Difference between revisions of "Team:Aachen/Description"

The reason for the load is the introduction of lye from the mining industry. At the Werra, potash salts are extracted, which are exported for fertilizer production worldwide. A waste product of this potash salt recovery is sodium chloride, however magnesium salts and others as well. These waste products are either disposed of on huge hills, pressed into the ground or led into the Werra as the already mentioned lye.

This leads to a chloride concentration in the Werra that is over 2000 mg / L high. This increased chloride content, together with other increased ion concentrations, leads to catastrophic damage to the ecosystem. Before the destruction through the introduction of industrial waste water without prior purification, the macrozoobenthos of the Werra was composed of 60-100 species.

Nowadays, the environment is dominated by only one species. The boosted river cancer, a brackish water species from North America, is a Neozoe (unnaturally introduced species) and became the dominant living being. In addition, the New Zealand dwarf snail, also a Neozoe, occurs frequently. There is only a small amount of mosquitos, fly larvae and some other species from the originally occurring species in the body of water with an extremely small share of the individual species of the ecosystem.

To tackle the significant damage salt stress causes in rivers like the Werra, but also to find a solution for ion burden in industrial process wasters, we developed "SALT VAULT".

The aim of our "SALT VAULT" system is to absorb as many sodium and chloride ions as possible to reduce the salt content in water. The system developed by us is based on the yeast Saccharomyces cerevisiae, which is not only a model organism, but also has a vacuole, which is of central importance for our project (details of why we chose this organism, see the >> design section).

S. cerevisiae has a large number of ion transporters and channels involved in the transport of sodium, potassium and chloride ions. These proteins are controlled by various regulatory mechanisms and work together to maintain the ion homeostasis in the cytosol of the yeast when the external salt concentrations vary, which is of central importance for metabolism.

There are multiple transport systems in the plasma membrane that lead to an influx of sodium, but there are no specific sodium importers in the plasma membrane [8]. The influx is only significant when sodium has much higher extracellular concentration than potassium [9,8] which, in our application, is the case:

- Phosphate influx into the cytoplasm is coupled to sodium and carried out by the cotransporter Pho89, contributing to sodium import [8,10].
- Low affinity nonspecific cation importers in the plasma membrane do not discriminate between K+ and Na+, leading to an import of both [11].
- Trk1, actually a potassium importer, transports Na+ in addition to K+, although with lower affinity [12].

Sodium ions play an important role for the function of macromolecules [8]. On the other hand, an increased intracellular concentration can lead to toxic effects because sodium ions can disturb the correct function of specific cellular pathways [9]. The most important transporters forming this system are shown in Figure 4.

Fig.4: ENA1 and NHA1 form the native efflux system responsible for the extrusion of sodium from the cytosol. NHX1 and GEF1 lead to sequestration of sodium and chloride in the prevacuolar compartments confering salt tolerance while also reducing toxic effects of sodium in the cytosol.

When there is an increased concentration of sodium chloride in the environment, S. cerevisiae reacts essentially in two ways to the entrance of sodium (and chloride) into the cytosol:

1. An efflux system consisting of two proteins encoded by the ENA1 and NHA1 genes transports sodium back into the extracellular space [15–18]:

Because the natural abundance of sodium ions in the environment is higher than the relatively low intracellular concentration, most eukaryotic organisms developed mechanisms to remove sodium ions from the cytosol. Especially at high external sodium concentrations, Saccharomyces cerevisiae relies on the ability to extrude the cation from the cytosol to maintain a low intracellular concentration. To achieve this, yeast developed two extrusion mechanisms.

The H+/Na+ antiporter, encoded by the Nha1 gene, can extrude Na+, Li+ and K+ using the proton gradient across the plasma membrane. However, the primary pathway for the extrusion of sodium ions is the plasma membrane P-type Na+ -ATPase encoded by Ena1 (for exnus natru) [17,18]. The ENA1 ATPase couples the hydrolysis of ATP to the transport of cations against the electrochemical gradients out of the cell [19]. This is because in yeast, the ENA1 gene plays an important role in salt tolerance. It encodes the most important protein for the extrusion of Na+ and Li+ out of the cell under saline stress conditions [20,18]. In response to saline or alkaline pH stress, the expression of ENA1 increases significantly, amongst other things, through the activation of calcineurin [21–23,17].

