Proteins Conferring Salinity Tolerance In Plants Biology Essay

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Soil salinity is one of the most serious problems faced by the agricultural sector in recent times . The primary cause of soil salinity is related to natural factors such as salt accumulation, weathering of rocks and deposition of oceanic salts . Soil is considered to be saline when the concentration of NaCl is more than 40 mM .

Saline environments stress plants primarily in two ways. Firstly, the high accumulation of salts in soil induces osmotic effects which reduce the ability of plant roots to extract water. Saline environments also stress the plant when Na+ ions accumulate in plant shoots and eventually cause toxicity to the plant by competing with potassium (K+) for enzyme binding sites, thereby inhibiting enzyme function and protein synthesis (Munns and Tester, 2008).

Na+ influx into plants

Na+ or other cation entry into the root primarily via passive movement; uniporter or ion channel type transporters . Under normal physiological condition plants maintain a high K+/Na+ ratio in the cytosol with concentration of K+ (100-200 mM) and Na+ (1-10mM). With the negative electrical membrane potential difference at the plasma membrane at -140 mV, an increase in extracellular Na+ concentration will cause a large Na+ electrochemical potential gradient that will induce passive transport of Na+ from the environment to cytosol. The second route which Na+ will enter root is via ion channel type transporters; high affinity cation transporter (HKT), low affinity cation transporter (LCT1) and non-selective cation channels (NSCC) . All transporters mediate Na+ influx into roots despite the slight variation that may exist within different species. After entering the root, Na+ transported through the epidermis, cortex and root xylem parenchyma cells. Na+ is then loaded into the xylem vessels and transported upward the plant via the transpiration stream . After that, ion are unloaded by shoot/leaf parenchyma cells will be transported into phloem sieves via symplastic diffusion

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The excessive accumulation of Na+ in plants is not favourable as it causes several adverse effects such as growth retardation , chlorosis and inhibits K+ absorption which vital for cellular function in plants such as enzyme activation . Despite the adverse effect of Na+, plants vary in their level of tolerance towards salt as depicted in figure 1.

Figure 1: Diversity in the salt tolerance of various species, shown as increases in shoot dry matter after growth in solution or sand culture containing NaCl for at least 3 weeks, relative to plant growth in the absence of NaCl . Data including rice (Oryza sativa), durum wheat (Triticum turgidum ssp durum), bread wheat (Triticum aestivum), barley (Hordeum vulgare), tall wheatgrass (Thinopyrum ponticum, syn. Agropyron elongatum), Arabidopsis (Arabidopsis thaliana), alfalfa (Medicago sativa), and saltbush (Atriplex amnicola)

As salinity stress in plants is multifactorial, plants utilize several mechanisms to ensure protection from salinity stress . One of the primary mechanisms is the utilisation of sodium transport processes which function as organellar Na+ sequestration , Na+ extrusion by plasma membrane Na+ - H+ exchange transporter, exclusion of Na+ from leaves and shoot and increasing the level of cytoplasmic potassium levels relative to sodium . A variety of sodium transporter proteins have been identified such as the high affinity potassium transporters (HKT), cyclonucletide-gated channels (CNGCs), glutamate-activated channels (GLR) and salt overly sensitive (SOS) proteins .

Among the entire family of transporters, HKT has received a significant attention from early 60's until recent times. The term 'high-afinity potassium transporter' was initially mentioned by based on the experiments using roots of barley seedlings that had been germinated in a diluted CuSO4 solution. The results of the experiments showed that roots of barley acquired a high-affinity K+ influx with a Km of 10-20 mM K+ which was unaffected with Na+ concentration. This system was then named as high affinity 'Mechanism 1' The identification of the gene that is involved in the high-affinity K+ influx was conducted on Arabidopsis and rice using BLAST searches and RT-PCR which led to the isolation its cDNA that encoded transporters which exhibit a great sequence similarity to potassium transporet (HAK) which was previously detected in fungi . The expression of these cDNAs in yeast mutants exhibit its characteristics: high efficiency in depleting external K+ content to a very low concentration (<1um) and low affinity towards Na+ uptake . Based on these evidences, it is hyphothesized by that HAK transporters function to mediate high-affinity K+ uptake or at least are involved significantly in the process.

