Long‐term net H+ influx in maize roots and its potential role in salt tolerance

Salinity is a severe world-wide threat to agriculture. The arable land available for crop production progressively decreases due to salt accumulation in soil (Isayenkov & Maathuis, 2019). So far, > 10% of cultivated land is affected, with numbers increasing steadily due to irrigation with brackish water. Ten years ago, salinity in China affected 36 million ha, or 4.88% of usable land, increasing at a rate between 1.4% and 3% p.a. (Li et al., 2014). Despite decades of research into mechanisms allowing crops to cope with potentially toxic NaCl concentrations, knowledge of the multiple traits conveying salt tolerance is still incomplete (Shabala et al., 2015; Isayenkov & Maathuis, 2019). Most crops of high economical importance are glycophytes, which tend to suffer from excess uptake of the potentially toxic Na+ and require mechanisms to keep cytosolic Na+ concentration in a tolerable range (10–30 mM; Munns et al., 2020a). Na+ entering the cytosol of root cells needs to be dumped in the vacuole or, more effectively, sequestered back into the apoplast. Therefore, SOS1, a Na+-H+ antiporter in the plasma membrane (PM) of root epidermis and cortex cells, has received much interest. Uphill Na+ efflux is energized by coupling to passive H+ influx along a trans-membrane pH gradient. Influx, for example via nonselective cation channels (NSCCs) and subsequent back-transport into the apoplast constitute circular Na+ flux, which is however highly controversial (Britto & Kronzucker, 2015; Munns et al., 2020a, b). According to textbooks, the trans-membrane H+ gradient required to drive Na+ efflux is provided by the activity of P-type H+ ATPases. However, under many conditions, the rate of ATP production by root respiration seems insufficient to energize Na+ detoxification of the cytosol. Extensive modeling by Foster & Miclavcic (2020) confirmed the apparent ‘energy deficit’, which is rather reflecting our knowledge gaps. A possible way out of this dilemma is provided by the ‘biochemical pH clamp’ hypothesis forwarded by Wegner & Shabala (2020). According to this hypothesis, a continuous net H+ influx not relying on the activity of the H+ ATPase can be maintained by a direct coupling to metabolism. This involves intracellular metabolic processes ‘consuming’ H+ (termed ‘active buffering’), thus keeping the cytosolic pH in the narrow range slightly above 7 required for cell survival. Apoplastic H+ can be mobilized by ‘passive’ buffering (the extracellular compartment which is in contact with soil solution outsizes the cytosol by several orders of magnitude) and by bicarbonate formation from CO2 (for details, see Wegner & Shabala, 2020; Wegner et al., 2021; Wegner, 2022). Imposing mild, moderate and severe salinity stress, and using the noninvasive microelectrode ion flux estimation (MIFE) technique, we showed that salinity can induce a steady net H+ influx across the root surface, providing first evidence for a key role of the biochemical pH clamp in salt tolerance in maize. Maize (Zea mays L.), the third most important crop world-wide, is known as a moderately salt-sensitive species, being highly susceptible to elevated NaCl concentrations. As a salt excluder (Fortmeier & Schubert, 1995), it relies on an effective Na+ export from the symplast back into the apoplast via SOS1 for minimizing Na+ uptake. We compared two cultivars commonly grown in China, Suketian1506 (SKT1506) and Pinnuo28 (PN28), which differ with respect to salt tolerance. The phenotype of both maize cultivars investigated in this study (see Supporting Information Methods S1) was affected by mild (60 mM NaCl) and even more severely by moderate (120 mM NaCl) salt stress, but to a different degree (Fig. 1a). Mild stress clearly retarded root growth of PN28, whereas shoot growth was less affected. Doubling the concentration entailed further growth reduction. Shoot and root length of SKT1506 progressively decreased with mild and moderate salt stress; particularly at 120 mM NaCl, SKT1506 seedlings were both tiny compared with controls, and to PN28 at the same treatment. Within limits of accuracy root and shoot length, fresh weight and dry weight attained similar values for both cultivars when exposed to ¼ Hoagland medium up to Day 12 (Fig. 1b–g). Shoot length differed for both cultivars at 60 and 120 mM NaCl, indicating that SKT1506 was more salt-sensitive than PN28 with respect to shoot growth; shoot fresh and dry weight were also lower in SKT1506, but the difference was not significant. However, root dry weight was significantly lower in SKT1506 than in PN28 at 120 mM NaCl. Taken together, these results suggest better salt tolerance of PN28 than that of SKT1506. For each of the cultivars, an increase in salt concentration entailed a significant decrease in shoot length and dry weight (for the fresh weight, there was no significant difference between salt treatments). A significant reduction was found for all root parameters when comparing moderate salt treatment with controls, confirming previous reports on salt-induced growth inhibition in maize (Farooq et al., 2015). Recent repetition of this phenotyping including also severe stress treatment (180 mM NaCl) confirmed these results (data not shown). As