Fig.5: The two proteins responsible for the efflux of sodium as a stress response are highlighted in pink. ENA1 is a primary active sodium ATPase while NHA1 is a secondary active antiporter relying on the proton gradient over the plasma membrane

Because the Δena1 mutant lacks most of the sodium efflux ability, the intracellular sodium content is increased when compared to that in the wild-type [24]. This organism then heavely relies on the internal detoxification system to survive salt stress.

2. Nhx1 transports sodium into prevacuolar compartments [25,26]. The GEF1 channel causes the follow-up of chloride ions:

This system is described with the sequestration model, characterizing the sequestration of sodium in the prevacuolar compartments and thereby lowering the toxic cytosolic concentration in the Δena1 mutant [25,27]. Nhx1 is a Na+/H+ exchanger and mediates the sequestration of sodium into the prevacuolar compartments, but is not contributing to the efflux of Na+ to the extracellular room [26]. It has been shown that NHX1 is localized exclusively in unique endosomal/prevacuolar compartments [25]. Through enhancing the sequestration of sodium in the endosomal membrane system, it was possible to restore the salt tolerance of the Δena1 mutant [24]. This is due to the reduced salt concentration in the cytosol but increased concentration in the prevacuolar compartments. Responsible for that is Nhx1 that uses the proton gradient over the membrane of this acidic compartments generated by the vacuolar ATPase, to sequestrate sodium [26].

Fig.5 b: 2D Topology of the GEF1 chloride channel

Thus, and due to other mechanisms, the organism is able to maintain a healthy ratio of sodium and potassium in the cytosol, even if the environmental conditions strongly fluctuate. If a hyperosmotic shock occurs, S.cerevisiae can reduce its intracellular water potential by importing these ions into the compartments via NHX1 and GEF1 and consequentially survive, making the organism capable of a certain natural salt tolerance [26].

In order to achieve our goal of accumulating as much sodium chloride as possible in the cell, we have modified some of these channels to prevent the salt from leaving the cell. At the same time, it is to be ensured that as much salt as possible is stored in the prevacuolar compartments.

The changes we made in the yeast native channel system are shown in Figure 6. They can be subdivided into two areas:

- The destruction of the two most important efflux mechanisms ENA1 and NHA1, so that as little salt as possible leaves the cell.

- Overexpression of NHX1 to maximize the sodium (and therefore chloride) uptake into the prevacuolar compartments.

Fig.6: We knocked out the two efflux mechanisms ENA1 and NHA1 and overexpressed NHX1 to increase the sequestration of ions in the intracellular compartments.

These mutations were performed individually as well as in various combinations to identify the most efficient mutant (>> results). To learn more about the methodology we used for these changes, check out our Workflow and Protocols pages.

But while working on and modifying the native transport system of S. cerevisiae, we have also integrated a new system which offers further possibilities of increasing the salt intake. For this purpose, we used a combination of ion transporters that are found in the plant Arabidopsis thaliana. The system differs from that in S. cerevisiae, since plants react comparable but not similar to salt stress.

AtNHX1

Plant vacuolar membrane Na+/H+ antiporter genes play an important role in cellular ion homeostasis, especially the sequestration of Na+ in the vacuole to confer salt stress resistance in plants [28–30]. Vacuolar membrane-bound and H+ translocating enzyme H+ adenosine triphosphatase (ATPase) and H+ inorganic pyrophosphatase (PPiase) generate an electrochemical potential across the membrane through a proton gradient [31,32] that is used by the Na+/H+ antiporters to move Na+ against its electrochemical potential and accumulate it inside the vacuole [31].

In Arabidopsis thaliana the most abundant Na+/H+ antiporter AtNHX1 is bound to the vacuolar membrane [33] . It mediates the import of not only Na+, but also K+ into the vacuole and with that contributes to the accumulation of cations significantly [28,29]. AtNHX1 is a channel which is homologous to the yeast NHX1 gene that encodes a Na+/H+ antiporter in the prevacuolar compartment [34]. When AtNHX1 is knocked out in Arabidopsis thaliana the plant loses its ability to sequestrate sodium and thus is more sensitive to salt stress [35].

Fig.7:The Arabidopsis thaliana proton sodium antiporter is integrated into the vacuolar membrane of S.cerevisiae in addition to the alteration in the native system of the organism.

