渗透胁迫调节基因——Na^+/H^+ Antiporter基因与植物耐盐性

Na+/H+Antiporter基因与植物耐盐性密切相关,其编码产物Na+/H+逆向转运蛋白通过Na+外排和Na+区隔化来维持植物细胞内较低的Na+水平,降低Na+的毒害,从而对植物的耐盐性起重要作用。

Involvement of Plasma Membrane Ca^(2+)/H^+ Antiporter in Cd^(2+) Tolerance

Cation exchangers （CAXs） belong to the cation/Ca2＋exchanger superfamily which have been extensively investigated in plant tonoplasts over the last decade. Recently, the roles of CAXs involved in heavy metal accumulation and tolerance in plants have been studied for phytoremediation and food security. In this mini review, we summarize the roles of the Ca2＋/H＋ antiporter in Ca2＋ signal transduction, maintaining ion homeostasis and sequestering heavy metals into the vacuole. Moreover, we present a possible role of the plasma membrane Ca2＋/H＋ antiporter in heavy metal detoxification.

Whole-genome identification and expression analysis of K^+ efflux antiporter(KEA) and Na^+/H^+ antiporter(NHX) families under abiotic stress in soybean

Sodium toxicity and potassium insufficient are important factors affecting the growth and development of soybean in saline soil. As the capacity of plants to maintain a high cytosolic, K^＋/Na^＋ ratio is the key determinant of tolerance under salt stress. The aims of the present study were to identify and analyse expression patterns of the soybean K^＋ efflux antiporter（KEA） gene and Na^＋/H^＋ antiporter（NHX） gene family, and to explore their roles under abiotic stress. As a result, 12 soybean Gm KEAs genes and 10 soybean Gm NHXs genes were identified and analyzed from soybean genome. Interestingly, the novel soybean KEA gene Glyma16g32821 which encodes 11 transmembrane domains were extremely up-regulated and remained high level until 48 h in root after the excessive potassium treatment and lack of potassium treatment, respectively. The novel soybean NHX gene Glyma09g02130 which encodes 10 transmembrane domains were extremely up-regulated and remained high level until 48 h in root with Na Cl stress. Imaging of subcellular locations of the two new Glyma16g32821-GFP and Glyma09g02130-GFP fusion proteins indicated all plasma membrane localizations of the two novel soybean genes. The 3D structures indicated that the two soybean novel proteins Glyma09g02130（NHX） and Glyma16g32821（KEA） all belong to the cation/hydrogen antiporter family.

Role of Na^+/H^+ antiporter in pulmonary artery smooth muscle of hypoxic pulmonary hypertension in the rat

Objective: To investigate the role of Na+ /H+ antiporter in the hypoxic pumonary hypertension ofrats. Methods: Thirty Wistar rats were randomly divided into 3 groups with 10 in each group: controlgroup, 3--week hypoxia group and 8--week hypoxia group. After the isolation of pulmonary artery smoothmuscles, pHi was determined by fluorescence measurement of the pH--sensitive dye BCECF and theexpression of NHE--1 mRNA was detected with reverse transcription--polymerase chain reaction. Results: ThepHi and expression of NHE-1 mRNA of pulmonary artery smooth muscle cell in the hypoxia groups weresignificantly increased than those in the normal group (P < 0. 01 ). There was no remarkable differencebetween the hypoxia groups. Conclusion: With the function of regulation pHi., NHE--1 may play an importantrole in the pulmonary vascular remodeling of pulmonary hypertension. The result provides a new therapeuticmethod with NHE--1 inhibitors and/or gene therapy for the hypoxic pulmonary hypertension.

