Co-expression of SpSOS1 and SpAHA1 in transgenicArabidopsisplants improves salinity tolerance

Background

Na⁺extrusion from cells is important for plant growth in high saline environments. SOS1 (salt overly sensitive 1), an Na⁺/H⁺antiporter located in the plasma membrane (PM), functions in toxic Na⁺extrusion from cells using energy from an electrochemical proton gradient produced by a PM-localized H⁺-ATPase (AHA). Therefore, SOS1 and AHA are involved in plant adaption to salt stress.

Results

In this study, the genes encoding SOS1 and AHA from the halophyteSesuvium portulacastrum(SpSOS1andSpAHA1, respectively) were introduced together or singly intoArabidopsisplants. The results indicated that either SpSOS1 or SpAHA1 conferred salt tolerance to transgenic plants and, as expected,Arabidopsisplants expressing bothSpSOS1andSpAHA1grew better under salt stress than plants expressing onlySpSOS1orSpAHA1. In response to NaCl treatment, Na⁺and H⁺in the roots of plants transformed withSpSOS1orSpAHA1effluxed faster than wild-type (WT) plant roots. Furthermore, roots co-expressingSpSOS1andSpAHA1had higher Na⁺and H⁺efflux rates than singleSpSOS1/SpAHA1-expressing transgenic plants, resulting in the former amassing less Na⁺than the latter. As seen from comparative analyses of plants exposed to salinity stress, the malondialdehyde (MDA) content was lowest in the co-transgenicSpSOS1andSpAHA1plants, but the K⁺level was the highest.

Conclusion

These results suggest SpSOS1 and SpAHA1 coordinate to alleviate salt toxicity by increasing the efficiency of Na⁺extrusion to maintain K⁺homeostasis and protect the PM from oxidative damage induced by salt stress.

盐,尤其是生理盐水,可以有毒植物through inhibition of important biochemical and physiological processes, such as protein synthesis, photosynthesis, and enzymatic reactions, after moving into the cytosol from soils [1］.While salt stress can inhibit plant growth and development, many types of plants are able to grow in high salinity environments because they have complex mechanisms that facilitate adaptation to salinity stress [2］.Of these mechanisms, the ability to transport excess Na⁺out of cells is critical to salt tolerance. SOS1 (salt overly sensitive 1) is a Na⁺/H⁺antiporter and the only Na⁺efflux protein present in plant plasma membranes (PMs) characterized to date. SOS1 mediates extrusion of Na⁺through a proton gradient generated by the H⁺-ATPase (AHA) in the PM [3］.Therefore, SOS1 and AHA are two key plant halotolerance factors.

PM H⁺-ATPase is encoded by a large family of genes [4,5］.Bioinformatics analyses ofArabidopsis and genomic sequences of rice revealed the presence of 11 and 10 PM AHAs, respectively [6,7］.Of these AHAs, NaCl treatment induced expression of three, AtAHA1, AtAHA2,andAtAHA3, inArabidopsis[8］.The transcript levels of PM AHA were found to be higher in a salt-tolerant poplar than a salt-sensitive poplar [9］.In addition, PM AHA mRNA is more abundant in halophytes than glycophytes [10,11］.Salinity causes upregulation of PMAHAgene expression, as well as accelerates protein biosynthesis and H⁺-pumping activity in some plants [12,13,14］.AHA in a salt-tolerant rice species has higher activity than in a salt-sensitive rice species [15］.AnArabidopsisPM AHA4 mutant has dramatically reduced growth when exposed to salt stress compared to WT [16］.Expression of a constitutively activated PM AHA lacking the autoinhibitory domain in transgenic tobacco plants increases salt tolerance compared to untransformed plants [17］.

SOS1genes have been found in many plants [18,19,20,21,22,23,24,25］.Of these,ArabidopsisSOS1 (AtSOS1) was the first PM Na⁺/H⁺antiporter to be thoroughly physiologically, biochemically, and molecularly characterized [18,26］.Exposure to salinity stress increasesSOS1transcript abundance in wheat plants [19], induces the accumulation ofSOS1mRNA in rice plants [27], and causes upregulation ofSOS1transcription inArabidopsis[28］.Under high salt conditions,SOS1mRNA levels are higher inThellungiella salsuginea(a halophyticArabidopsis-relative plant) thanArabidopsis[20］.MutantArabidopsisplants lacking SOS1 are extremely sensitive to salt stress [18,29］.Thellungiella salsuginealines expressing SOS1-RNAi (RNA interference) are sensitive to salt [20］.The salt sensitivity of anArabidopsis sos1mutant can be overcome by transforming in native or other plantSOS1genes [27,28］.ArabidopsisoverexpressingAtSOS1is more salt tolerant than WT plants [30］.Expression of wheat SOS1 (TaSOS1) in transgenic tobacco plants improves their growth following NaCl treatment [31］.SOS1 uses the proton gradient established by PM AHA to exchange Na⁺for H⁺across the PM [3,27］.The aforementioned data indicate the PM Na⁺/H⁺antiporter SOS1 and H⁺-ATPase AHA are involved in plant salt tolerance, where an Na⁺/H⁺antiporter utilizes the proton gradient generated by H⁺-ATPase to move Na⁺from the cytoplasm to the external medium and help plant cells maintain non-toxic cytosolic concentrations of Na⁺. Therefore, theoretically, coordinating SOS1 and AHA could enhance Na⁺extrusion, where co-expression of these two genes should confer better tolerance to salinity to transgenic plants. However, it has not been reported whether SOS1 and AHA1 function cooperatively in transgenic plants to more efficiently improve salinity tolerance.