Overexpression of AtNHX1 led to an increased vacuolar Na+/H+ transport rate that was higher than the relative increase in AtNHX1 protein abundance and increased accumulation of sodium in the vacuole of Arabidopsis thaliana. Through that, the salt tolerance was increased, confirming AtNHX1 plays an important role in salt stress response [28,29,36]. We decided to use this channel and integrate it into the vacuolar membrane of S.cerevisiae. But while overexpression of AtNHX1 increased its Na+/H+ transport rate, the still relatively low Vmax of this transporter is limiting the application in terms of fast hyperaccumulation of sodium in the vacuole [30].

Through DNA shuffling, it was possible to generate a mutated channel - called AtNHXS1 - that showed an about two-fold increased Na+/H+ Vmax while the K+/H+ exchange activity was not significantly altered when compared to the native form of AtNHX1. The channel is still localized in the vacuolar membrane but the plant cell accumulated more sodium than the native strain with AtNHX1 [30]. AtNHXS1 consists only of 4 transmembrane helices, the C-terminus and the 5 nearest transmembrane helices of AtNHX1 where deleted, resulting in the increase in Na+/K+ selectivity ratio described previously [30].

AtNHXS1 also shows two mutations (L29P and S158P) in its amino acid sequence that might be responsible for the increased sodium transport activity and thus enable increased sodium sequestration in the vacuole (Figure 8) [30]. We integrated this synthetic transporter into the vacuolar membrane to use the yeast´s vacuole as an additional compartment for salt storage with higher volume than the prevacuolar compartments (Figure 7).

Fig.8: Comparison of AtNHXS1 and AtNHX1. AtNHXS1 was generated through DNA shuffeling and lost 5 transmembrane helices and the regulatory C-terminus. This mutant of AtNHX1 is more efficient and is not negatively regulated, resulting in higher sequestration.

AVP1

To increase the efficiency of this channel we will additionally integrate the protein encoded by AVP1 into the vacuolar membrane of S.cerevisiae.

Cation antiporters like AtNHX1 or NHX1 mediate the transport of solutes from the cytosol into the vacuolar lumen against their concentration gradient using the proton gradient over the vacuolar membrane (or prevacuolar compartment membrane in case of NHX1) [37]. In yeast, this proton gradient is generated by the membrane bound H+-ATPase that uses ATP to translocate protons into the vacuole and thus acidifies this compartment [38]. In principle, by increasing the availability of protons through an enhanced expression of the vacuolar H+-ATPase the sequestration of ions in the vacuole should increase [39]. But the overexpression of vacuolar H+-ATPase would be difficult because the yeast vacuolar H+-ATPase is a multi-subunit protein [40]. Each of the subunits would have to be overexpressed exactly at the right level at the same time to get an increased activity of the complex protein [41].

Fig.9: AVP1 uses pyrophosphate to pump protons into the vacuole thus causing acidification of this compartment. The proton gradient can be used by secondary active transporters like AtNHXS1 to pump ions into the vacuole against their concentration gradient.

In contrast to yeast, the proton electrochemical gradient across the vacuolar membrane (tonoplast) of Arabidopsis thaliana is generated by two proteins, the plant vacuolar H+-ATPase and AVP1, the vacuolar membrane bound H+-pyrophosphatase, which is encoded by only a single gene [42]. AVP1, as a vacuolar H+-pyrophosphatase, hydrolyses cytosolic inorganic pyrophosphate (PPi) to orthophosphate (Pi) to use the derived energy to actively pump protons into the vacuole (Figure 9) [43–45]. This process not only reduces the cytosolic PPi concentration, but more importantly acidifies the vacuole [46,47]. The difference in electrochemical potential for protons across the tonoplast, generated by the vacuolar H+-ATPase and AVP1, can then be used by vacuolar transporters, e.g. AtNHX1, to pump ions into the vacuole [46,48,49].

Resulting from that, there will also be a compensatory transport of anions to maintain electroneutrality [50]. Through expression of the Arabidopsis thaliana H+-PPi- ase AVP1 in yeast it is possible to increase the translocation of protons and anions over the tonoplast and thus acidify the vacuole [44]. This increased proton-influx leads to more cytosolic Na+ being sequestered inside the vacuole via the NHX1 exchangers and in general leads to a higher solute accumulation through H+-syn- and antiporters [40,39,43]. This shows that the vacuole acidification through AVP1 plays a key role in facilitating vacuolar Na+ sequestration [51] and increases the concentration of cations in the vacuole [39].