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The discovery of HAK transporters as high-affinity K+ uptake would pose a question on the function of HKT that was initially detected as high-affinity K+ transporter by . The uncertainty was answered as recent research on plant transporters reveals that wheat HKT1 is involved in high-affinity Na+ uptake . Similar findings by (Uozami, 2000) and which reported that Arabidopsis AtHKT1 and the rice OsHKT1 also mediate Na+ uptake that was not coupled to K+ uptake. Additionally, molecular studies conducted in rice by have demonstrated that K+-starved rice mediates the high affinity Na+ uptake. The findings would exhibit an accurate representation on the function of HKT transporter in plants.

Thus, evidences from later research had proven that high affinity 'Mechanism 1' that was proposed by was essentially made up of two transporters, HAK and HKT where HAK responsible for high-affinity K+ transporter with low affinity to Na+ and HKT is a high-affinity Na+ transporter that does not transport K+.

The HKT transporters have a structure consisting of four (membrane-pore-membrane) MPM motifs . It also contains binding sites for two cations that must be occupied before the ions allow to cross the membrane . Further explanation of this characteristic is demonstrated in figure 2.

The HKT transporters exist in different parts of plants. In Arabidopsis thaliana, AtHKT1;1 is expressed at several locations. It is reported by that the AtHKT1;1 was detected in root stele and vascular tissues in leaves using promoter-GUS expression analysis. The analysis involved the amplification of 837 bp fragment of genimuc DNA located upstream of AtHKT1 start codon using PCR with sense and antisense primers. It was then cloned and transformed into Arabidopsis thaliana using Agribacterium tumefacien. The observation on GUS activtity revealed its expression in the root stele and leaf vasculature. A consistent result was reported by . Moreover, a similar results was also found for rice and wheat . Using the same method; promoter-GUS expression analysis, has also affirmed that the expresssion of AtHKT1 gene in vascular tissue but suggested a specific location in the phloem. However, contrary to , did not found any expression of AtHKT1 in roots of Arabidopsis thaliana??.

The HKT sodium transporter functions both as a selective Na+ transport or Na+-K+ symporter depending on the low or high affinity towards Na+ . The HKT proteins consist of two subfamilies where each subfamily varies by having either serine/glycine residue in the first pore loop domain of the protein . Subfamily 1 contains a serine substitution at the domain's position while subfamily 2 contains a glycine at this position . Such differences account for different functional property for each subfamily; subfamily 1 proteins are Na+ selective transporters while subfamily 2 proteins are Na+ and K+ symporters . The division of two subfamilies is described in Figure 3

Figure 2: Representation of theoritical model that explains the activity of plant HKT transporters. Theouter part of the pore has to bind to two cations before these can move inside and cross to the other side of membrane. The first two models are Na+ or K+ transporters, which bind two identical cations and allow them to move across the pore. The third model is a Na+K+ symport which accomodates one Na+ and one K+, but does not accommodate two Na+ or two K+. The foruth model is the Na+ transporeter presented in the first one but in a form that inhibited by K+ bceasise K+ binds the transporter but is not transported. Abbreviations: Os, Oryza sativa; Sc, Saccharimyces cerevisiae; Ta, Triticum aestivum Adapted from

Figure 3: Evolutionary tree of the HKT genes depicting the division of two major clades . Abbreviations: At, Arabidopsis thaliana; Ec, Eucalyptus camaldulensis; Hv, Hordeum vulgare; Mc, Mesembryanthemum crystallinum; Os, Oryza sativa; Pa, Phragmites australis; Pt, Populus trichocarpa; Sm, Suaeda maritima; Ta, Triticum aestivum.

HKT sodium transporters play several roles in regulating Na+ contents in plants. This review will describe the function of HKT sodium transporters in unloading Na+ from the xylem, in regulating of Na+ influx into the roots and in controlling Na+ recirculation from phloem to root. Throughout the discussion, the evidences and examples that will be cited is related to Arabidopsis, wheat and rice.

The role of HKT sodium transporter in regulating the efflux of Na+ ion from xylem vessel to xylem parenchyma cell.