Effect of salt stress on the expression of NHX-type Na^+/H^+ antiporters in Populus euphratica and P.pruinosa calli

Populus euphratica and Populuspruinosa, sister species in the Turanga Section （Salicaceae）, growing in semi-arid saline areas are known for their high salinity tolerance. In this study, by combining growth level with Na＋ and K＋ contents, the expression level of vacuolar Na＋/H＋ antiporters was investigated for NaCl-induced changes in P. euphratica and t3. pru- inosa calli. Compared to R euphratica, P. pruinosa calli grew well in 200 mM NaC1 stress from 14. to 21 days. Increasing the stressed time caused an increase in Na＋ content concomitant with a decrease in K＋ content in P. euphratica calli, whereas, with the presence of 200 mM NaCI, K＋ content has a less increase in 14 and 21 days than in 7 days which was detected in R pruinosa calli. The transcript levels of six genes coding for NHX-type Na＋/H＋ antiporters suggest that vacuolar NHX1-NHX6 antiporters play important roles in responding to salt stress in R pruinosa. Our data suggest that there exists a higher salt tolerance for P. pruinosa than P. euphratica at the cellular level, Na＋ avoidance or accumulation is observed in cellular compartments, and that expression of NHX antiporters is linked to the accumulator phenotype.

Cloning of Three Antiporter Genes from Arabidopsis and Rice for Over-Expressing Them in Farmer Popular Tomato Varieties of Bangladesh

Salinity is one of the most critical environmental problems, which causes plant growth retardation by disturbing intracellular ion homeostasis. The Na+/H+ antiporter plays an important role in resistance to salt stress by sequestering Na+ in exchange for H+ across the vacuolar membranes. In the current study, the coding regions of two Arabidopsis antiporters (AtNHX1 and AtNHX2) and one rice antiporter (OsNHX1) were amplified by target specific PCR. PCR amplicons were first cloned into pENTR/D-TOPO and later recombined with a destination vector (pK7WG2.0) by LR reaction. Positive clones were selected by PCR, restriction digestion (RD) and sequencing. They were then transformed into Agrobacterium tumefaciens (LBA4404 strain) for subsequent transformation of farmer popular tomato varieties.

Plastid KEA-type cation/H+ antiporters are required for vacuolar protein trafficking in Arabidopsis

Arabidopsis plastid antiporters KEA1 and KEA2are critical for plastid development, photosynthetic efficiency, and plant development.Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles(PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1(VSR1) subcellular localizations, VSR–cargo interactions, and p24 distribution on the endoplasmic reticulum(ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K+homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K+homeostasis.

Dynamic changes of phosphatidylinositol and phosphatidylinositol 4-phosphate levels modulate H^(+)-ATPase and Na^(+)/H^(+) antiporter activities to maintain ion homeostasis in Arabidopsis under salt stress

Plant metabolites are dynamically modified and distributed in response to environmental changes.How-ever,it is poorly understood how metabolic change functions in plant stress responses.Maintaining ion ho-meostasis under salt stress requires coordinated activation of two types of central regulators:plasma membrane(PM)H^(+)-ATPase and Na^(+)/H^(+) antiporter.In this study,we used a bioassay-guided isolation approach to identify endogenous small molecules that affect PM H^(+)-ATPase and Na^(+)/H^(+) antiporter activities and identified phosphatidylinositol(PI),which inhibits PM H^(+)-ATPase activity under non-stress conditions in Arabidopsis by directly binding to the C terminus of the PM H^(+)-ATPase AHA2.Under salt stress,the phosphatidylinositol 4-phosphate-to-phosphatidylinositol(PI4P-to-PI)ratio increased,and PI4P bound and activated the PM Na^(+)/H^(+) antiporter.PI prefers binding to the inactive form of PM H^(+)-ATPase,while PI4P tends to bind to the active form of the Na^(+)/H^(+) antiporter.Consistent with this,pis1 mutants,with reduced levels of PI,displayed increased PM H^(+)-ATPase activity and salt stress toler-ance,while the pi4kβ1 mutant,with reduced levels of PI4P,displayed reduced PM Na^(+)/H^(+) antiporter activity and salt stress tolerance.Collectively,our results reveal that the dynamic change between PI and PI4P in response to salt stress in Arabidopsis is crucial for maintaining ion homeostasis to protect plants from un-favorable environmental conditions.

