbon (CO2 and ) in natural seawater. Ethoxyzolamide, the plasma membrane-permeable carbonic anhydrase inhibitor (), has been used in combination with CO2 to test for the effect of limiting any CO2-dependent generation. Since the first work in Chara corallina (), -selective microelectrodes have been used in different plant species, such as barley (; ), A. thaliana () and rice (), plus the liverwort Conocephalum conicum (). However, so far no direct measurement of cytosolic has been reported for marine vascular plants. Furthermore, external has been monitored, and the potential effects of elevated atmospheric CO2 on the hypothesized diminution on the biomass nitrogen content are also discussed.
Materials and Methods
Plant Material
Posidonia oceanica (L.) Delile plants were sampled in Punta de Calaburras (36°30′23.4′′N 4°38′37.6′′W) Málaga, southern Spain, at 2 m depth. Plants with 6 to 12 leaves attached to a piece of the rhizome were collected and transported to the laboratory in a thermos container in less than 30 min. Then, plants were placed in an aquarium filled with continuously aerated natural seawater (NSW). The air used for this purpose was obtained from the compressed air supply of the Faculty of Sciences building. Concentration of CO2 in the air was regularly monitored (390 ± 10 ppm) using an IRGA, LICOR LI-820, Li, Nebraska (USA). Temperature was held at 15°C, and illumination was at a light intensity of 150 µmol photons·m−2·s−1 with a photoperiod 16L/8D. Renewing the seawater every three days, plants were used for experiments within two weeks after sampling.
Cytosolic and Cl− Measurements
Cytosolic nitrate and chloride were measured by electrophysiological techniques using double-barreled intracellular microelectrodes. Glass capillary preparation details have been previously described (). In short, double-barreled capillaries with different diameter (1.5 and 0.75 mm o.d., respectively, Hilgenberg, Germany) were twisted before pulling using a Narishige PD-5 horizontal puller. Then, pulled double-barreled capillaries were heated for 30 min at 180°C and silanized by adding one drop of dichlorodimethylsilane dissolved in benzene (0.05% v/v) to the interior of the blunt end of the larger barrel, while the smaller barrel (that operates as voltage electrode) was not silanized. After that, the silanized double-barrelled capillaries were heated again for 60 min at 180°C. Once cool, the silanized barrel was backfilled with the appropriate chloride or nitrate sensor solution.
For Cl−-microelectrodes, the ionophore I (99408, Fluka), dissolved in a mixture of polyvinylchloride dissolved in tetrahydrofuran (PVC/THF, 4% w/v) was used and backfilled into the silanized barrel. Once THF evaporated, the remainder was filled with 0.5 M KCl (). As described previously (), Cl−-microelectrodes were calibrated against NaCl solutions (1–100 mM) that contained 5 mM NaNO3, the putative cytosolic concentration (), to minimize the interference between Cl− and in the cytosol (). Calibration showed a linear relationship of 37 mV/pCl.
-microelectrodes were backfilled using a sensor (Fluka 72549) based on the quaternary ammonium compound, methyltridodecylammonium nitrate (MTDDA.) containing the PVC/THF solution. These liquid ion-exchange based microelectrodes are highly selective for , maintaining a nitrate detection limit of 0.5 mM in the presence of 100 mM Cl−, which means any interference will not be important in a physiological situation, as described in . Once THF was evaporated from the nitrate-sensor cocktail and before use, the -selective barrel was backfilled with 0.1 M NaNO3 and 0.1 M KCl. Then, -selective electrodes were calibrated against NO3K solutions (0.1–20 mM) containing 10 mM KCl, to saturate the presumed interference using the Cl− concentration reported previously in P. oceanica mesophyll leaf cells (), and KH2PO4 (from 15 to 50 mM), to give a constant background ionic strength in the calibration solutions (). calibration showed a linear relationship of 53 mV/pNO3.
For measurements, the microelectrode voltage barrel was backfilled with 0.5 M KCl (; ). Then, the - or Cl−-selective microelectrode and the reference electrode (containing agar 0.03% (w/v) in 0.5 M KCl) were fixed to Ag/AgCl electrode holders and connected to a high-impedance differential amplifier (FD223a, WPI, Sarasota, Florida, USA). Amplifier signals were continuously monitored on a double pen chart recorder (Linseis L250E).