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It is essential for plants to maintain low Na+ levels in shoot tissues in order to avoid ionic stress . During an increase in Na+ concentration in the xylem, the HKT sodium transporter function by unloading Na+ from the transpiration stream (xylem) into xylem parenchyma cell confine the amount of Na+ reaching the shoot of plants and thus allowing a high K+/Na+ ratio in leaves that would contribute towards salt tolerance in plant .

The HKT sodium transporter in Arabidopsis exhibits the ability to unload Na+ from the xylem as reported by . This has been demonstrated by comparing the rate of Na+ retrieval in hkt1;1 mutants compared to the wild type (control) using 22NaCl radioactive tracers . The wild type succeeded in withdrawing 44% of Na+ content from xylem vessel to xylem parenchyma cell while only 22% of Na+ ion withdrawn by hkt1;1 mutant thus suggesting the lack of function hkt1;1 mutant in withdrawing Na+ from the xylem vessel. Moreover, the growth of athkt1;1 mutant and wild type Arabidopsis in stress environments (50 Mm NaCl) for the time course of 60 minutes revealed that the wild type took up more than twice of the level of Na+ into the shoot (figure 4). This result would indicate that Arabidopsis containing the HKT1;1 would be responsible to unload Na+ from the xylem vessel into xylem parenchyma cells . Thus, the ability of HKT sodium transporter to unload Na+ from xylem vessel enable the reduction of Na+ in the shoots and allow Arabidopsis to confer better salt tolerance .

Furthermore, reported that the usage of n enhancer trap system to overexpress AtHKT1;1 in the root pericycle that including parenchyma cells would enhance salt tolerance of Arabidopsis. The enhancer trap system was based on the use of a yeast transcription activator GAL4 and GAL4 upstream activation sequence (UAS GAL 4) to drive the specific and high level of expression of a gene of choice which in this instance is the AtHKT1;1 . The expression achieved by the transformation with another DNA construct including the UAS GAL 4 sequence and the coding sequence of the gene of choice . Ultimately, it will increase Na+ transport expression in the stellar root cells which will increase Na+ retrieval from the transpiration stream.

The mechanism manages to elevate Na+ influx into parenchyma cells from the xylem vessel. The overexpression leads to a reduction of 47% of Na+ level in shoot. To further test the efficiency of AtHKT1;1 overexpression, it was grown in a stress environment (100 mM NaCl for 5 days). Phenotypic observation and dry mass measurement of the AtHKT1;1 overexpression in comparison with hkt1;1 mutant proved that the AtHKT1;1 overexpression is less affected by salt stress as the wild type exhibit healthier growth and higher dry mass value . Thus this would account for greater salt tolerance in Arabidopsis. Moreover, findings by revealed that athkt1;1 mutants grown in stress environment (75 mM NaCl) has resulted in an increase of Na+ contents in xylem vessels and ultimately resulting higher Na+ content in the phloem incomparison to AtHKT1;1. This would infer that the disruption of AtHKT1;1 represented by athkt1;1 mutants would impair the unloading activity of Na+ from xylem vessels and at the same time affirming the role of AtHKT1 in Na+ transport that leads to salt tolerance capability of Arabidopsis

Figure 4: Na+ uptake from 50 mM NaCl into shoot measured using 22Na+ for the time course of 60 minutes. Na+ uptake was linear during the first 50 minutes but in the last 10 minutes control plant showed a significant decline in Na+ uptake but hkt1;1 mutant continue to uptake more Na+ .

Additionally, immunological detection using AtHKT1 epitope-GST (glutathione-S-transferase) confirmed the presence of the HKT sodium transporter at xylem parenchyma cell. The detection would reinforce the function of HKT sodium transporter to unload Na+ from xylem vessel to xylem parenchyma cell. Similar mechanism as previously discussed by occurs in wheat but involving different HKT gene family. In wheat (Triticum aestivum) two genes previously were identified which are Nax1 and Nax2 that function to regulate Na+ content in the xylem . It was proposed by that the transporters expressed by Nax1 and Nax2 are HKT Na+ transporters due to their Na+ selective profile and Na+ unloading capacity from the xylem. HKT transporters expressed in root and leaf vasculature tissues were postulated by to contribute to xylem unloading activity in roots and shoots respectively.

