Research in the field of abiotic stress for plants has become more and more popular in the last years. Especially drought and increased salt concentrations in soil are a serious problem for cultivation of crops in many regions of the world, primarily in face of climate change . Therefore a lot of genetically engineered plants with a better salt and drought resistance have been developed. [2–4]
One of the most important model organisms in plant physiology is Arabidopsis thaliana, so it does not surprise that there are many papers regarding improved salt tolerance in this plant [5–7]. Responsible for a higher salt tolerance in many cases the overexpression of channels responsible for the accumulation of ions in the vacuole of the cell. [6,7]
In our project we chose four channels, occurring native in A. thaliana, to work with. Two of these channels, named AtNHX1 and AVP1, are localized in the vacuole membrane of plants and play an important role regarding Na+ and K+ influx as well as maintenance of a proton gradient across the vacuolar membrane. We showed that the integration of each AVP1 and AtNHXS1, a genetically modified mutant of AtNHX1 (see below), in the Sacharomyces cerevisiae strain BY4742 leads to a two times higher accumulation of Na+ in the yeast cell. [See results]
Fig.1: AVP1, together with AtNHXS1 leads to an increased sequestration of sodium in the vacuole. This is the seqeuestration system in Arabidopsis. We used this system to increase the salt tolerance of our model organims and to promote ion sequestration inside the vacuole
The third ion channel, named AtAKT1, is localized in the plasma membrane of plant cells and is responsible for the influx of K+ into the intracellular room . The integration of AtAKT1 in the yeast strain Sacharomyces cerevisiae BY4742 combined with the knockout of the native efflux channels NHA1 and ENA1 lead to higher accumulation of K+ in the cell. [see results] In total, our mutant yeast, called NAKT, accumulated 2,4 mg/ml K+ from a media containing 19 mg/ml K+. This corresponds to a 12,4% uptake of K+ of total concentration.
Atsultr1;2, the name of the fourth channel, is a sulfate transporter, localized in the plasma membrane of root cells of A. thaliana and plays an important role regarding sulfate uptake. [9,10]
During our project we performed biobricks and sent them to the iGEM headquarter, so future teams working with plants can use our designed DNA in their projects.
We submitted genes encoding for the high affinity sulfate transporter AtSultr1;2 and for the genetically modified version of AtNHX1, which is named ATNHXS1.
AtNHX1 is a Na+/H+ antiporter, transporting Na+ inside the vacuole while pumping H+ out in the cytoplasm  . It mediates the import of not only Na+, but also K+ into the vacuole and with that, probably contributes to the accumulation of cations significantly. [12,13]When AtNHX1 is knocked out in Arabidopsis thaliana the plant loses its ability to sequestrate sodium and thus is more sensitive to salt stress [14,14].
Overexpression of AtNHX1 led to 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 has an important role in salt stress response [12,13]. 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 . AtNHX1 is built out of 9 transmembrane helices and 3 hydrophobic regions that do not span the tonoplast membrane but are still associated to it. The N-terminus is facing the cytosol while the hydrophilic C-terminus domain is facing the vacuolar lumen and moreover is involved in the regulation of the ion selectivity and efficiency of the Na+ and K+ transporter .
The protein interacting with the C-terminus and through that regulating the selectivity of AtNHX1 is the Arabidopsis thaliana calmodulin-like protein 15 (AtCaM15), which is localized in the vacuolar lumen and is transported there via cytoplasm-to vacuole-targeting pathway . The small CaM proteins bind Ca2+ and are often transducing secondary messenger signals that lead to different cellular responses [18,19]. When AtCaM15 binds to the AtNHX1 C-terminus it changes the Na+/K+ selectivity by decreasing the Na+/H+ Vmax while not significantly altering the K+/H+ exchange efficiency . The binding of AtCaM15 to the C-terminus of AtNHX1 is Ca2+ and pH dependent. When the pH lowers the binding of AtCaM15 increases so at lower pH value the Na+/K+ ratio went down . At physiological conditions with a high intravacuolar Ca2+ concentration and a low acidic pH this would result in a low Na+/H+ transport rate respective to the K+/H+ exchange rate .
The deletion of Arg-496 to Gly-518 of the C-terminus - that can form the typical positively charged amphiphilic α-helix of CaM targeting peptides - doubled the Na+/K+ selectivity ratio of the transporter which could mean that this is the specific binding site for AtCaM15 on the C-terminus . When a plant is exposed to salt stress its vacuolar pH rises [21,22] which releases AtCaM15 from the C-terminus resulting in an increased Na+/H+ exchange activity that leads to an increased vacuolar Na+ accumulation as a salt stress response .
Through DNA shuffling it was possible to generate a mutated channel - called AtNHXS1 - that showed an about 1 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 to the vacuolar membrane and when expressed in a yeast strain the cell accumulates more sodium than the native stain with AtNHX1 . AtNHXS1 consists only of 4 transmembrane helices, the C-terminus and the 5 nearest transmembrane helices were deleted, resulting in the increase in Na+/K+ selectivity ratio described previously .
Fig.2: 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.Integrating this transporter into the vacuolar membrane of a plant could increase the salt tolerance.
AtNHXS1 also shows 2 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 .
AVP1 is a vacuolar membrane bound H+-pyrophosphatase, which is encoded by only a single gene . It hydrolyses cytosolic inorganic pyrophosphate (PPi) to orthophosphate (Pi) to use the derived energy to actively pump protons into the vacuole [24–26]. This process not only reduces the cytosolic PPi concentration, but more importantly acidifies the vacuole [27,28]. 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 [27,29,30].
Fig.3: 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.
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 [31–34]. While there are multiple other families of transporters involved in potassium uptake, AtAKT1 specifically is a high affinity transporter in the plasma membrane [35–37]. Since sodium has a negative effect on the efficiency of AtAKT1 , 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 NHA1 already knocked out.
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. Overexpression of is channel not only increases the uptake on potassium (a nutrient) but also increases salt and drought resistance.
AtSultr1;2 encodes for a high-affinity sulfate transporter that naturally occurs in Arabidopsis thaliana and is expressed at epidermis and cortex of its roots . 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 Sultr1;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 [10,9].
To reach our goal of desalinating waste water with genetically modified microorganisms we chose the yeast strain Sacharomyces cerevisiae as our preferred biological system. The wide range of genetic tools that make it possible to easily modify genes in yeast in addition to its fast growth and simple cultivation conditions make S. cerevisiae a perfect model organism . Furthermore, the natural ability of yeast to sequestrate different molecules in its large vacuole and resist huge osmotic pressure made us pick out yeast as our favourite microbe. 
But this system of locking up salts in the vacuole is not restricted to yeast, but may be implemented for other organisms. Our far-reaching aim is to transfer our system of overexpressed ion channels and eliminated efflux mechanisms to an algae species. Algae also own a vacuole to accumulate salts and do not rely on an external carbon source, because of their ability to run photosynthesis. Transferring our system to these algal cells, we would create a biological desalination system which not only is independent of external carbon sources but also reduces the carbon-dioxide concentration in its environment.
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