Measurements were performed on leaf pieces (≈ 1 cm long), longitudinally peeled to remove part of the epidermis and fixed with paraffin wax on a Plexiglas transparent chamber (1.1 ml volume). A gravity-based flow-through system permitted controlled changes of the assay medium at a rate of 10 ml·min−1, which renewed chamber volume approximately 10 times every minute. This system kept the temperature, the ionic concentration, and gases constant during the experiments. Measurements were made under a microscope light of 150 µmol photons·m−2·s−1.
Quantification in Assay Solutions
In order to monitor the net efflux and/or uptake of from assay medium, plants were previously adapted to N-sufficiency by incubation in NSW enriched with 100 µM NaNO3 during 2 days. Then, excised leaves (2–3 g fresh weight) were placed separately in 250 ml flasks and incubated in 100 ml NSW (control) or NSW supplemented with 3 mM NaHCO3. The assay was carried out at 25°C with gentle and constant agitation. For each treatment, samples of assay medium were taken at 0, 1, 2, 3, 5, 7, 10, 15, 20, and 30 min. The highly sensitive method for determination, based on vanadate reduction and the subsequent spectrophotometric determination of NO2− () was used to quantify concentration in each sample. Net efflux and uptake rates were estimated as the slope of the linear phase of -concentration time course, as a function of fresh weight (FW). Six replicates were conducted for each assay.
Assay Solutions
Natural seawater (NSW) supplemented with 3 mM was prepared by adding the appropriate volume of a 0.5 M NaHCO3 stock solution (pH 8.2). CO2-enriched NSW was obtained by bubbling with artificial air (Air Liquide, Spain, 1000 ppm CO2-air). A stock solution of the plasma membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide (EZ, 10 mM) was prepared in 0.05 M NaOH. The addition of equivalent volumes of NaOH without the inhibitor had no effect on measurements. All chemicals were analytical grade and were purchased from Sigma-Aldrich.
Data Presentation and Statistical Analyses
Time-course measurements are shown as single traces, representative of a number of equivalent experiments carried out under the same conditions, as stated in the figure legends. Data are presented as means, and error bars are standard deviations. Number of repetitions (n) is indicated in every experiment. Data were analyzed using SPSS Statistics, version 21. The significance level was set at P < 0.05.
Results
Effect of Light-Dark Transitions on P. oceanica Cytosolic
In NSW, mesophyll leaf cells of P. oceanica showed a stable plasma membrane potential of −174 ± 8 mV (as in ) and a cytosolic concentration of 5.7 ± 0.2 mM (n = 10). The transition from light to darkness evoked a fast depolarization of approximately 14 mV followed by a transient hyperpolarization of 22 mV and a subsequent, more prolonged, depolarization to level of at a lower membrane potential of −140 ± 5 mV (n = 12, P = 0.003, Student t test) after 25 min in the dark. With a delay of a few minutes, light-dark transition promoted the gradual increase of cytosolic concentration that stabilized at a higher value (7.2 ± 0.6 mM ; n = 10, P = 0.02, Student t test) after 30 min in the dark (Figure 1).
Figure 1
Effect of -Enriched NSW on Cytosolic
We have previously reported that in P. oceanica mesophyll leaf cells, incubated in light conditions, the addition of NSW enriched with 3 mM evoked an initial and transient plasma membrane depolarization that turned into a transient hyperpolarization to stabilize at a depolarized value (). The simultaneous measurement of cytosolic chloride showed a delayed but significant decrease of the cytosolic concentration of this anion concomitant with the extent of membrane depolarization (). The partial sensitivity of Cl−-selective microelectrodes to (; ) allows us to hypothesize that enrichment not only produces the cytosolic Cl− decrease but could also evoke a cytosolic shift. To test this hypothesis, cytosolic was measured in P. oceanica mesophyll leaf cells in the same conditions that we had reported for cytosolic chloride, the monitoring of which was used as a control in this work. Figure 2 shows the membrane potential response to the enrichment of NSW with 3 mM (black trace) and the simultaneous measurements of cytosolic Cl− (green trace) or (blue trace), values and stats are presented in Table 1. As found previously (), the addition of 3 mM caused an initial and transient membrane depolarization (black trace) of approximately 5 mV, which turned into a transient hyperpolarization (reaching a minimum membrane potential of −181 ± 2 mV, n = 5, P = 0.012, Student t test), followed by a prolonged depolarization that stabilized within approximately 40 min at a membrane potential of −168 ± 3 (n = 5). Also in agreement with the previous study, the addition of 3 mM evoked a decrease of cytosolic Cl− (after approximately 4 min) which continued progressively from 9.7 ± 0.2 mM to 6.3 ± 0.3 mM (green trace, n = 5; P = 0.004, Student t test) 20 min after addition. Supporting the hypothesis of enrichment's producing a cytosolic shift, P. oceanica mesophyll leaf cell cytosolic also decreased in response to the enrichment of NSW with 3 mM (Figure 2, blue trace). In common with the response of cytosolic Cl−, the diminution of cytosolic started approximately 4 min after treatment. Cytosolic diminished gradually from 5.7 ± 0.2 mM to a steady lower value of 4.7 ± 0.1 mM after 35 min of addition, resulting in a significant cytosolic shift of 0.9 ± 0.06 mM (n = 5; P = 0.03, Student t test). In both cases, time courses of cytosolic and cytosolic Cl− decreases aligned with the recovery of the transient membrane hyperpolarization and profound membrane depolarization, supporting a cytosolic leak of negative charge induced by the addition.