Control of shoot Na+ uptake could be due to either tight control of xylem loading or high rate of withdrawal of Na+ from the transpiration stream into the upper part of the roots. Evidence for xylem withdrawal of Na+ in the roots of both [+] Nax1 and [+] Nax2 lines was obtained in a separate compartmental loading experiment. When the lower part of the root was exposed to 22 Na+, the [+] Nax1 line withdrew more of the total transported 22 Na+ into the upper roots (88%) than the [-] Nax1 line (51%; Fig. 4). Similarly, the [+] Nax2 line withdrew more 22 Na + into the upper roots (91%) than the [-] Nax2 line (44%). These differences were associated with a 4-fold higher shoot 22 Na + content in both the [-]Nax1 and [-]Nax2 lines than their respective isogenic pairs

Figure x: Withdrawal of 22Na+ from the xylem by roots of Nax1 and Nax2 which grown in 25 mM NaCl. Withdrawal rate was calculated as he amount of 22Na+ in the unlabelled upper roots as a percentage of total 22Na + transported from the labelled lower roots after two hours .

Homology searches of rice genomic databases conducted by identified seven HKT genes and two pseudogenes. SKC1, a major (quantitative trait locus) QTL for K+ content which related to OsHKT8. Moreover, OsHKT8 was reported to shows significant sequence similarities to HKT-type transporters found in wheat TaHKT1, Arabidopsis AtHKT1 and in both rice OsHKT1 and OsHKT4. HKT sodium transporters have been widely reported to be expressed around xylem parenchyma cells . Furthermore, several evidences have been postulated to support the SKC1 activity in unloading of Na+ from xylem such as its capacity as Na+ transporter and notably the rise of Na+ content in the xylem with the expression of weaker SKC1 allele.

Since sodium uptake in Arabidopsis and rice mainly govern by the similar HKT transporter, has proposed a model to illustrate the mechanim in unloading Na+ from xylem as depicted in figure 6.

Figure 6: Xylem Na+ exclusion model . The model include the xylem Na+ unloading function in both AtHKT1;1 and OsHKT1;5 transporter identify in rice . The exclusion of Na+ enables plants to sustain high K+/Na+ ratio in shoots during salinity stress as both AtHKT1;1 and OsHKT1;5 transport Na+ from xylem vessel to xylem parenchyma cell

The overwhelming evidences on the function of HKT sodium transporters in unloading Na+ from the xylem has established a robust profile on the role of HKT sodium transporter in plants. The research conducted on AtHKT1;1 in Arabidopsis was more thorough and prevalent in comparison to the HKT gene family in rice and wheat. However, since many of the HKT family genes from wheat and rice are orthologues to AtHKT1;1 Arabidopsis , it solidifies the fact that it may confer a similar function as sodium transporters.

HKT sodium transporter regulate the Na+ infux into the roots.

Another significant function of HKT sodium transporters was as Na+ mediator in roots. In Arabidopsis it is most likely that the AtHKT1;1 gene would express a HKT1;1 sodium transporter that function as a Na+ transporter in plant . Findings by revealed that AtHKT1 has the ability to regulate Na+ uptake into plant roots. The claim was made based on the experiment when the Arabidopsis thaliana mutant of sos3-1 hkt-1 exhibit lower Na+ accumulation than the wild type as depicted in figure x . Furthermore, reverse transcription polymerase chain reaction (PCR) analysis conducted on AtHKT1 revealed that the AtHKT1 transcript was expressed mainly in roots . The findings was also consistent with reports by who detected high levels of AtHKT1 mRNA in roots.

Figure x: Accumulation of Na+ in different variants of Arabidopsis. Plants grown in 100 mM NaCl and without salt stress (No NaCl). From left to right, sos3-1 HKT, sos3-1 hkt1-1 and SOS3 HKT1. The disruption of HKT gene cause a significant drop in level of Na+ uptake (less than 20 Mm) while normal expression of HKT gene recorded more than 30 mM of Na+ uptake by plant.