Plant and Yeast NHX Antiporters: Roles in Membrane Trafficking

The plant NHX gene family encodes Na＋/H＋ antiporters which are crucial for salt tolerance, potassium homeostasis and cellular pH regulation. Understanding the role of NHX antiporters in membrane trafficking is becoming an increasingly interesting subject of study. Membrane trafficking is a central cellular process during which proteins, lipids and polysaccharides are continuously exchanged among membrane compartments. Yeast ScNhxlp, a prevacuole/ vacuolar Na＋/H＋ antiporter, plays an important role in regulating pH to control trafficking out of the endosome. Evidence begins to accumulate that plant NHX antiporters might function in regulating membrane trafficking in plants.

Ectopic Expression of a Bacterium NhaD-type Na^+/H^+ Antiporter Leads to Increased Tolerance to Combined Salt/Alkali Stresses

AaNhaD, a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica, encodes a Na＋/H＋ antiporter crucial for the bacterium＇s resistance to salt/alkali stresses. However, it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses. To investigate the use of extremophile genetic resources in higher plants, transgenic tobacco BY-2 cells and plants harboring AaNhaDwere generated and their stress tolerance was evaluated. Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner. Compared to wild-type controls, the transgenic cells exhibited increased Na＋ concentrations and pH levels in the vacuoles. Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts. Similar to the transgenic BY-2 cells, AaNhaD.overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil. These results indicate that AaNhaD functions as a pH-dependent tonoplast Na＋/H＋ antiporter in plant cells, thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.

A Putative Chloroplast-Localized Ca2＋/H＋ Antiporter CCHA1 Is Involved in Calcium and pH Homeostasis and Required for PSII Function in Arabidopsis

Calcium is important for chloroplast, not only in its photosynthetic but also nonphotosynthetic functions. Mul- tiple Ca2＋/H＋ transporters and channels have been described and studied in the plasma membrane and organ- elle membranes of plant cells; however, the molecular identity and physiological roles of chloroplast Ca2＋/H＋ antiporters have remained unknown. Here we report the identification and characterization of a member of the UPFO016 family, CCHA1 （a chloroplast-localized potential Ca2＋/H＋ antiporter）, in Arabidopsis thaliana. We observed that the ccha I mutant plants developed pale green leaves and showed severely stunted growth along with impaired photosystem II （PSII） function. CCHA1 localizes to the chloroplasts, and the levels of the PSII core subunits and the oxygen-evolving complex were significantly decreased in the ccha I mutants compared with the wild type. In high Ca2＋ concentrations, Arabidopsis CCHA1 partially rescued the growth defect of yeast gdtl3 null mutant, which is defective in a Ca2＋/H＋ antiporter. The cchal mutant plants also showed significant sensitivity to high concentrations of CaCI2 and MnCI2, as well as variation in pH. Taken these results together, we propose that CCHA 1 might encode a putative chloroplast-localized Ca2＋/H＋ antiporter with critical functions in the regulation of PSII and in chloroplast Ca2＋ and pH homeostasis in Arabidopsis.

DL-3-n-butylphthalide alleviates motor disturbance by suppressing ferroptosis in a rat model of Parkinson’s disease

DL-3-n-butylphthalide(NBP)-a compound isolated from Apium graveolens seeds-is protective against brain ischemia via various mechanisms in humans and has been approved for treatment of acute ischemic stroke.NBP has shown recent potential as a treatment for Parkinson’s disease.However,the underlying mechanism of action of NBP remains poorly understood.In this study,we established a rat model of Parkinson’s disease by intraperitoneal injection of rotenone for 28 successive days,followed by intragastric injection of NBP for 14-28 days.We found that NBP greatly alleviated rotenone-induced motor disturbance in the rat model of Parkinson’s disease,inhibited loss of dopaminergic neurons and aggregation ofα-synuclein,and reduced iron deposition in the substantia nigra and iron content in serum.These changes were achieved by alterations in the expression of the iron metabolism-related proteins transferrin receptor,ferritin light chain,and transferrin 1.NBP also inhibited oxidative stress in the substantia nigra and protected mitochondria in the rat model of Parkinson’s disease.Our findings suggest that NBP alleviates motor disturbance by inhibition of iron deposition,oxidative stress,and ferroptosis in the substantia nigra.