Fig.10: AVP1, together with AtNHXS1 leads to an increased sequestration of sodium in the vacuole. This system is added to the altered native one

We have integrated the gene AVP1 together with AtNHXS1 into the yeasts vacuolar membrane (Figure 10). Together with the changes we carried out in the native transporter system of S. cerevisiae this can be described as an expanded and optimized sequestration model to maximize the salt concentration in the yeast and remove it from the wastewater. You can compare the salt uptake of different combinations of transporters we integrated into S.cerevisiae in our results page. We also compared the salt tolerance of the different mutants because this is a good indicator for the functionality of our laboratory work and model. Before we integrated the different combinations of channels we created a mathematical model of our system to determine the maximal amount of salt our system can take up.

Following our intensive Integrated Human Practice work, we realized that contamination of wastewater through salts is not restricted to Na+ and Cl- but that ions like K+ and SO4- lead to problems, too. Read more about the negative effects of these ions in our integrated HP section. Potassium contamination is discussed in the section about the Werra while details about problems with sulphate can be found in the interviews at the municipal and chemical wastewater treatment plants.

Following the input we got from these cooperations we decided to identify possible channels for alteration to increase the accumulation of potassium and sulphate.

AtAKT1

We identified AtAKT1 as a promising gene to increase the potassium accumulation. AtAKT1 is a potassium channel originally from Arabidopsis thaliana root tissue. It mediates potassium uptake over a wide range of potassium concentration in the extracellular room [52–55]. While there are multiple other families of transporters involved in potassium uptake, AtAKT1 specifically is a high affinity transporter in the plasma membrane [56–58]. Since sodium has a negative effect on the efficiency of AtAKT1 [59], we did not combine a sodium accumulating mutant with this channel, but rather created a separate mutant called NAKT. Therefore, we integrated the channel in the plasma membrane of a mutant with ENA1 and NHA already knocked out. We did this, because the genes not only mediate efflux of sodium but also transport potassium back into the extracellular room.

Compared to the system we created to accumulate sodium inside a cell, the AtAKT1 system does not struggle with the toxicity of the accumulated ions, since potassium is a nutrient and indispensable for many physiologic functions and does not inhibit metabolism [8]. We were able to accumulate potassium in this mutant. Details can be found on our results page.

Fig.11: 2D topological structure (left) and function (right) of AtAKT1. AtAKT1 uses ATP to actively import potassium into the cytosol. CYCLN and ANKY are intracellular regulation domains.

AtSultr1;2

Since we want to increase the uptake of sulfate into the vacuoles of Saccharomyces cerevisiae, we decided to integrate the AtSultr1;2-gen. The gene encodes for a high-affinity sulfate transporter that naturally occurs in Arabidopsis thaliana and is expressed at epidermis and cortex of its roots [60].

AtSultr1;2 belongs to a family of sulfate transporters and due to studies with mutants the AtSultr1;2 seems to be the most important one. The mutant having a AtSultr1;1-knockout showed up a phenotype similar to the wildtype while the AtSultr1;2-mutant showed differences in the phenotype compared to the wildtype relating to root length, growth and sulphate uptake. This confirms the assumption that AtSultr1;2 plays a major role in the sulfate uptake system [61] [62].

References

[1] R. W. Sleigh, H. Aral, L. Simons et al., “Sustainable management of salty waste streams from cheese manufacture,” Australian journal of dairy technology, 2006.

[2] Z. BIAN, H. I. INYANG, J. L. DANIELS et al., “Environmental issues from coal mining and their solutions,” Mining Science and Technology (China), vol. 20, no. 2, pp. 215–223, 2010.

[3] C. Zhang, L. Zhong, X. Fu et al., “Managing Scarce Water Resources in China's Coal Power Industry,” Environmental management, vol. 57, no. 6, pp. 1188–1203, 2016.

[4] N. F. Gray, “Acid mine drainage composition and the implications for its impact on lotic systems,” Water Research, vol. 32, no. 7, pp. 2122–2134, 1998.