Figure 2
Table 1
| Membrane Potential (mV) | Cytosolic (mM) | Cytosolic Cl− (mM) | |
|---|---|---|---|
| NSW | −170 ± 8 initial value | 5.7 ± 0.2 initial concentration | 9.7 ± 0.2 initial concentration |
| +3 mM | −181 ± 2* initial hyperpolarization −168 ± 3 final value | 4.7 ± 0.1* final concentration | 6.3 ± 0.3* final concentration |
| +1000 ppmCO2 | −174 ± 3 initial hyperpolarization −165 ± 3 final value | 5.3 ± 0.6 final concentration | |
| +1000 ppmCO2 (0.1 mM EZ) | −178 ± 2 final value | 5.6 ± 0.1 final concentration |
Membrane potential (mV), cytosolic (mM), and cytosolic Cl− (mM) measured in mesophyll leaf cells of P. oceanica incubated in natural seawater (NSW) supplemented with different inorganic carbon concentrations.
EZ is the carbonic anhydrase inhibitor, ethoxyzolamide. Values are mean ± SD of a number of experiments indicated in the text. Asterisks (*) denote significant differences with respect to control conditions (NSW), the statistical values are indicated in the text.
Effect of CO2 Increase on Cytosolic
As we have previously reported, in P. oceanica mesophyll leaf cells the direct uptake of and the subsequent use of CO2 for photosynthesis has consequences for anion homeostasis (). In this work, we show that use in this plant has an effect not only on cytosolic Cl− but also on cytosolic (Figure 2). The match of membrane depolarization and the onset of cytosolic Cl− and diminution suggests the involvement of plasma membrane S-type anion channels, which are permeable to both anions and whose activation takes place when cells depolarize (; ; ). In terrestrial angiosperm guard cells, these channels are activated when the concentration of (not CO2) increases and the cytosolic pH (pHc) is alkaline (). These are similar conditions to those observed in P. oceanica in the presence of high (). In order to rule out the role of CO2, cytosolic was measured in mesophyll leaf cells of P. oceanica incubated in NSW supplemented with 1000 ppm CO2, in the absence or in the presence of the plasma membrane-permeable carbon anhydrase inhibitor ethoxyzolamide (0.1 mM EZ; ). As shown in Figure 3 (data are summarized in Table 1), the increase of CO2 in NSW evoked a gradual plasma membrane hyperpolarization, reaching a value of −174 ± 3 mV (n = 5) after 7 min of 1000 ppm CO2 treatment. This maximum hyperpolarization was lower and almost 2 min delayed compared to that induced by the treatment with 3 mM , included in Figure 3 as control (black trace). Cytosolic also decreased after the addition 1000 ppm CO2; however, in these conditions cytosolic diminution was lower than that induced by 3 mM treatment. After 15 min in the presence of 1000 ppm CO2, cytosolic stabilized at a concentration of 5.3 ± 0.6 mM, a statistically non significant shift (n = 5; P = 0.37, Student t test). Furthermore, in the presence of 0.1 mM EZ, the enrichment of NSW with 1000 ppm CO2 evoked a marked plasma membrane hyperpolarization that reached a steady maximum value of −178 ± 2 mV after 12 min treatment. In the presence of EZ, cytosolic showed a minimum, statistically non-significant, shift from 5.7 ± 0.2 to 5.6 ± 0.1 mM (n = 4; P = 0.53, Student t test). That neither the long-term depolarization nor the drop in cytosolic content evoked by were evident with CO2 in the presence of EZ (thus restricting production from the CO2 source) points to the need for substantial cytosolic accumulation to effect ionic fluxes. Accordingly, these results strongly indicate that the cytosolic decrease is caused by the enrichment and point to the S-type anion channel activation by the cytosolic (not CO2) increase.