Sesuvium portulacastrumis a halophyte that grows optimally in the presence of 200–300 mM NaCl [32］.When growing in a saline environment,S. portulacastrumcells accumulate large amounts of Na⁺despite salt glands and bladders not being present in all tissues [33,34,35], suggestingS. portulacastrummay have a unique ability to remove Na⁺from cells. The SOS1 protein functions as a PM Na⁺/H⁺antiporter driven by the proton gradient that is produced by the PM H⁺-ATPase AHA, so they are considered as superior salt tolerance determinants [3,36］.TheSpAHA1andSpSOS1genes encode a PM H⁺-ATPase and Na⁺/H⁺antiporter, respectively, and are more highly transcribed inS. portulacastrumplants exposed to salt stress. SpSOS1 more efficiently mediates Na⁺removal using a proton gradient created by SpAHA1 inSpAHA1-SpSOS1co-transgenic yeast cells, where yeast cells co-expressingSpSOS1andSpAHA1grow better following NaCl treatment than cells transformed with onlySpSOS1orSpAHA1[3］.Over-expression ofSpAHA1conferred salt tolerance to transgenicArabidopsis[37］.SpSOS1 complemented the salt sensitivity of transgenicArabidopsis sos1mutant plants [38］.These results suggest that SpSOS1 and SpAHA1 are involved in salt tolerance ofS. portulacastrum, and co-expression ofSpAHA1andSpSOS1may improve transgenic plant salt tolerance. To test this hypothesis,SpAHA1andSpSOS1genes were co-transformed intoArabidopsisplants. Functional analyses indicate thatArabidopsisplants co-expressingSpSOS1andSpAHA1had better salt tolerance than plants expressing either gene alone due to efficient Na⁺removal mediated by SpSOS1 using the extra proton gradient generated by SpAHA1. Therefore, genetic evidence may significantly guide development of more salt tolerant crops using PM-localized Na⁺/H⁺antiporters and H⁺-ATPases.

Transgenic plant identification

SpSOS1andSpAHA1were transformed alone or together intoArabidopsisplants usingAgrobacteriacarrying pCAMBIA1304-SpSOS1, pCAMBIA1304-SpAHA1,or pCAMBIA1304-SpSOS1-SpAHA1. PCR analyses of genomic DNA performed usingSpAHA1/SpSOS1andhygBgene-specific primers revealed 12SpSOS1-, 11SpAHA1-, and 10SpSOS1-SpAHA1-transgenic lines were obtained (Additional file1: Figure S1).Total RNA was isolated from the above transgenic plant lines and RT-PCR analyses were used to study theSpAHA1andSpSOS1expression levels. TheSpAHA1gene was significantly expressed in all singleSpAHA1-transgenic lines, except for SpAHA1- lines 5 and 8. Of theSpSOS1-expressing single transgenic plants,SpSOS1-line 1 had the highestSpSOS1expression of theSpSOS1-transgenic lines. InSpAHA-SpSOS1co-expressing plants, the clearest expression of bothSpAHA1andSpSOS1在10号线(额外的文件吗2: Figure S2). Therefore, the T3 generation transgenic plants of the homozygousSpSOS1-line 1,SpAHA1-line 1, andSpAHA1-SpSOS1-line 10 were used to characterize the functions of SpSOS1 and SpAHA1.