利用转基因技术创造甜菜耐盐新种质

采用农杆菌介导法将来源于E coli的胆碱脱氢酶 (CDH)基因 (betA)和来源于拟南芥的Na+ /H+antiport基因分别转入 5个糖甜菜品种的丛生芽细胞 ,获得了转基因植株。在耐盐性筛选中 ,转基因植株存活率高 ,部分植株生长较好 ,表现出比对照植株明显提高的耐盐性。在 2 %～ 3%NaCl胁迫下 ,少数转基因植株仍生长正常 ,显示出高耐盐性。本研究表明 ,转入在不同盐胁迫反应途径中起关键作用的酶基因均可提高甜菜耐盐性 。

Neuroprotective effect of deferoxamine on erastin-induced ferroptosis in primary cortical neurons

The iron chelator deferoxamine has been shown to inhibit ferroptosis in spinal cord injury.However,it is unclear whether deferoxamine directly protects neurons from ferroptotic cell death.By comparing the survival rate and morphology of primary neurons and SH-SY5Y cells exposed to erastin,it was found that these cell types respond differentially to the duration and concentration of erastin treatment.Therefore,we studied the mechanisms of ferroptosis using primary cortical neurons from E16 mouse embryos.After treatment with 50μM erastin for 48 hours,reactive oxygen species levels increased,and the expression of the cystine/glutamate antiporter system light chain and glutathione peroxidase 4 decreased.Pretreatment with deferoxamine for 12 hours inhibited these changes,reduced cell death,and ameliorated cellular morphology.Pretreatment with the apoptosis inhibitor Z-DEVD-FMK or the necroptosis inhibitor necrostain-1 for 12 hours did not protect against erastin-induced ferroptosis.Only deferoxamine protected the primary cortical neurons from ferroptosis induced by erastin,confirming the specificity of the in vitro ferroptosis model.This study was approved by the Animal Ethics Committee at the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences,China(approval No.DWLL-20180913)on September 13,2018.

Overexpression of the Suaeda salsa SsNHX1 gene confers enhanced salt and drought tolerance to transgenic Zea mays

Maize is one of the most important crops worldwide, but it suffers from salt stress when grown in saline-alkaline soil. There is therefore an urgent need to improve maize salt tolerance and crop yield. In this study, the SsNHX1 gene of Suaeda salsa, which encodes a vacuolar membrane Na~+/H~+ antiporter, was transformed into the maize inbred line 18-599 by Agrobacterium-mediated transformation. Transgenic maize plants overexpressing the SsNHX1 gene showed less growth retardation when treated with an increasing NaCl gradient of up to 1%, indicating enhanced salt tolerance. The improved salt tolerance of transgenic plants was also demonstrated by a significantly elevated seed germination rate(79%) and a reduction in seminal root length inhibition. Moreover, transgenic plants under salt stress exhibited less physiological damage. SsNHX1-overexpressing transgenic maize accumulated more Na~+ and K~+ than wild-type(WT) plants particularly in the leaves, resulting in a higher ratio of K~+/Na~+ in the leaves under salt stress. This result revealed that the improved salt tolerance of SsNHX1-overexpressing transgenic maize plants was likely attributed to SsNHX1-mediated localization of Na~+ to vacuoles and subsequent maintenance of the cytosolic ionic balance. In addition, SsNHX1 overexpression also improved the drought tolerance of the transgenic maize plants, as rehydrated transgenic plants were restored to normal growth while WT plants did not grow normally after dehydration treatment. Therefore, based on our engineering approach, SsNHX1 represents a promising candidate gene for improving the salt and drought tolerance of maize and other crops.