Figure 3
Effect of Increase on External
The activation of S-type anion channels may allow the efflux of from mesophyll leaf cells; such a phenomenon of efflux should be higher in the case of -replete cells. Thus, in order to investigate if the enrichment of NSW promoted efflux from P. oceanica leaves, the time course of external concentration change was monitored in assay medium containing leaves from plants pre-incubated for 2 days in NSW containing 100 µM . -supplied leaves were then incubated in NSW containing the standard concentration (10 µM). After the addition of 3 mM , external concentration increased significantly (10.8 ± 0.04 µM ; n = 6; P <0.001, Student t test) within the first minute of incubation. Maximum net efflux rate was 18 ± 2 nmol ·gFW−1·min−1. Then, external decreased at a net rate of 8 ± 1 nmol ·gFW−1·min−1 until 5 min of incubation to stabilize at a concentration of 9.2 ± 0.1 µM (Figure 4). Under control conditions (no addition), no external increase was detected, but concentration progressively decreased at a net rate of 11 ± 2 nmol ·gFW−1·min−1 during the first 10 min of incubation to reach a steady lower value of 7.6 ± 0.2 µM (n = 6; P = 0.002, Student t test). External concentrations showed higher values in all samples from leaves incubated in NSW enriched with 3 mM suggesting a net efflux from P. oceanica leaves under these conditions.
Figure 4
Discussion
Cytosolic Changes During Light–Dark Transitions in P. oceanica Leaf Cells
The concentration of in the cytosol () depends on a series of highly regulated processes at the cellular level that includes sensing and transport at the plasma membrane (uptake and efflux), subcellular compartmentalization and metabolism. Depending on the technique and the plant system used, a wide range of cytosolic values has been reported with also a large degree of variability. Therefore some authors suggest that would not be subject to homeostasis, as would be the case for cytosolic pH or Ca2+ for example (for discussion see or ). After the development of intracellular -selective microelectrodes by it is possible to perform continuous measurements of the cytosolic free ion activity of with the possibility to obtain instantaneous responses to changes in the experimental conditions. This approach has been used here to report the first cytosolic values of a marine vascular plant.
Under light conditions the cytosolic measured using intracellular -selective microelectrodes in P. oceanica mesophyll leaf cells was 5.7 ± 0.2 mM (n = 10), almost double the value reported for both epidermal and mesophyll leaf cells of A. thaliana (2.2 and 2.8 mM , respectively; ). This cytosolic in mesophyll leaf cells of P. oceanica is also much higher than that reported for the intermodal cells of the freshwater alga C. corallina (1.6 mM ; ) or in the liverwort C. conicum (0.63 mM ; ), using the same technique. However, the value found in P. oceanica is similar to those observed in root cells of barley (5.4 mM ; ) or maize (3.1 mM ; ).
The value in mesophyll leaf cells of P. oceanica is lower than the cytosolic Cl− concentration (Cl−c) of 9.7 mM Cl−, yielding a /Cl−c of 0.6. This ratio is higher than that calculated for C. conicum,/Cl−c ≈ 0.1, taking 7.4 mM as the Cl−c (), but it is in the range of the estimates for root cells of barley (/Cl−c ≈ 0.9) or A. thaliana mesophyll leaf cells (/Cl−c ≈ 0.3). Those values have been calculated considering Cl−c as 6 mM for barley root cells () and assuming that the reported cytosolic Cl− in A. thaliana root cells (8.7 mM Cl−c; ) would be the same in mesophyll leaf cells.