SpSOS1 and SpAHA1 functioned together to more efficiently improve transgenic plant salt tolerance

In plant cells, the PM Na⁺/H⁺antiporter SOS1 mediates Na⁺excretion using a proton gradient created by PM H⁺- - - - - - atp酶. Therefore, both of these proteins are involved in plant salt tolerance. Much evidence indicates that overexpressingSOS1orAHAincreases the salt tolerance of transgenic plants [39］.In addition, our recent investigation found SpSOS1 and SpAHA1 function cooperatively in transgenic yeast cells, where yeast cells co-expressingSpSOS1andSpAHA1are better growers than cells transformed with onlySpAHA1orSpSOS1[3］.Therefore, we hypothesized co-expression ofSpSOS1andSpAHA1would increase the salt tolerance of transgenic plants compared to plants transformed with onlySpSOS1orSpAHA1. To examine the influence ofSpSOS1-SpAHA1co-expression on the salt tolerance of transgenic plants, 5-day-oldArabidopsisWT,SpSOS1-expressing,SpAHA1-expressing, andSpSOS1-SpAHA1co-expressing seedlings were grown on MS plates containing 0, 50, 75, or 100 mM NaCl. Two weeks post-NaCl treatment, the seedlings were photographed and their fresh weight, root length, and lateral root number were measured.Upon exposure to salinity stress, the growth of all tested plants decreased, but expression of eitherSpSOS1orSpAHA1ameliorated this growth inhibition from NaCl treatment compared to WT plants. Furthermore, among all the transgenic plants, salt tolerance improved the most in plants co-expressingSpSOS1andSpAHA1based on growth in MS medium containing different concentrations of NaCl (Fig.1).

Similarly, the growth of transgenic and WT plants was inhibited in soil supplemented with 200 mM NaCl. However,Arabidopsisplants expressing bothSpSOS1andSpAHA1grew the best among the different experimental cohorts under these conditions (Fig.2a).SpAHA1-SpSOS1-line 10 displayed 26, 33, and 67% greater fresh weights thanSpSOS1-line 1,SpAHA1-line 1, and WT plants, respectively (Fig.2b). The percent reduction in growth of plant lines treated with NaCl was ordered:SpSOS1-SpAHA1co-expressing plants <SpSOS1-expressing plants<SpAHA1-expressing plants 2c). These findings indicate the PM-localized Na⁺/H⁺antiporter SpSOS1 and H⁺-ATPase SpAHA1 function cooperatively to improve the salt tolerance of transgenic plants.

SpSOS1-SpAHA1co-expressingArabidopsisplants had higher H⁺efflux rates thanSpSOS1-orSpAHA1-expressing plants under high saline conditions

Net H⁺flux at the roots of WT plants was close to the mock control (Fig.3A), which is in full agreement with the recent report that both transient and long-term salinity exposure did not induce H⁺efflux fromArabidopsisroots [8］.These results suggested that H⁺efflux might be balanced by H⁺influx at the roots exposed to salinity stress. PM H⁺-ATPase activity is a major factor in H⁺excretion at the PM [40］.It was recently reported that SpAHA1 can function as an H⁺-ATPase on vesicles isolated from yeast cells expressingSpAHA1[3］.Roots expressing SpAHA1 had a faster net H⁺efflux than the WT plants under saline conditions, suggesting SpAHA1 is responsible for the extra H⁺efflux, i.e., SpAHA1 pumped more protons out of the cells. It is not expected that protons were extruded faster in roots transformed withSpSOS1relative to WT plants and the phenomenon might be from feedback regulation of Na⁺extrusion mediated by SpSOS1. This hypothesis is also supported by the H⁺flux in the roots ofSpSOS1-SpAHA1co-expressing transgenic plants (Fig.3) being the highest among all the transgenic plants, where the H⁺efflux rates in the roots co-expressingSpSOS1-SpAHA1were 49 and 52% greater thanSpSOS1- andSpAHA1-expressing roots, respectively.

Plants co-expressingSpSOS1-SpAHA1had higher Na⁺efflux in roots and less Na⁺accumulation after NaCl treatment

SOS1 mediates Na⁺excretion from cells and is a key halotolerance factor. SpSOS1 has been shown to be a PM-localized Na⁺/H⁺antiporter and capable of improving the growth of transgenic yeast cells under salt stress by decreasing the cellular Na⁺content [3］.In this scenario, the roots from all tested plants grown in medium without NaCl displayed Na⁺uptake characteristics, but no significant differences in Na⁺flux activities at roots of transgenic and untransformed plants under unstressed condition were observed (Additional file3: Figure S3). On the contrary, NaCl treatment stimulated Na⁺effluxes at all tested roots.SpSOS1-expressing roots had faster Na⁺efflux relative to WT plants in saline conditions (Fig.4a, b), suggesting the extra Na⁺extrusion may be mediated by SpSOS1, which would result in the observed lower Na⁺content in theSpSOS1-transgenic plants than WT plants under salt stress (Fig.5a). SOS1-mediated Na⁺/H⁺exchange is powered by a proton gradient generated by an H⁺-ATPase. Therefore, a proton gradient generated by SpAHA1 (Fig.3) might catalyze native SOS1 (AtSOS1) to transport more Na⁺out of cells, which may be one reason for the higher Na⁺efflux rate in roots transformed withSpAHA1compared to WT plants in saline conditions (Fig.4). Roots co-expressingSpAHA1andSpSOS1had the highest Na⁺射流速度在所有的转基因植物tested, where the Na⁺efflux rate inSpSOS1-SpAHA1co-transgenic roots was 53 and 72% greater than the plants expressingSpSOS1orSpAHA1singly, respectively (Fig.4b). Correspondingly, the Na⁺levels in the transgenic plants were lower than in WT plants (Fig.5a). Therefore, it is reasonable thatSpSOS1-SpAHA1co-expression quickened Na⁺extrusion in the roots of and decreased Na⁺accumulation in transgenic plants compared toSpSOS1-orSpAHA1-expressing plants under saline conditions (Fig.4,5a). These results indicate SpAHA1 produced an additional proton gradient and, thus, promoted SpSOS1-mediated Na⁺extrusion inArabidopsisplants co-expressing both theSpAHA1andSpSOS1genes.

