Difference between revisions of "Part:BBa K2225000"

Latest revision as of 19:57, 21 October 2021

AtNHXS1 for S. cerevisiae (Na+/H+ antiporter)

AtNHXS1 is a Na+/H+ antiporter which is located in the membrane of vacuoles and that original comes from Arabidopsis thaliana. This part consists of the prefix, the AtNHXS1-gene directly starting with the start-codon ATG till stop-codon TAG (-> UAG) followed by suffix. For using this part you need to assemble with promoter and terminator.

Sequence and Features

Design Notes

We performed some changes of the sequence of the AtNHXS1 gene in order to eliminate illegal restriction sides and to optimize the codon-usage for Saccharomyces cerevisiae.

Source

The gene original comes from Arabidopsis thaliana, but here is used a mutant of AtNHX as a basis for this gene called AtNHXS1 to increase the uptake of Na+ into the vacuole.

Description

Plant vacuolar membrane Na+/H+ antiporter genes play an important role in cellular ion homeostasis, especially the sequestration of Na+ into the vacuole to confer salt stress resistance [12–14]. Vacuolar membrane bound and H+ translocating enzyme H+-adenosine triphosphatase (ATPase) and H+-inorganic pyrophosphatase (PPiase) generate an electrochemical potential over the membrane through a proton gradient [15,16] that is used by the Na+ /H+ antiporters to move Na+ against its electrochemical potential and accumulate it inside the vacuole [15]. In Arabidopsis thaliana the most abundant Na+/H+ antiporter AtNHX1 is bound to the vacuolar membrane [17] . 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]. AtNHX1 is a homologous channel to the yeast Nhx1 gene product that encodes a Na+ /H+ antiporter in the prevacuolar compartment [18]. When AtNHX1 is knocked out in Arabidopsis thaliana the plant loses its ability to sequestrate sodium and thus is more sensitive to salt stress [19]. 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 [12,13,20]. 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 [14]. AtNHX1 is build 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[21]. 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 [22]. The small CaM proteins bind Ca2+ and are often transducing secondary messenger signals that lead to different cellular responses [23,24]. When AtCaM15 binds to the AtNHX1 Cterminus it changes the Na+/K+ selectivity by decreasing the Na+/H+ Vmax while not significantly altering the K+ /H+ exchange efficiency [22]. The binding of AtCaM15 to the C-terminus of AtNHX1 is Ca2+ and pH dependent. When the pH lowered the binding of AtCaM15 increases so at lower pH value the Na+ /K+ ratio went down [22]. 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 [22]. 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 [21]. When a plant is exposed to salt stress its vacuolar pH rises [25,26] 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 [22]. 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 [14]. 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 [14]. AtNHXS1 consists only of 4 transmembrane helices, the C-terminus and the 5 nearest transmembrane helices where deleted, resulting in the increase in Na+/K+ selectivity ratio described previously [14]. 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 [14].

Improvement from iGEM 2018 Kyoto

We iGEM Kyoto 2018 improved this part and added a new part BBa_K2665005 [1] , SseNHX1, which is an improved version of AtNHXS1. SseNHX1 was also created by DNA shuffling, with a combination of Salicomia europaea enzyme SeNHX1 and Suaeda salsa enzyme SsNHX1. It was reported that SseNHX1 show faster kinetics on the import of Na+ to the vacuoles than other relatives.

We characterized SseNHX1 by in vivo colony formation assay for the salt tolerance and flame photometry for the Na+ uptake into the cells.

Figure1. Colony formation assay

Measurement of intracellular Na+ concentration by flame photometry

We evaluated the Na+ uptake efficiency by SseNHX1 and AtNHXS1 by measuring intracellular Na+ concentration. When SseNHX1-expressing strain was grown in 400 mM NaCl containing media, the average concentration of intracellular Na+ was 50.6 mM. On the other hand, when AtNHX1 was used, the intracellular Na+ was 49.2 mM. We concluded that SseNHX1 is slightly superior to AtNHXS1 in this assay.

Figure2 Intracellular Na+ concentration (mM) of S. cerevisiae ena1- strain expressing AtNHXS1 or SseNHX1.