Changes in environmental conditions have been related to alterations, supporting the idea that the cytosol operates a strong ion homeostasis not only for pH, Pi or Ca2+, but also for (reviewed by ). Light-dark transition triggers a slight increase of in the liverwort C. conicum () and a transient increase, from 2 to 3.5 mM, which peaked after 7 min of darkness in the case of A. thaliana leaf cells (). A similar response has been found here in P. oceanica mesophyll leaf cells, in which cytosolic reached a maximum value of 7.2 mM after 25 min of dark treatment. In the case of A. thaliana, since the effect was not observed when measurements were performed in the nitrate reductase (NR) mutant (nia1nia2) leaf cells, the increase was explained because the shift to the dark inactivates NR, leading to a transient build-up of due to a slower reduction rate (). A higher in A. thaliana nia1nia2 mesophyll leaf cells and a similar time course of cytosolic increase to the rate of NR activity change in response to illumination transitions (half-life of 2 to 15 min in spinach; ; ; ) support the evidence for the role of NR in regulating (). In P. oceanica, the increase observed in the dark could also explain the one-half diminution of the maximum high affinity uptake observed previously in mesophyll leaf cells of this plant (), due to the apparent substrate inhibition of the transporter.
Inorganic Carbon Increase Triggers Cytosolic Decrease
In a previous work we demonstrated that a plasma membrane nH+/ symporter mediates the uptake of in P. oceanica mesophyll leaf cells. Further, the direct uptake of followed by its internal dehydration renders CO2 (used for photosynthesis) and hydroxyl anions, promoting membrane potential and cytosolic pH and Cl− variations (). In the present work, we have also verified that uptake of promotes the decrease of in P. oceanica mesophyll leaf cells. The increases of cytosolic and cytosolic pH have been related to promoting the opening of plasma membrane S-type anion channels in guard cells (). A similar pathway seems to explain the diminution observed in response to addition in P. oceanica mesophyll leaf cells, as was previously proposed in the case of Cl−c (), used as a control in this work. In both cases, the onset of and Cl−c decreases matched that of the plasma membrane depolarization, and indeed depolarization is a required initial phase to activate S-type anion channels operating at the plasma membrane of guard cells (; ; ).
S-type anion channels are encoded by the small SLAC/SLAH gene family, that share homology to transport systems found in different kingdoms (). In A. thaliana, apart from SLAC1 that is exclusively expressed in guard cells, four additional homologs (SLAH1-4) are present (). SLAC1 and SLAH3 channels exhibit a permeability preference for and Cl− but not for malate (; ; ). The SLAH3 channel has the highest permeability for (/Cl− permeability ratio of 20) and in contrast to SLAC1, SLAH3 also requires extracellular to induce its activity (). Furthermore, a role in -dependent alleviation of ammonium toxicity in A. thaliana roots has been proposed for SLAH3 ().
Although seagrasses have lost stomata differentiation genes () the presence of S-type anion channels cannot be ruled out in those plants since, presumably, these anion channels have evolved as emergency valves, rapidly releasing excess osmolytes under stress conditions (). The Zostera marina genome, the first one available from a seagrass (), contains two homologs for the A. thaliana SLAC/SLAH gene family (Phytozome v12.1, data base), one SLAH1 homolog (Zosma91g00860.1) and one SLAC1 homolog (Zosma76g00610.1). The presumed homologs of these channels in P. oceanica should be good candidates for the proposed channels for the leak of and Cl− ions from the cytosol, but further molecular analyses are needed to characterize the specific role of these anion channels to mediate anion efflux in response to high .
In guard cells, stomatal closure in response to high CO2 is mediated by the activation of S-type (SLAC1/SLAH3) anion channels (reviewed by ). Interestingly, guard cells do not sense CO2 themselves, but instead HCO3– synthesized from CO2 within the cytosol by carbonic anhydrases (). The same case could be proposed in P. oceanica mesophyll leaf cells, since no significant decrease was found in response to 1000 ppm CO2 treatment in either the absence or the presence of the plasma membrane-permeable carbon anhydrase inhibitor ethoxyzolamide, suggesting that , not CO2, is the inorganic carbon species that triggers efflux from P. oceanica mesophyll leaf cells (Figure 5). Furthermore, probably due to the activation of the H+-pump in response to the cytosolic accumulation of CO2, a weak acid, the 1000 ppm CO2 treatment renders the hyperpolarization of the plasma membrane and the cytosolic acidification (see ). Both responses are prolonged in the presence of ethoxyzolamide, that impairs production from the CO2 source, leading to conditions that not support the activation of S-type anion channels described above and could explain the non-significant diminution observed in response to CO2 increase.