Similar findings were reported in wheat where the down regulation of HKT1 gene expression in wheat resulted in a decrease in Na+ uptake thus inducing better salt tolerance . Qualitative analysis affirms that the down regulated transgenic line exhibit a smaller membrane depolarisation value of root cortical cells in comparison to the control wheat plants . Thus, it is suggested that the down regulated transgenic line would decrease the membrane Na+ conductance in cortical root cells . Moreover, quantitative evidence was also presented through the measurement of short-term unidirectional Na+ influxes in roots of transgenic line and control when growing in 20, 50 and 100 mM concentration. A significant increase in Na+ uptake was recorded in control wheat particularly in a high concentration (100 mM) environment . Additionally, the data on the Na+ content of root sap when grown in 200 mM NaCl suggested that the transgenic lines would accumulate four fold lower Na+ in comparison to the control (figure 7). Additionally, It is also reported by that the expression of HKT1 in Xenopus oocytes shows the role as Na+-driven high-affinity K+ uptake and low-affinity Na+ uptake.

Figure 7: Sodium content of root exudate. Seedlings were grown for 14 d in FNSN and then transferred to high stress conditions (FNS - K+200mM NaCl). After 5 d growth at high salt, shoots were excised and root exudate was collected for 5min using a pressure bomb. Measurements are means of three samples .

Despite the evidence of HKT Na+ transporter functioning as a Na+ regulator in roots, it is mentioned by that the HKT transporter in wheat expressed by Nax1 and Nax2 genes manages to seclude 98% of the Na+ from entering the root. With the absence of both genes, 94% of Na+ was secluded. The data produced by calculating the net Na+ transport rate and the transpiration rate which measured over 24 hours (table x). Thus, with reduction of Na+ seclusion in the absence of both genes, there are possibilities that other sodium regulator may be involved in the Na+ seclusion process.

Table x: Na+ and K+ net transport rates to the shoot, Na+ and K+ concentration in the xylem stream, and percentage exclusion of Na+ by the roots in line 149. Nax1 and Nax2 near-isogenic lines grown in 50mM NaCl. The values are calculated over a 6 to 10 days period.

Unlike AtHKT1;1, which is a single-copy gene in Arabidopsis thaliana, seven full-length OsHKT genes were identified in the Japonica rice genome based on the completed genome sequence (Garciadebla´s et al, 2003). However, the most prominent gene that functions to mediate Na+ from soil into root is OsHKT2;1 . This is proved by the activity of oshkt2;1 mutant alleles that recorded a staggering reduction of Na+ influx into rice rootsin comparison to wild type.The concentration of Na+ influx was measured in xylem sap via inductively coupled plasma-optic emission spectroscopy (ICP-OES)(figure 8) . The ICP-OES involvesquantitative measurement of the optical emission from excited atoms produced when material is heated which in this instance, the Na+. Lower level of Na+ influx in oshkt2;1 mutant alleles is inferred from the lower level of emission spectrum. Thus, from the ICP-OES measurement, it can be deduced that OsHKT2;1 functions to accumulate Na+ in both roots and shots. Additionally, it was reported by that OsHKT1 was also involved in the Na+ uptake via the roots as the in situ PCR analysis depicts the expression of OsHKT1 sodium transporter in xylem.

Figure 8: oshkt2;1 mutant accumulate less Na+ in roots and shoots. Plants grown hydroponically for 19 days under 0.5 mM Na+. The content of roots (A) and shoots (B) were measured by inductively coupled plasma-optic emission spectroscopy (ICP-OES)

However, the Na+ uptake from the soil into the roots by OsHKT2;1 transporter was only limited to nutritional requirements as surplus intake of Na+ that would generate toxicity in rice would trigger a rapid respond of down regulation OsHKT2;1 transporter . Thus, OsHKT2;1 contributes to salt tolerance in rice by downregulating its expression during high Na+ influx to avoid excessive Na+ uptake that could cause toxicity. Recent studies have shown that AtHKT1;1 in Arabidopsis and its closest homologue, SKC1 or OsHKT1;5 in rice, functions by removing Na+ from the xylem sap, thus reducing Na+ accumulation in leaves (Ren et al,2005; Sunarpi et al, 2005; Horie et al, 2006).

Evidence on the function of HKT genes in rice mostly related to OsHKT2;1 sodium transporter as suggested by With many more genes of the family in rice have not been studied and tested for their function to mediate Na+ into roots, the overall function of HKT genes in rice is still indistinct. There is a probability that the rest of HKT gene family would have certain influence in the regulation of Na+ into the root as OsHKT2;1 did not seclude all of the Na+ uptake by plants.