Based on the above two observations, we carefully compared the two homologous genes, SseNHX1 and AtNHXS1, and concluded that SseNHX1 is an improved version of AtNHXS1 in some features.

Panel 1-3: NaCl 0 mM, Panel 4-6: NaCl 300 mM Overnight cultures of the S. cerevisiae ena1- strain carrying each plasmid were serial diluted and spotted on a salt-containing SD plate. Colonies were photographed after 2 days incubation at 30oC. SseNHX1 and AtNHX1, separately cloned into pRS313, a low-copy-number plasmid, were expressed from TDH3 promoter. Control strain carrys empty vectors.

As shown in Figure1, both SseNHX1 and AtNHXS1 show salt tolerance effect on ena1- strain when compared to the wild type. Remarkably, the effect was much greater in SseNHX1 strain than AtNHXS1 strain.

References

[12] 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.

[13] 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.

[14] 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.

[15] 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.

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

[17] 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.

[18] 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.

[19] 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.

[20] 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.

[21] T. Yamaguchi, M. P. Apse, H. Shi et al., “Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 21, pp. 12510–12515, 2003.

[22] T. Yamaguchi, G. S. Aharon, J. B. Sottosanto et al., “Vacuolar Na+/H+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca2+- and pH-dependent manner,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 44, pp. 16107–16112, 2005.

[23] W. A. Snedden and H. Fromm, “Calmodulin as a versatile calcium signal transducer in plants,” New Phytologist, vol. 151, no. 1, pp. 35–66, 2001.

[24] R. E. Zielinski, “CALMODULIN AND CALMODULIN-BINDING PROTEINS IN PLANTS,” Annual review of plant physiology and plant molecular biology, vol. 49, pp. 697–725, 1998.

[25] M. L. Gruwel, V. L. Rauw, M. Loewen et al., “Effects of Sodium Chloride on plant cells; a 31P and 23Na NMR system to study salt tolerance,” Plant Science, vol. 160, no. 5, pp. 785–794, 2001.

[26] Y. OKAZAKI, M. KIKUYAMA, Y. HIRAMOTO et al., “Short-term regulation of cytosolic Ca2+, cytosolic pH and vacuolar pH under NaCl stress in the charophyte alga Nitellopsis obtusa,” Plant, Cell and Environment, vol. 19, no. 5, pp. 569–576, 1996.

More Information (added by WVHS_San_Diego 2021)

Summary: Provided background information of the AtNHX1 protein from literature regarding its origin and function in Na+ transportation

Documentation:

AtNHX1, the first plant vacuole Na+/H+ antiporter was cloned from A. thaliana. It showed significant sequence homology with Na+/H+ antiporters ScNHX1, an encoding vacuolar membrane from S. corniculata whose expression led to significantly higher vacuolar alkalization under salt stress. Plant vacuolar antiporters have been found to play important roles in maintaining ion homeostasis and transporting Na out of the cytosol and into the vacuole, which is quite effective in helping plants respond to high salinity environments. In fact, Xu et al determined that the overexpression of AtHNX1 conferred salt tolerance in transgenic plants, which was associated with increased vacuolar Na+/H+ exchange, and thus higher vacuolar sodium accumulation. Plants use two mechanisms to remove excess Na+ from the cytosol - SOS1 type plasma membrane antiporters and Na+/H+ antiporters (NHX1-type). Parts like AtNHKX1 are able to generate force to transporte Na because of H+/ATP in the plasma membrane and vacuole as well as H+/inorganic pyrophosphate. Removing the Na+ content from the cytosol is essential in maintaining osmotic balance and cell turgor. The novel AtNHKX1 has currently been studied with DNA shuffling, in which the gene’s mutations are generated and recombined.

References Liu, Liang, et al. “Coexpression of ScNHX1 and ScVP in Transgenic Hybrids Improves Salt and Saline-Alkali Tolerance in Alfalfa (Medicago Sativa L.).” Journal of Plant Growth Regulation, Springer-Verlag, 24 Mar. 2012, link.springer.com/article/10.1007/s00344-012-9270-z. Xu, Kai, 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, American Society for Biochemistry and Molecular Biology, 23 July 2010, www.ncbi.nlm.nih.gov/pmc/articles/PMC2906293/