Figure 5
Cytosolic Efflux Under High Inorganic Carbon Could Contribute to Biomass N-Impoverishment in Seagrasses
As we showed previously, direct uptake has consequences for cytosolic ion homeostasis in P. oceanica mesophyll leaf cells (). The short-term net efflux of and the low net uptake rate observed in the present work also indicate that natural seawater enrichment may be important not only for cytosolic ion homeostasis but for the metabolic flux of in this seagrass. In natural seawater, containing 500 mM Cl− and 10 µM , the efflux of Cl− and from P. oceanica mesophyll leaf cells is driven by the outwardly directed anion electrochemical potential gradients for both anions (Figure 5). This electrochemical potential gradient (in mV) is almost five-fold higher for (+330 mV) than for Cl− (+71 mV). However, in response to high the Cl−c decrease observed in P. oceanica mesophyll leaf cells (ΔCl−c = 3.4 mM) was much higher than for (Δ = 1 mM). This lower leak than the expected from electrochemical potential gradient comparison with Cl−c could be explained by a different membrane permeability for both anions and/or different capability of compartmentalization. Nevertheless, even the observed decrease of , the short-term efflux, and the low uptake rate could impair N-assimilation in P. oceanica leaf cells in natural seawater containing high inorganic carbon.
An impaired metabolic flux of N should be relevant in the context of atmospheric CO2 rise. Oceans have been the sink for 30% of CO2 released during the industrial era, at a higher rate (3.8 GTons·year−1) than the 1.8 GTons·year−1 fixed by photosynthesis or the 2 GTons·year−1 removed by abiotic absorption (). Atmospheric CO2 is exchanged into aquatic environments rendering the dissolved inorganic carbon (DIC) equilibrium. Controlled by pH, this equilibrium generates the distribution of DIC species: dissolved CO2, bicarbonate () and carbonate (CO32−) ions. Consequently, elevated atmospheric CO2 concentration increases the total DIC and lowers the pH, shifting the relative proportion of each DIC species. Under current ocean pH (~8.04) and atmospheric CO2 (~ 410 ppm), the smallest pool of DIC is dissolved CO2, but this will have the greatest increase (> 250%) among the DIC constituents as the pH drops (~ 0.3–0.4 pH units) under the predicted rise in atmospheric CO2 (1000 ppm) for 2100. In contrast, the pool will only increase by 15% at that date (). However, in terms of absolute concentration (mol·kg−1) levels will rise more than dissolved CO2 (). Using the predicted data for 2100, calculations render 2.05 mmol·kg−1 and only 0.03 mmol·kg−1 CO2 (). Considering such a rise (1.2 fold higher than actual concentration) and that the addition of 3 mM corresponds to an increase by 2.3 fold of the concentration in natural seawater, a leak of 0.5 mM would be expected in P. oceanica mesophyll leaf cells by 2100. However, long-term effect of elevated atmospheric CO2 concentration on seagrasses N content needs further investigation, since N-deficiency induces high-affinity uptake, which contributes to homeostasis (reviewed by ).
Furthermore, the concomitant natural seawater pH decrease under elevated atmospheric CO2 could alter N availability in seawater and even the energy cost for nutrient uptake. A pH change from 8.1 to 7.8 evokes a decrease in the amount of NH3 in the NH4+/NH3 ratio (), whereas the amount of would not be affected (). Instead, the 0.3 external pH unit decrease renders a rise of the proton motive force at the plasma membrane of P. oceanica leaf cells of −18 mV, considering −174 mV as membrane potential, 7.3 as the cytosolic pH and constant temperature (). This amount represents, approximately, a 15% increase of the proton motive force (inwardly directed), which could prompt the activity of H+-dependent transport systems in this seagrass, including the plasma membrane H+/ symporter (). As discussed by , ocean acidification could increase both the H+ and even the Na+ motive force due to changes in the activity of the plasma membrane Na+/H+ antiporter found in seagrass species (). The activity of this antiporter generates a lower cytosolic Na+ concentration at acid external pH and, consequently, a rise of the inwardly directed Na+ motive force could be also expected, favoring high-affinity uptake based on Na+-dependent transport systems in seagrasses (reviewed by ). In fact, a recent study in P. oceanica beds from the Gulf of Naples (Italy) shows that the long-term exposure (9 months) to acidified seawater (pH 7.82) under constant DIC conditions promotes the diminution of the C/N molar ratio, due to the increase of N content by 21% and 70% in leaves and rhizome, respectively, whereas C content of those organs is not affected by external pH acidification (). Those authors suggest that seawater acidification promoted a feed-forward long-term effect on N accumulation in P. oceanica, especially at the rhizome, although they recognize the need to specify the effect of acidification on the nutrient availability in their study (). Interestingly, long-term nutrient enrichment seems to modulate the effects of ocean acidification on P. oceanica. Molecular analysis indicates that after 18 months at low pH (7.78) conditions the expression of nitrate transporter genes in P. oceanica leaves is altered; while NRT1_6.3 and NRT1_2.13 (involved in sensing and low-affinity transport, respectively) are overexpressed the high-affinity transporter gene NRT2 shows a down-regulated expression (). Thus, long-term overexpression of nitrogen transporter genes following nutrient additions at low pH suggests enhanced nutrient uptake and proposes that the effects of ocean acidification on P. oceanica depend upon local nutrient concentration ().