expected, the administration of NaCl resulted in a strong increase in root, stem and leaf Na+ content (Fig. 1h–j). Stepping from mild to moderate salinity treatment further increased the leaf and stem Na+ content in SKT1506, but not significantly in PN28. In all tissues, the Na+ content was higher in SKT1506 than in PN28, suggesting that tolerant PN28 was more efficient in excluding Na+ than SKT1506, an important trait for salt tolerance in maize (Fortmeier & Schubert, 1995). By contrast, K+ content of shoot, stem and root (Fig. 1k–m) decreased strongly with progressive salt application in both cultivars. At 120 mM NaCl, K+ content of the shoot and stem was significantly higher in PN28 than in SKT1506, indicating that the tolerant PN28 was more effective in retaining K+, which is beneficial for enzyme activities and photosynthesis (see also the Na+/K+ ratio, Fig. S1a–c). In order to relate better salt tolerance of PN28 compared with SKT1506 to root membrane transport, net H+ and K+ fluxes were measured at the root surface using the MIFE technique (Methods S1). In the first set of experiments, the effect of an acute salt shock was tested. To this end, net H+ and K+ fluxes were measured before and up to c. 25 min after adding 240 mM NaCl to the BSM medium in which the root was immersed. Upon salt administration, a rapid, transient K+ efflux was elicited (Fig. 2a) as reported previously for various species including maize (Zaki Mostafa Ali, 2011; Yun et al., 2018). After c. 15 min, a steady level of K+ efflux was reached. Interestingly, the initial amplitude of salt-induced K+ efflux and the steady flux was significantly higher in the salt-sensitive SKT1506 than in PN28, indicating a more severe K+ loss in the former. A negative correlation of salt tolerance with the amplitude of salt-induced K+ efflux was previously shown by Chen et al. (2005) for barley and identified as a trait for selecting salt-tolerant cultivars (but see also Britto et al., 2010). Our results are in agreement with those of Chen et al. (2005), but at variance with data of Zaki Mostafa Ali (2011) on two maize cultivars differing in susceptibility to Na+ stress, perhaps reflecting the fact that maize salt tolerance can depend on diverse traits (Farooq et al., 2015). Acute salt treatment activated the ATPases and enhanced net H+ efflux (Fig. 2b) independent of the cultivar, this time confirming the observations of Zaki Mostafa Ali (2011). The activation of proton pumps tends to hyperpolarize the membrane, but this effect is overruled by a massive Na+ influx resulting in a depolarization of epidermis and cortex cells instead (Hua et al., 2008). Depolarization, in turn, entails the activation of outward-rectifying K+ channels leading to K+ efflux (Shabala & Cuin, 2008). The extent of depolarization and subsequent K+ loss tends to correlate with sensitivity to salt stress. Subsequently, we studied the long-term effects of salinity on net ion fluxes at the root surface by incubating roots in ¼ Hoagland medium supplemented with 60, 120 and 180 mM NaCl. After 1 or 3 d, steady-state net H+ and K+ fluxes were recorded for ≥ 20 min after 1 h incubation in BSM supplemented with the respective NaCl concentration. Fig. 2(c) shows profiles of H+ fluxes after 1 d of treatment at all three levels of salinity for PN28 and SKT1506 in the mature zone (10 mm above root tip). Bar plots summarizing recordings taken 5 and 10 mm above the root tip and after 1 and 3 d of pre-incubation at the various NaCl concentrations are shown in Fig. 2(d,e) for both cultivars. Based on the rationale of this study, the finding that severe salt stress (180 mM NaCl) induced a continuous net H+ influx into root cells of PN28 after 1 and 3 d of incubation is most important and remarkable, implying that H+ is transported at a higher rate into the cell than it is pumped out by P-type ATPases (Fig. S2a,b). Long-term H+ influx was also previously reported by Sun et al. (2009) for NaCl-exposed Populus roots but not identified as a trait confering salt tolerance. The biochemical pH clamp hypothesis provides a conclusive explanation for this puzzling phenomenon. A prerequisite for a continuous net H+ influx is the maintenance of a trans-membrane pH gradient. ‘Passive’ buffering by the cytosolic matrix can maintain a physiological pH despite continuous H+ influx for c. 20 min at most (Kurkdjian & Guern, 1989). We extended the recording of steady influx up to 35 min, although it can apparently continue for several days (from d1 to d3 at least). This implies ‘active buffering’ with a key role for biochemical reactions consuming H+, such as the step catalyzed by the glutamate decarboxylase (GAD; EC4.1.1.15; see Fig. S2b) as part of the GABA shunt – in fact the GABA shunt bypassing part of the TCA cycle is activated by acidification of the cytosol and salinity (Wegner & Shabala, 2020). Consistently, H+ influx was observed with the salt-tolerant cultivar PN28, but not the salt-sensitive SKT1506 when the roots were challenged with 180 mM NaCl. In the latter cultivar, H+ fluxes were close to zero or directed outwardly (at 10 mm above the tip, 3 d of exposure). Net H+ efflux was also observed for PN28 at mild and moderate salt stress. It implies an ATPase-generated H+ efflux just partly balanced by a H+ influx