[5] S. S. Kaushal, P. M. Groffman, G. E. Likens et al., “Increased salinization of fresh water in the northeastern United States,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 38, pp. 13517–13520, 2005.

[6] Heike Zimmermann-Timm, “Salinization of inland waters,” Globale Change: Enough water for all?, 2007.

[7] D. Thomas and N. J. Middleton, “Salinization: New perspectives on a major desertification issue,” Journal of Arid Environments, vol. 24, no. 1, pp. 95–105, 1993.

[8] J. Ariño, J. Ramos, and H. Sychrová, “Alkali metal cation transport and homeostasis in yeasts,” Microbiology and molecular biology reviews : MMBR, vol. 74, no. 1, pp. 95–120, 2010.

[9] A. Rodríguez-Navarro and J. Ramos, “Dual system for potassium transport in Saccharomyces cerevisiae,” Journal of Bacteriology, vol. 159, no. 3, pp. 940–945, 1984.

[10] B. L. Persson, A. Berhe, U. Fristedt et al., “Phosphate permeases of Saccharomyces cerevisiae,” Biochimica et Biophysica Acta (BBA) - Bioenergetics, vol. 1365, 1-2, pp. 23–30, 1998.

[11] M. Gómez, “The capacity to transport potassium influences sodium tolerance in Saccharomyces cerevisiae,” FEMS Microbiology Letters, vol. 135, 2-3, pp. 157–160, 1996.

[12] J. Ramos, P. Contreras, and A. Rodríguez-Navarro, “A potassium transport mutant of Saccharomyces cerevisiae,” Archives of Microbiology, vol. 143, no. 1, pp. 88–93, 1985.

[13] M. J. Page and E. Di Cera, “Role of Na+ and K+ in enzyme function,” Physiological reviews, vol. 86, no. 4, pp. 1049–1092, 2006.

[14] R. Serrano, “Salt tolerance in plants and microorganisms: toxicity targets and defense responses,” International review of cytology, vol. 165, pp. 1–52, 1996.

[15] M. A. Bañuelos, H. Sychrová, C. Bleykasten-Grosshans et al., “The Nha1 antiporter of Saccharomyces cerevisiae mediates sodium and potassium efflux,” Microbiology (Reading, England), 144 (Pt 10), pp. 2749–2758, 1998.

[16] C. Prior, S. Potier, J. L. Souciet et al., “Characterization of the NHA1 gene encoding a Na+/H+-antiporter of the yeast Saccharomyces cerevisiae,” FEBS letters, vol. 387, no. 1, pp. 89–93, 1996.

[17] A. Ruiz and J. Ariño, “Function and regulation of the Saccharomyces cerevisiae ENA sodium ATPase system,” Eukaryotic cell, vol. 6, no. 12, pp. 2175–2183, 2007.

[18] R. Haro, B. Garciadeblas, and A. Rodríguez-Navarro, “A novel P-type ATPase from yeast involved in sodium transport,” FEBS letters, vol. 291, no. 2, pp. 189–191, 1991.

[19] P. Catty, A. de Kerchove d'Exaerde, and A. Goffeau, “The complete inventory of the yeast Saccharomyces cerevisiae P-type transport ATPases,” FEBS letters, vol. 409, no. 3, pp. 325–332, 1997.

[20] A. Ruiz, L. Yenush, and J. Arino, “Regulation of ENA1 Na+-ATPase Gene Expression by the Ppz1 Protein Phosphatase Is Mediated by the Calcineurin Pathway,” Eukaryotic cell, vol. 2, no. 5, pp. 937–948, 2003.

[21] S. Y. Park, S. B. Seo, S. J. Lee et al., “Mutation in PMR1, a Ca(2+)-ATPase in Golgi, confers salt tolerance in Saccharomyces cerevisiae by inducing expression of PMR2, an Na(+)-ATPase in plasma membrane,” Journal of Biological Chemistry, vol. 276, no. 31, pp. 28694–28699, 2001.

[22] I. Mendoza, F. J. Quintero, R. A. Bressan et al., “Activated Calcineurin Confers High Tolerance to Ion Stress and Alters the Budding Pattern and Cell Morphology of Yeast Cells,” Journal of Biological Chemistry, vol. 271, no. 38, pp. 23061–23067, 1996.