SpSOS1-SpAHA1co-transgenic plants had higher K⁺retention under saline conditions

Among the physiological and biochemical processes in plant cells influenced by high salinity, nutrient imbalance is among the most deleterious resulting effects [41］.The chemical and physical characteristics of sodium most resemble potassium among the nutrient elements. Therefore, excess Na⁺inhibits plant growth by interfering with cytosolic K⁺homeostasis. No differences in K⁺content was found among all the tested plants under normal conditions. However, upon salinity stress, the transgenic plants displayed less of a decrease in K⁺content than the WT plants. Furthermore, co-expression ofSpSOS1andSpAHA1most efficiently alleviated K⁺loss among the transgenic plants exposed to NaCl, where the K⁺content was highest in the leaves of plants co-expressingSpSOS1andSpAHA1(85 mg/g dry weight), followed by those fromSpSOS1-transgenic plants (73 mg/g dry weight), and thenSpAHA1-expressing plants (69 mg/g dry weight). WT plants had the lowest K⁺content (47 mg/g dry weight; Fig.5b).

SpSOS1-SpAHA1co-expression decreased malondialdehyde accumulation in transgenic plants

Salinity creates oxidative stress and excess reactive oxygen species can interfere with metabolism in the cytoplasm, such as by damaging membrane structures and destroying membrane integrity through lipid peroxidation [42］.An indicator of membrane lipid oxidation, malondialdehyde (MDA) represents membrane lipid damage to some extent. Upon exposure to NaCl, the amount of MDA in the leaves of all tested plants increased, but MDA accumulation in theSpSOS1-SpAHA1co-expressing leaves was the lowest (Fig.6) at only 84, 74, and 61% of that inSpSOS1-expressing,SpAHA1-expressing, and WT plants under saline conditions. These results indicateSpSOS1andSpAHA1coordination could more efficiently reduce oxidative damage to membranes from salinity stress in transgenic plants.