Contrary to the acidification effect in P. oceanica meadows, several lines of evidence show that the most common effect of elevated CO2 is a decrease in the dry mass concentration of N in plant tissue (; ; ; ; ; ). This suggests that physiological changes leading to decreased biomass N under elevated CO2 predominate in their effects over factors that would tend to increase N content (reviewed by ). Consequently, plants with non-saturated photosynthesis at actual atmospheric CO2, show an ionomic imbalance, with N the main nutrient that decreased at high C assimilation (). The physiological mechanisms responsible for this phenomenon have not been well established yet, although different hypotheses are proposed to account for it. In terrestrial vascular plants, the best-supported are the decrease of specific N uptake and assimilation due to a diminution of the transpiration-driven mass flow of N by a decreased stomatal conductance at elevated CO2 and the biomass dilution of N by increased photosynthetic assimilation of C (reviewed by ).
Seagrass meadows rank among the most productive ecosystems on Earth (), which largely contribute to C uptake in coastal waters and with most species capable of utilizing as a CO2 source for photosynthesis (). With the exception of Cymodocea nodosa (considered C4), seagrasses show C3 photosynthetic pathways and are not saturated at the current ocean DIC concentration (reviewed by ). As occurs in C3 terrestrial plants, a higher C assimilation occurs in seagrasses due to the rise of DIC discussed above (). The unpredicted effect of enrichment on anion homeostasis in P. oceanica mesophyll leaf cells, leading to the short-term efflux of from the cytosol, possibly through the activation of S-type anion channels, supports a new mechanism as a key consideration in understanding the expected biomass N-alteration in seagrasses under elevated DIC, and probably, for terrestrial plants growing in waterlogged alkaline environments.
Funding
This work was supported by the Spanish Ministerio de Economía y Competitividad (cofinanced by the European Regional Development Fund; grant BFU2017-85117-R) awarded to JF and LR and the University of Cambridge's Central Chest (JD).
Statements
Data availability statement
The datasets generated for this study are available on request to the corresponding author.
Author contributions
LR, DG-P, and JF performed the experiments. DG-P and LR accomplished data analysis. LR, JD, and JF discussed the results, wrote and edited the manuscript. LR and JF conceived the project. All authors contributed to the article and approved the submitted version.
Acknowledgments
This work has been done in the framework of BIO2016-81957-REDT (LR and JF) and Campus de Excelencia Internacional del Mar, CEIMAR (Andalucía Tech).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Summary
Keywords
cytosolic NO3−, elevated inorganic carbon, NO3− efflux, anion channels, intracellular nitrate-selective microelectrodes, seagrasses
Citation
Rubio L, García-Pérez D, Davies JM and Fernández JA (2020) Short-Term Response of Cytosolic to Inorganic Carbon Increase in Posidonia oceanica Leaf Cells. Front. Plant Sci. 11:955. doi: 10.3389/fpls.2020.00955
Received
28 February 2020
Accepted
10 June 2020
Published
25 June 2020
Volume
11 - 2020
Edited by
Francisco Rubio, Center for Edaphology and Applied Biology of Segura, Spanish National Research Council, Spain
Reviewed by
Juan Jose Rios, Center for Edaphology and Applied Biology of Segura, Spanish National Research Council, Spain; Santiago Alejandro, Martin Luther University of Halle-Wittenberg, Germany
Updates
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© 2020 Rubio, García-Pérez, Davies and Fernández.
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*Correspondence: Lourdes Rubio, Lrubio@uma.es
This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science
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