[23] J. Märquez and R. Serrano, “Multiple transduction pathways regulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast,” FEBS letters, vol. 382, 1-2, pp. 89–92, 1996.

[24] R. A. Gaxiola, R. Rao, A. Sherman et al., “The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast,” Proceedings of the National Academy of Sciences, vol. 96, no. 4, pp. 1480–1485, 1999.

[25] R. Nass and R. Rao, “Novel Localization of a Na + /H + Exchanger in a Late Endosomal Compartment of Yeast,” Journal of Biological Chemistry, vol. 273, no. 33, pp. 21054–21060, 1998.

[26] R. Nass, K. W. Cunningham, and R. Rao, “Intracellular Sequestration of Sodium by a Novel Na + /H + Exchanger in Yeast Is Enhanced by Mutations in the Plasma Membrane H + -ATPase,” Journal of Biological Chemistry, vol. 272, no. 42, pp. 26145–26152, 1997.

[27] R. A. Gaxiola, D. S. Yuan, R. D. Klausner et al., “The yeast CLC chloride channel functions in cation homeostasis,” Proceedings of the National Academy of Sciences, vol. 95, no. 7, pp. 4046–4050, 1998.

[28] M. P. Apse, “Salt Tolerance Conferred by Overexpression of a Vacuolar Na+/H+ Antiport in Arabidopsis,” Science, vol. 285, no. 5431, pp. 1256–1258, 1999.

[29] H. X. Zhang and E. Blumwald, “Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit,” Nature biotechnology, vol. 19, no. 8, pp. 765–768, 2001.

[30] K. Xu, H. Zhang, E. Blumwald et al., “A novel plant vacuolar Na+/H+ antiporter gene evolved by DNA shuffling confers improved salt tolerance in yeast,” The Journal of biological chemistry, vol. 285, no. 30, pp. 22999–23006, 2010.

[31] E. Blumwald, “Tonoplast vesicles as a tool in the study of ion transport at the plant vacuole,” Physiologia Plantarum, vol. 69, no. 4, pp. 731–734, 1987.

[32] P. A. Rea and D. Sanders, “Tonoplast energization: Two H+ pumps, one membrane,” Physiologia Plantarum, vol. 71, no. 1, pp. 131–141, 1987.

[33] A. Hernández, X. Jiang, B. Cubero et al., “Mutants of the Arabidopsis thaliana cation/H+ antiporter AtNHX1 conferring increased salt tolerance in yeast: the endosome/prevacuolar compartment is a target for salt toxicity,” The Journal of biological chemistry, vol. 284, no. 21, pp. 14276–14285, 2009.

[34] R. Nass, K. W. Cunningham, and R. Rao, “Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. Insights into mechanisms of sodium tolerance,” The Journal of biological chemistry, vol. 272, no. 42, pp. 26145–26152, 1997.

[35] M. P. Apse, J. B. Sottosanto, and E. Blumwald, “Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter,” The Plant journal : for cell and molecular biology, vol. 36, no. 2, pp. 229–239, 2003.

[36] H. X. Zhang, J. N. Hodson, J. P. Williams et al., “Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 22, pp. 12832–12836, 2001.

[37] M. P. Apse and E. Blumwald, “Na+ transport in plants,” FEBS letters, vol. 581, no. 12, pp. 2247–2254, 2007.

[38] T. H. Stevens and M. Forgac, “Structure, function and regulation of the vacuolar (H+)-ATPase,” Annual review of cell and developmental biology, vol. 13, pp. 779–808, 1997.

[39] R. A. Gaxiola, J. Li, S. Undurraga et al., “Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 20, pp. 11444–11449, 2001.

[40] R. A. Gaxiola, R. Rao, A. Sherman et al., “The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 4, pp. 1480–1485, 1999.

[41] E. Fischer-Schliebs, R. Ratajczak, P. Weber et al., “Concordant Time-Dependent Patterns of Activities and Enzyme Protein Amounts of V-PPase and V-ATPase in induced (Flowering and CAM or Tumour) and Non-Induced Plant Tissues*,” Botanica Acta, vol. 111, no. 2, pp. 130–136, 1998.

[42] H. Sze, “Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis,” THE PLANT CELL ONLINE, vol. 11, no. 4, pp. 677–690, 1999.