Plants grown under K⁺deficiency substitute it by Na⁺, especially some halophyte species can use Na⁺for stomata operation instead of K⁺[43］.However, when there is excess sodium in the cytosol, it interferes with some key metabolic processes and eventually inhibits the growth and development of the plant. To tolerate high Na⁺levels, plant cells must be capable of removing Na⁺from the cytoplasm through some physiological processes. In one of these processes, cytoplasmic Na⁺can be imported into vacuoles through tonoplast Na⁺/H⁺antiporter NHXs using an electrochemical gradient established by a vacuolar H⁺-ATPase and H⁺-PPase (AVP/VP) using protons. The sequestration of Na⁺into vacuoles not only prevents the deleterious effects resulting from Na⁺in the cytoplasm, but also lets the plants use Na⁺as an osmoticum, which helps maintain the osmotic potential that drives water into the cells [39,44］.Therefore, tonoplast NHX antiporters and H⁺-pumps have important functions in plant responses to salt stress. In transgenic plants, overexpression of genes encoding vacuolar Na⁺/H⁺antiporters or H⁺-PPases enhances salt tolerance [39,42］.Furthermore, co-expression of AVP and NHX may better improve the growth of transgenic plants exposed to salt stress through more efficient compartmentalization of Na⁺into vacuoles than when NHX or AVP are expressed alone. Co-expression ofZxNHXandZxVP1–1confers better salt tolerance to transformed sugar beet and lotus plants [42,45］.NHX1-AVP1co-transgenic rice grows better under salt stress than rice plants expressing only one of these genes [46,47］.Tobacco plants co-expressingTNHXS1andTVP1have higher salt tolerance than transgenic plants expressingTNHXS1orTVP1alone [48］.Another mechanism contributing to Na⁺extrusion is the PM Na⁺/H⁺antiporter SOS1. The Na⁺extrusion mediated by SOS1 is also driven by electrochemical gradients of protons generated by a PM H⁺-pump (H⁺-ATPase, AHA). The overexpression ofSOS1significantly improves the salt tolerance of transgenic grapevine compared to WT plants [49］.Overexpression of theSOS1gene in tobacco plants increases salt tolerance by maintaining a lower Na⁺content [50] and the growth ofArabidopsisplants overexpressingSOS1is better than that of WT plants under salt stress [51］.It has been reported that overexpression ofPeHA1(H⁺-ATPase 1),a poplar gene encoding a PM-localized H⁺-ATPase, enhances the salt tolerance of transgenicArabidopsis[52］.These studies suggest that co-expression of bothSOS1andAHAin transgenic plants should more effectively increase salinity tolerance just as co-expression of vacuolar NHX and AVP results in higher salt tolerance. In the present investigation,SpAHA1-transgenic roots had faster H⁺efflux than WT plants under salt stress (Fig.3), suggesting SpAHA1 enhanced proton efflux and generated an additional proton gradient that acted as a driving force for Na⁺/H⁺exchange mediated by SpSOS1. More interestingly, the magnitude of net H⁺flux is in 3 to 4 pmol•cm^− 2•s^− 1range (Fig.3), while Na⁺flux is around 1000 pmol•cm^− 2•s^− 1; the stoichiometric ratio for Na⁺/H⁺交换的年代OS1 protein is 1H⁺:1Na⁺, so such tiny increase in H⁺flux (from 2 to 4 pmol•cm^− 2•s^− 1) may not cause such a massive flux of Na⁺. Net H⁺fluxes at roots was determined in the present study, that is, the data for net H⁺efflux is equal to total H⁺efflux minus total H⁺influx at the roots, suggesting that H⁺-ATPase mediated H⁺efflux is likely balanced by H⁺uptake through SOS1 transporters. These suggest that SpAHA1 might provided more H⁺gradient than the shown data of net H⁺efflux in transgenic plant exposed to salinity stress, resulting in faster Na⁺efflux from cells, consequently Na⁺content in transgenic plants was lower than that in wild type plants (Fig.5a). Overexpression ofSpSOS1in transgenicArabidopsisaccelerated Na⁺efflux in the roots (Fig.4a, b), resulting in decreased Na⁺content in the transgenic plants compared to the WT plants (Fig.5a)。有趣的是,H⁺efflux in theSpSOS1-expressing roots was faster than in the WT orSpAHA1-transgenic roots (Fig.3), suggesting that increased Na⁺extrusion mediated by SpSOS1 might regulate H⁺-pumping activity via feedback at the PM. This is because the Na⁺/H⁺exchange mediated by SOS1 is dependent on energy and driven using a PM-localized H⁺-ATPase-driven proton motive force [53］.The H⁺and Na⁺efflux rates in the roots ofArabidopsisplants co-expressingSpAHA1andSpSOS1were highest among all the transgenic plants (Figs.3and4), leading to the lowest Na⁺content in the co-transformed plants relative to other transgenic plants expressing onlySpSOS1orSpAHA1(Fig.5a). In response to NaCl treatment, the biomass of the transgenicArabidopsisplants co-expressingSpSOS1andSpAHA1was greater than the biomasses of the single-gene expressing plants (Fig.2). Taken together, the higher rate of Na⁺extrusion, lower Na⁺levels, and better growth of theSpAHA1-SpSOS1co-expressing plants compared to the singleSpAHA1orSpSOS1gene transgenic plants provides direct genetic evidence that SOS1 and AHA function in a cooperative manner to inhibit Na⁺accumulation in the cytosol and play important roles in plant adaption to highly saline conditions.