Comparative Study on Growth Performance of Transgenic(Over-Expressed OsNHX1) and Wild-Type Nipponbare under Different Salinity Regimes

Transgenic Nipponbare which over-expressed a Na＋/H~ antiporter gene OsNHX1 was used to compare its growth performance, water status and photosynthetic efficiency with its wild type under varying salinity regimes. Chlorophyll content, quantum yield and photosynthetic rate were measured to assess the impact of salinity stress on photosynthetic efficiency for transgenic and wild-type Nipponbare. Effects of salinity on water status and gas exchange to both lines were studied by measuring water use efficiency, instantaneous transpiration rate and stomatal conductance. Dry shoot weight and leaf area were determined after three months of growth to assess the impacts of salinity on the growth of those two lines. Our study showed that both lines were affected by salinity stress, however, the transgenic line showed higher photosynthetic efficiency, better utilization of water, and better growth due to low transpiration rate and stomatal conductance. Reduction of photosynthetic efficiency exhibited by the wild-type Nipponbare was correlated to its poor growth under salinity stress.

Differential expression of PeSOS1, PeNhaD1 and PeNHX1 genes between the salt-tolerant Populus euphratica and salt-sensitive P. hopeiensis calli

Populus euphratica Oliv. is of high salinity tolerance and used as a model species for investigating molecular mechanisms of trees＇ responses to salt stress. In the work presented here we found that calli of P. euphratica grew more rapidly and accumulated less Na＋, but more K＋, under salt stress than those of salt-sensitive poplar, Populus hopeiensis. Different types of Na＋/H＋ antiporters （SOS1, NhaD1 and NHX1） were isolated from P. euphratica; all of these genes have been shown to play important roles in plant salt tolerance mechanism in previous studies. Expression profiles of these three genes were compared between P. hopeiensis and P. euphratica in the presence and absence of salt stress by real-time PCR. The three genes were induced in both P. euphratica and P. hopeiensis by salt. Transcript levels of PeNHX1 were lower in P. euphratica than in P. hopeiensis under 150 mM NaCl stress. In addition, transcript levels of PeNhaD1 were lower, while PeSOS1 were higher in P. euphratica than in P. hopeiensis under both stressed and unstressed conditions. The results indicated that P. euphratica up-regulates different genes and consistently maintains both effluxes of Na＋ and high K＋ levels. Our data suggests that differences in gene expression patterns may contribute to the dif-ference in salt tolerance between these two poplars.

Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na^+/H^+Antiport Activity of Three Malus species with Different Salt Tolerance

Salt stress contains osmotic and ionic stress, while iso-osmotic polyethylene glycol （PEG） has only osmotic stress. This study aimed to compare the different effects on the activity of H＋-ATPase, proton pump and Na＋/H＋antiport in Malus seedlings between osmotic and ionic stress. Species of salt tolerant Malus zumi, middle salt tolerant Malus xiaojinensis and salt sensitive Malus baccata were used as experimental materials. Malus seedlings were treated with NaCl and iso-osmotic PEG stress. The activity of H＋-ATPase, proton pump and Na＋/H＋antiport of plasmolemma and tonoplast in Malus seedlings were obviously increased under salt stress, and those in salt-tolerant species increased more. Under the same NaCl concentration, the activity of H＋-ATPase, proton pump and Na＋/H＋antiport of plasmolemma and tonoplast in salt-tolerant species were all obviously higher than those in salt-sensitive one. Higher Na＋/H＋antiport activity of plasmolemma and tonoplast in salt-tolerant species could help to extrude and compartmentalize sodium in roots under salt stress. The ascent rate of activity of H＋-ATPase, proton pump and Na＋/H＋antiport in Malus seedlings under the three salt concentration stress was all obviously higher than that under the iso-osmotic PEG stress. It indicated that the sodium ion effect had more stimulation on the activity of H＋-ATPase, proton pump and Na＋/H＋antiport in salt-tolerant species, and salt-tolerant species has higher capability of sodium extrusion and compartmentalization in roots and is therefore more salt tolerant.

Cloning of Na^+/H^+ antiport gene from Dunaliella salina

1 Introduction Dunaliella Salina,which taxi Dunaliella,Volvocales,Chlorophyceae Chlorophyta,is unicell algae with double flagllum at top,and cup shaped chloroplast without cell wall.Dunaliella Salina is the most salt tolerance eucaryotes.It can grow at the range of salt concentration