[43] X.-G. Duan, A.-F. Yang, F. Gao et al., “Heterologous expression of vacuolar H(+)-PPase enhances the electrochemical gradient across the vacuolar membrane and improves tobacco cell salt tolerance,” Protoplasma, vol. 232, 1-2, pp. 87–95, 2007.

[44] E. J. Kim, R. G. Zhen, and P. A. Rea, “Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 13, pp. 6128–6132, 1994.

[45] S.-M. Lin, J.-Y. Tsai, C.-D. Hsiao et al., “Crystal structure of a membrane-embedded H+-translocating pyrophosphatase,” Nature, vol. 484, no. 7394, pp. 399–403, 2012.

[46] M. Maeshima, “Vacuolar H(+)-pyrophosphatase,” Biochimica et biophysica acta, vol. 1465, 1-2, pp. 37–51, 2000.

[47] A. Ferjani, S. Segami, G. Horiguchi et al., “Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis,” The Plant cell, vol. 23, no. 8, pp. 2895–2908, 2011.

[48] A. Kriegel, Z. Andrés, A. Medzihradszky et al., “Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase,” The Plant cell, vol. 27, no. 12, pp. 3383–3396, 2015.

[49] M. Krebs, D. Beyhl, E. Görlich et al., “Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 7, pp. 3251–3256, 2010.

[50] R. A. Gaxiola, D. S. Yuan, R. D. Klausner et al., “The yeast CLC chloride channel functions in cation homeostasis,” Proceedings of the National Academy of Sciences, vol. 95, no. 7, pp. 4046–4050, 1998.

[51] R. K. Schilling, M. Tester, P. Marschner et al., “AVP1: One Protein, Many Roles,” Trends in plant science, vol. 22, no. 2, pp. 154–162, 2017.

[52] R. E. Hirsch, B. D. Lewis, E. P. Spalding et al., “A role for the AKT1 potassium channel in plant nutrition,” Science (New York, N.Y.), vol. 280, no. 5365, pp. 918–921, 1998.

[53] D. Lagarde, M. Basset, M. Lepetit et al., “Tissue-specific expression of Arabidopsis AKT1 gene is consistent with a role in K+ nutrition,” The Plant journal : for cell and molecular biology, vol. 9, no. 2, pp. 195–203, 1996.

[54] H. Sentenac, N. Bonneaud, M. Minet et al., “Cloning and expression in yeast of a plant potassium ion transport system,” Science (New York, N.Y.), vol. 256, no. 5057, pp. 663–665, 1992.

[55] E. P. Spalding, R. E. Hirsch, D. R. Lewis et al., “Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium,” The Journal of general physiology, vol. 113, no. 6, pp. 909–918, 1999.

[56] H. H. Fu and S. Luan, “AtKuP1: A dual-affinity K+ transporter from Arabidopsis,” The Plant cell, vol. 10, no. 1, pp. 63–73, 1998.

[57] G. E. Santa-María, F. Rubio, J. Dubcovsky et al., “The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter,” The Plant cell, vol. 9, no. 12, pp. 2281–2289, 1997.

[58] F. Rubio, W. Gassmann, and J. I. Schroeder, “Response: High-affinity potassium uptake in plants,” Science (New York, N.Y.), vol. 273, no. 5277, pp. 978–979, 1996.

[59] J. K. Zhu, “Regulation of ion homeostasis under salt stress,” Current opinion in plant biology, vol. 6, no. 5, pp. 441–445, 2003.

[60] N. Yoshimoto, E. Inoue, A. Watanabe-Takahashi et al., “Posttranscriptional regulation of high-affinity sulfate transporters in Arabidopsis by sulfur nutrition,” Plant physiology, vol. 145, no. 2, pp. 378–388, 2007.

[61] M. Barberon, P. Berthomieu, M. Clairotte et al., “Unequal functional redundancy between the two Arabidopsis thaliana high-affinity sulphate transporters SULTR1;1 and SULTR1;2,” The New phytologist, vol. 180, no. 3, pp. 608–619, 2008.

[62] T. Kataoka, A. Watanabe-Takahashi, N. Hayashi et al., “Vacuolar Sulfate Transporters Are Essential Determinants Controlling Internal Distribution of Sulfate in Arabidopsis,” The Plant cell, vol. 16, no. 10, pp. 2693–2704, 2004.