High soil salinity is characterized by high soluble salt concentrations, of which sodium salt is the most soluble and widespread salt [44］.Excessive sodium ions in soils can enter into plant cells and then interference with some critical biochemical and physiological processes. The most deleterious effect of salinity is ion toxicity [41］.K⁺is a necessary macronutrient that has a critical role in the growth and development of plants [54］.Due physicochemical similarities between Na⁺and K⁺, Na⁺can compete with K⁺for binding sites important in critical cytoplasmic physiological and biochemical processes [55］.In particular, Na⁺inhibits the activity of many K⁺-dependent enzymes [56] and, therefore, excess Na⁺can inhibit K⁺-associated activities in the cytosol [55］.It is hypothesized that plant survival in the presence of salt stress requires a high K⁺/Na⁺ratio in the cytoplasm [57］.Therefore, limiting Na⁺influx into cells may facilitate plant growth under salt stress [58,59］.Under high salt conditions, the PM potential becomes depolarized, which encourages passive Na⁺influx into cells and K⁺efflux out of cells. H⁺-ATPase-generated electrochemical potential gradients across PMs can repolarize PMs following NaCl-induced depolarization [39］.Therefore, maintenance of the PM potential using H⁺-ATPases can reduce the Na⁺influx via depolarization-activated non-selective cation channels (NSCCs) and K⁺efflux via K⁺outward rectifiers (KORs) and NSCCs [60］.The net H⁺efflux from root cells inSpAHA1-transgenic plants occurred at a higher rate relative to WT plant roots, suggesting SpAHA1 increased the H⁺-ATPase activity and electrochemical potential and, thus, the H⁺gradient across the PM. This may reduce Na⁺influx and K⁺efflux and, correspondingly, theSpAHA1-transgenic plants had less Na⁺and more K⁺than WT plants (Fig.5a, b). In addition to Na⁺extrusion, SOS1 is also involved in K⁺homeostasis in plant cells under high salt conditions. Transgenic tobacco plants expressingSbSOS1contain less Na⁺, but more K⁺, in their roots than WT plants under high salt stress [50］.Horie et al. [61] suggested SOS1 plays a primary role facilitating high-affinity absorption of K⁺into roots. SOS1 is necessary for protecting K⁺uptake and is involved in K⁺homeostasis maintenance in cells under salinity stress [19,62］.Overexpression ofTaSOS1confers salt tolerance to transgenic tobacco plants by decreasing the Na⁺and increasing the K⁺levels [31］.Arabidopsisroots expressingSpSOS1displayed faster Na⁺efflux than WT plant roots under saline condition (Fig.4), suggesting SpSOS1 was responsible for the extra Na⁺extrusion. The faster H⁺efflux in the roots of plants expressingSpSOS1may aid repolarization following NaCl-induced depolarization of the PM, thus decreasing Na⁺influx and K⁺efflux [60］.These actions may have ledSpSOS1-transgenic plants to contain less Na⁺and more K⁺relative to WT plants under salt treatment. Therefore, faster H⁺and Na⁺efflux in the roots also resulted in retention of more K⁺and a lower Na⁺concentration in cytosol ofSpSOS1-SpAHA1co-transgenicArabidopsisplants compared to plants expressingSpSOS1orSpAHA1alone (Figs.3and4). This led toArabidopsisplants co-expressingSpSOS1andSpAHA1to have higher a K⁺/Na⁺level than the transgenic plants with onlySpSOS1orSpAHA1, which is strong evidence of salt tolerance. These results suggest SOS1 and AHA1 facilitate more efficient prevention of K⁺loss and enhance Na⁺extrusion and thereby contribute to better salt tolerance.

Another deleterious effect of salinity stress in plants is associated with oxidative stress [39］.Accumulation of ROS (reactive oxygen species) is toxic in cells. Therefore, intracellular ROS levels are tightly regulated under normal conditions through a number of intracellular peroxidative and antioxidative reactions within the cell. Salinity can disrupt the ROS production and scavenging balance, resulting in ROS accumulation, which can negatively affect cellular structures and metabolism [63,64］.In order to protect cells from salinity-induced oxidative damage, excess ROS is scavenged by antioxidant molecules and enzymes. RCD1 (Radical-induced cell death) is a regulator of responses to oxidative stress and protects cells from oxidative damage caused by H₂O₂, diamide, and tert-butyl peroxide [65,66,67］.SOS1 functions in tolerance to oxidative stress by interacting with RCD1 and regulating expression of certain genes associated with oxidative-stress tolerance inArabidopsis[66］.血红素氧合酶(HO)是一个重要的因素lant antioxidant defense systems. Overexpression of theAtHOgene enhancesArabidopsistolerance to salt by increasing PM H⁺-ATPase activity and expression [68］.过量的活性氧会破坏膜结构由牛idizing lipids in the PM, leading some key metabolites abnormally leak out of cells. ROS could disturb ion homeostasis in cells by inducing the efflux of several cations [69,70,71］.Coskun et al. [53] found NaCl-induced efflux of K⁺was a result of a lack of PM integrity in rice. This indicates the maintenance of PM stability has a key role in plant salt tolerance. In the present investigation, both SpSOS1 and SpAHA1 prevented the accumulation of MDA in transgenic plants following NaCl treatment, but plants co-expressingSpSOS1andSpAHA1had a more drastic decrease in MDA content under salt stress than plants expressing only one of these genes. This suggests SpSOS1 and SpAHA1 coordinate in transgenicArabidopsisand ameliorate salt toxicity by more efficiently alleviating oxidative damage to the PM generated by salinity stress.