Na+ recirculation to root through phloem

The recirculation activity of Na+ from the shoot into the phloem and then unloading it into the roots is hypothesized by to be one of the essential function of the AtHKT1;1 sodium transporter. It assists Arabidopsis in gaining a certain degree of salt tolerance as it reduces the amount of Na+ in the shoot . The hypothesis was supported by the results of allelic (sas2-1) recessive mutation of Arabidopsis that reduced AtHKT1 Na+ transport activity. It was observed that the Na+ concentration in the phloem sap emanating from leaves reduced. As the results, the Na+ content in the shoots remain high and low in the roots. The findings was also supported by which proposed that the decline in uptake of Na+ into xylem parenchyma cells in athkt1 mutants would contribute to the reduced Na+ loading of the phloem as depicted by figure 9.

Figure 9: AtHKT1 mutation causes an increase in the Na content of xylem

sap and a decrease in the Naþ content of phloem sap under salt stress.

Soil-grown plants were subjected to 75 mM NaCl with one-twentieth MS salts

for 2 days after bolting.

Furthermore, has reported that the disruption of the AtHKT1 gene would decrease the Na+ content in roots and increase the Na+ content in shoots thus supporting the hypothesis that the AtHKT1;1 sodium transporter was involved in recirculation of Na+. Additionally also proposed that AtHKT1 sodium transporter was part of the synergistic mechanism with AtNHX1 sodium transporter in ensuring that the rate of Na+ ions withdrawal from shoots is greater than the uptake level. This would both support the recirculation function of HKT1 sodium transporter and mechanisms on how plants achieve their salt tolerance.

However, in the recent research conducted by who disputed the HKT1 sodium transporter function in recirculating Na+ through phloem. The research reported that the measurement of Na+ ions in the shoots of Arabidopsis using radioisotopes 22Na+ for the period of 52 hours resulting only 13% decline in 22Na+. The result suggested that the 13% decline is not significant enough to imply that the recirculation of Na+ would affect the net shoot Na+ accumulation in Arabidopsis (figure 10)

In rice, the HKT sodium transporter is expressed by the SKC1 (OsHKT1;5) gene which does not exhibit any difference in the Na+ phloem content thus suggesting the Na+ recirculation through the phloem is possibly mediated by other transporters belonging to the OsHKT family . Another family member of OsHKT, which is OsHKT2 was reported by to be down regulated in phloem thus suggesting the lack of function in recirculating Na+ through phloem.

Figure 10: Measurement of Na+ content in shoot using 22Na+ over the period of 52 hours. Black bar represents the hkt1;1 while the white bar represents the wild type

Significant amount of discussion on the function of HKT sodium transporter in recirculating Na+ through phloem has been related to Arabidopsis and rice. There is a lack of attention on the study of HKT sodium transporter function to recirculate Na+ through phloem in wheat. Although some evidence disputed that the function HKT sodium transporter is to recirculate Na+ through phloem in Arabidopsis, the evidence provided by was convincing enough to support the idea that HKT sodium transporter involved in recirculating Na+ through phloem and thus contributing to salt tolerance in plants.

The summary of various locations HKT sodium transporter expression in plants is depicted in figure x.

Figure x: Summary of Na+ movement in plants. The Na+ uptake initiated from the root, passing through xylem and finally reaching shoot . Consequently, the Na+ ion were recirculating back to root via phloem. . Arabidopsis and rice share the same expression of HKT in xylem.

Future research

One of the interesting fields to study the HKT sodium transporter in plants is the role of Na+ recirculation from the shoots to roots via the phloem. This area received lack of focus and depth in analysing the function of HKT sodium ???What?? . Work conducted by should be extended further to gain more comprehensive understanding regarding the HKT role not only in Arabidopsis but other plants as well such as wheat and rice.

Another possibility in the future research is to expand the scope of analysing the activity of other members of the HKT family gene in rice and wheat. This is because, current research only highlighted the functions of few HKT family genes whilst many more that lack of attention. By doing so, more functions of HKT family genes could be revealed.

As the HKT is not the only sodium transporter, further research to understand the interaction of HKT sodium transporters with other relevant genes that also express sodium transporter in plants such as Na+ /H+ exchanger NHX1 and saltly over sensitive SOS would provide a better view on the physiological mechanisms of plants in acquiring salt tolerance.