via other transporters, for example SOS1. ATP was apparently not rate-limiting under these conditions. In SKT1506, net H+ efflux was generally much smaller and at 5 mm above the tip turned to influx already with 120 mM NaCl (1d of treatment), indicating a less favorable energy status. The somewhat complex net H+ flux patterns shaped by NaCl concentrations and exposure time (Fig. 2d,e) can be interpreted in terms of two antagonistic effects: Salinity tends to activate PM H+ pumps by a selective increase in the expression of a P-type ATPase isoform, MHA4 (Kovalenko & Palladina, 2016), and an increase in the coupling rate of H+ flux and ATP hydrolysis from 1 to c. 3 (Janicka-Russak & Kabała, 2015). This can explain enhanced net H+ efflux with mild NaCl treatment compared with controls, most clearly observed with PN28 (significant at P = 0.001). Net maximum H+ efflux is observed in PN28 at 60 (d3) or at 120 mM NaCl (d1). On the other hand, energy costs for Na+ detoxification increase with the external NaCl concentration (Munns et al., 2020a), and ATP availability is compromised. Therefore, irrespective of enhanced H+ transport capacities, efflux is strongly reduced when moving from moderate to severe stress (in SKT1506) or efflux even turns to influx (in PN28), possibly with a delay related to progressive ATP depletion. More effort will be needed to disentangle the contributions of the various transporters to H+ fluxes, their regulation by salinity stress and the involvement of metabolism. This will include the use of blockers targeted at the ATPases, SOS1 as well as the NSCCs, which provide the pathway for Na+ influx, and the use of mutants lacking at least one of these transporters (e.g. Zhou et al., 2022). The interplay of membrane transport and metabolism, particularly the GABA shunt, also has to be addressed by a concerted use of inhibitors and knockout mutants. Net fluxes of K+, a major osmoticum and key Na+ antagonist for protecting cells against salinity stress (Shabala et al., 2010), were also measured in this study (Fig. 2f,g). In the framework of the biochemical pH clamp hypothesis, K+ efflux was considered to provide for charge balance. Even though H+/Na+ antiport via SOS1 itself is electroneutral, the whole process of Na+ influx and subsequent re-translocation back into the apoplast is associated with a net transfer of positive charge into the cell, raising the question how electroneutrality is established (Fig. S2b). Note that influx of positive charges coincides with that of H+ since circular Na+ flux across the membrane is electroneutral. K+ efflux (Fig. 2f) would even over-compensate for the accumulation of positive charges associated with H+ influx (Fig. 2d) in severely stressed PN28 roots (except for flux 5 mm above the tip and 3d of treatment). Surprisingly, K+ efflux tended to be still larger at mild and moderate salinity stress than at severe stress conditions, and larger than in control plants. Consistently, the K+ content of all tissues decreased strongly with aggravating salinity stress, in sensitive SKT1506 more than in tolerant PN28. Probably, the kernel is the main K+ source for the growing tissues and efflux from the root at this early stage of development. Note further that net K+ and H+ fluxes were both outwardly directed at 60 and 120 mM NaCl under most conditions. One candidate for keeping charge balance is net influx of Na+ accumulating in all tissues (Fig. 1h–j). Moreover, the release of deprotonated organic acids required for maintaining long-term net H+ efflux (Wegner et al., 2021) is likely to contribute. We compare the impact of salinity on two maize cultivars differing in salt tolerance. The administration of NaCl tends to stimulate net H+ efflux in roots of both cultivars persisting at mild and intermediate stress (60 and 120 mM NaCl, respectively), particularly in the more tolerant PN28. Remarkably, net H+ efflux turns to influx at severe stress (180 mM) in that cultivar, but not in the salt-sensitive SKT1506. According to the biochemical pH clamp hypothesis, net H+ influx is directly energized by metabolism, provides additional capacities for Na+ secretion and is an hitherto overlooked trait imparting salinity tolerance. An acute salt shock also triggers large, transient K+ efflux; a residual K+ efflux persists for 3 d at least and could provide for charge balance during H+ influx. We acknowledge financial support by the National Science Foundation of China (grant nos. 32070277, 31961143001, 3187011551) and from Guangdong Province (Higher Education Department, grant 2020KCXTD025; Science and Technology Department, grant no. 2022B1212010015). None declared. LHW and ZW designed the research project. ZW performed the experiments and evaluated the data. XH and LHW supervised the experimental work. LHW wrote a first draft. XH, XL and MY discussed the data and edited the manuscript. All authors approved the final version of the manuscript. The raw data that support the findings of this study have been deposited at https://www.researchgate.net/publication/380910478. Fig. S1 Relative Na+/K+ ratios. Fig. S2 Na+ secretion back into the apoplast by ATP hydrolysis and according to the biochemical pH clamp. Methods S1 Materials and Methods. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.