Arabidopsisplants co-expressingSpSOS1andSpAHA1had higher K⁺and lower MDA levels than plants transformed with onlySpSOS1orSpAHA1and, thus, grew better under salt stress. The coordinated action of these genes might be a novel and effective method for increasing the salt tolerance of crops.

Plasmid construction

TheSpAHA1andSpSOS1genes were separately cloned fromS. portulacastrumand inserted into plasmids p414 (p414-SpAHA1) and p416 (p416-SpSOS1) in our recent investigation [3］.Plant vectors expressingSpAHA1orSpSOS1alone or together bicistronicly were constructed as described in Additional file4: Figure S4. (1) Amplification of theSpSOS1gene was performed using the p416-SpSOS1plasmid as the template and the primers SpSOS1-F and SpSOS1-R (Additional file5: Table S1). The amplified gene was inserted into the pCAMBIA1300 vector betweenSalI andKpnI restriction sites, generating pCAMBIA1300-SpSOS1. (2) A fragment containing a constitutive promoter (cauliflower mosaic virus 35S promoter), theSpSOS1gene, and the NOS terminator was excised from the pCAMBIA1300-SpSOS1plasmid usingPstI andEcoR I restriction enzymes and transferred into the plant expression vector pCAMBIA1304 between the same restriction sites. The resulting plasmid was named pCAMBIA1304-SpSOS1. (3) TheSpAHA1gene was amplified using the p414-SpAHA1plasmid as the template and the primers SpAHA1-F and SpAHA1-R (Additional file5: Table S1) and inserted into the pCAMBIA1304 and pCAMBIA1304-SpSOS1plasmids between theSpeI andEco72 I restriction sites to replace theGUS(β-glucuronidase) gene. The resulting plasmids were designated pCAMBIA1304-SpAHA1and pCAMBIA1304-SpSOS1-SpAHA1, respectively. The plasmids were all verified by sequencing.

Arabidopsistransformation and identification

The three recombinant plasmids described above were added to a 100 mM CaCl₂solution containing competentAgrobacterium tumefaciensGV3101 cells. The plasmids were then introduced into theAgrobacteriumcells via heat shock (42 °C). Finally, the above three expression cassettes were transformed intoArabidopsis thaliana(Col-0) by infecting flower buds with theAgrobacteriumcells containing the recombinant plasmids [72］.T0-generation seeds were screened initially on MS (Murashige & Skoog) medium supplemented with 50 μg/mL hygromycin B. DNA was purified from the candidate lines and used as template for PCR (polymerase chain reaction) amplification with specific primer pairs to identify the different transgenic plants. The primers are listed in Additional file5: Table S1. All the transgenic lines were furthermore verified by PCR using primers specific for thehygBmarker gene, HygB-TF and HygB-TR (Additional file5: Table S1). Total RNA was purified from the transgenic lines andSpAHA1andSpSOS1expression was assessed by RT-PCR (reverse transcription PCR) with a housekeeping gene,Actin,as an internal control. The primers for theSpAHA1(SpAHA1-RT-F and SpAHA1-RT-R),SpSOS1(SpSOS1-RT-F and SpSOS1-RT-R), andActin(Actin-RT-F and Actin-RT-F) genes are listed in Additional file5: Table S1.PCR条件如下:94°C 2米n, followed by 28 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. The resulting PCR products were assessed by agarose gel electrophoresis.

Cultivation and salt treatment of transgenic and WT plants

To analyze the salt tolerance of transgenic and WT plants, seeds from T3 homozygous transgenic lines (expressing singleSpSOS1, singleSpAHA1,and bothSpSOS1andSpAHA1) and untransformed plants were germinated on MS plates in a growth chamber (22 °C with a 16 h light / 8 h dark cycle and a light intensity of 100 μmol•m^− 2•s^− 1). After 5 days, the seedlings grown on MS plates were transferred onto MS plates containing 0, 50, 75, and 100 mM NaCl and allowed to grow for 2 weeks. Then the root length, number of lateral roots, and fresh seedling weights were measured. In addition, 10-day-old seedlings were transferred to a mixture of organic soil and sand (3:1,v/v) in pots (4 seedlings/pot) and grown in a greenhouse with long-day conditions (16 h light/8 h dark at 22 °C and a light strength of 150 μmol•m^− 2•s^− 1) for 4 weeks. The pots containing the plants were then put into water containing 0 or 200 mM NaCl. Ten days post-NaCl treatment, the treated plants were photographed and their fresh weights were determined.

Determination of Na⁺and K⁺content inArabidopsisplants

At the end of the NaCl treatment, the WT and transgenic plants were separately collected. The Na⁺and K⁺in the samples were measured using atomic absorption spectrometry as described in a previous work [31］.

Measurement of Na⁺and H⁺flux in roots

Seven-day-old uniform T3 seedlings, which had been grown on MS plates, were transferred to MS medium containing 100 mM NaCl and grown for 3 days, and then the roots of salt stressed seedlings were put into measurement buffer to balance for 10 min, after that net H⁺and Na⁺fluxes were measured in the YoungerUSA Xuyue (Beijing) BioFunction Institute by using Non-invasive Micro-test Technology (NMT100 Series, Xuyue (Beijing) Sci. & Tech. Co., Ltd., Beijing, China) Software.

H⁺, Na⁺-selective microsensors were prepared as described previously [60］.Pre-pulled and silanized microsensor (Φ4.5 ± 0.5 μm, XY-CGQ-01, YoungerUSA) were first filled with a backfilling solution (H⁺: 15 mM NaCl + 40 mM KH₂PO₄, pH 7.0; Na⁺: 250 mM NaCl) to a length of approximately 1.0 cm from the tip. Then the microsensors were front filled with 40–50 μm columns of selective liquid ion-exchanger (LIX) (H⁺: XY-SJ-H; Na⁺: XY-SJ-Na; all from YoungerUSA). An Ag/AgCl wire microsensor holder YG003-Y11 (YoungerUSA) was inserted in the back of the microsensor to make electrical contact with the electrolyte solution. YG003-Y11 (YoungerUSA) was used as the reference microsensor. Prior to the flux measurement, the microsensor were calibrated with cultural media having different concentrations of H⁺(pH 6.0 and pH 7.0) or Na⁺(5 mM and 0.5 mM), respectively. Only microsensor with a Nernstian slope >50 mV/decade were used in our study. The same microsensors were calibrated again according to the same procedure and standards after each test. After that, net ion fluxes were recorded on the root meristematic zones, 120 μm from the tip where SOS1 activity was the highest [73], in 5 mL measurement buffer (0.1 mM KCl, 0.1 mM CaCl₂, 0.1 mM MgCl₂, 0.5 mM NaCl, 0.3 mM MES (2-(N-Morpholino) ethanesulfonic acid) and 0.2 mM Na₂SO₄, pH 6.5). Net H⁺and Na⁺flux was calculated by Fick’s law of diffusion [60］.Six biological repeats were performed for each analysis.

Assays of malondialdehyde content

Two-week-old T3 transgenic and untransformedArabidopsisseedlings were grown in the presence of 200 mM NaCl for 7 days. Malondialdehyde (MDA) content in the leaves was measured using the thiobarbituric acid method previously described by Dhindsa and Matowe [74］.

Statistical analysis

Two-tailed Student’s t-tests were used to analyze the data. The results are expressed as mean ± SE and differences with aP-value < 0.05 were considered statistically significant. At least three biological replicates were performed for each experiment.

AHA:

H⁺-ATPase

AVP/VP:

Vacuolar H⁺-PPase

GUS:

β-glucuronidase

HA:

H⁺-ATPase

HO:

Haem oxygenase

KOR:

K⁺outward rectifier

MDA:

Malondialdehyde

MES:

2-(N-Morpholino) ethanesulfonic acid

MS:

Murashige & Skoog

NHX:

Na⁺/H⁺antiporter

NSCC:

Non-selective cation channels

PCR:

Polymerase chain reaction

PM:

Plasma membrane

RCD:

Radical-induced cell death

RNAi:

RNA interference

ROS:

Reactive oxygen species

RT-PCR:

Reverse transcription polymerase chain reaction

SOS:

Salt overly sensitive

WT:

Wild-type

We thank Boston Professional Group (BPG) Editing for English language editing. We also thank the reviewers and editors for helpful comments on earlier drafts of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (31660253 to XJ), the Scientific and Technological Foundation of Hainan Province (HNGDhs201502 to XJ), the Foundations of Hainan University and Shandong Normal University (hdkytg201706 to XJ and JS), and Startup funding from Hainan University (KYQD(ZR)1845 to YZ). The above funding was used for the design of the study and collection, analysis, and interpretation of data in writing the manuscript.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from corresponding authors on reasonable request.

Author notes

1. Yafei Fan and Xiaochang Yin contributed equally to this work.

Affiliations

1. Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources /Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, 570228, China

   Yafei Fan, Xiaochang Yin, Qing Xie, Youquan Xia, Zhenyu Wang, Yang Zhou & Xingyu Jiang

2. Shandong Key Laboratory of Plant Stress/College of Life Science, Shandong Normal University, Jinan, 250014, China

   Jie Song

Contributions

XJ, YZ and ZW conceived and designed the experiments. YF, XY, QX and YX performed the experiments. YZ and JS made substantial contributions to the data analysis. XJ and JS supervised, wrote and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence toYang ZhouorXingyu Jiang.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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