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.

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.

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.

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 。

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。

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.

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.

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.

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.

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.

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.

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

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.

A nanoagent for concurrent therapy of breast cancer bone metastasis and cancer-induced bone pain through SLC7A11 interruption and photodynamic therapy

Bone metastasis,a life-threatening complication of advanced breast cancer,is often accompanied by debilitating pain(cancer-induced bone pain,CIBP)that severely impairs life quality and survival.The concurrent treatment of bone metastases and CIBP remains a clinical challenge because the therapeutic options are limited.In this study,we construct a near-infrared light-activated nano-therapeutic system to meet this conundrum.In detail,sorafenib(SRF)and photosensitizer(chlorin e6,Ce6)are encapsulated into mesoporous hydroxyapatite nanoparticles(HANPs),which are further functionalized with hyaluronic acid(HA)to obtain HA-SRF/Ce6@HANPs system.The designed nanoplatform destroys tumor cells in vitro and in vivo via the synergism of SRF(interrupting the exchange of cystine/glutamate by inhibiting SLC7A11)and photodynamic therapy(PDT,inducing reactive oxygen species generation).The decrease in tumor burden and reduction of extracellular glutamate significantly attenuate CIBP in mice model with developing bone cancer.Moreover,the combination of HA-SRF/Ce6@HANPs and PDT inhibit osteoclasts activation,promote osteoblast differentiation and accelerate bone repair.Overall,the nanoagent with good biocompatibility may provide an effective therapy method for the concurrent treatment of breast cancer bone metastasis and CIBP.

High-level production of𝛾γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system in Escherichia coli

Biosynthesis of the functional factor𝛾γ-aminobutyric acid(GABA)in bacteria involves two key proteins an intra-cellular glutamate decarboxylase(GadB)and a membrane-bound antiporter(GadC).Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA productivity.In this study,gadBΔC11 of Lacti-plantibacillus plantarum and gadCΔC41 of Escherichia coli were inserted into the designed double promoter(P T7lac and P BAD)expression system.Then,E.coli Lemo21(DE3)was chosen as the host to minimize the toxic effects of GadCΔC41 overexpression.Furthermore,a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3)cells as biocatalysts was developed.The total GABA yield reached 829.08 g/L with a 98.7%conversion ratio within 13 h,when engineered E.coli Lemo21(DE3)cells were concentrated to an OD 600 of 20 and reused for three cycles in a 3 M L-glutamate solution at 37℃,which represented the highest GABA productivity ever reported.Overall,expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in E.coli.

甘蓝型油菜钙离子转运蛋白CAX家族基因生物信息学及其对镉胁迫响应表达分析

CAXs(Cation/H+ exchanger antiporter)是一类重要的阳离子跨膜转运蛋白,在调控植物Ca2+平衡,参与转运金属离子Cd2+和Mn2+中发挥了重要作用,但在甘蓝型油菜中尚缺乏系统研究。本文利用生物信息学的方法对甘蓝型油菜CAX家族成员的基因拷贝数、系统进化、进化选择压力、分子特征、保守基序、染色体定位、基因结构及其启动子区域所能结合的顺式作用元件进行了预测和分析;同时采用荧光定量PCR分析了甘蓝型油菜BnaCAXs的组织表达模式及其对Cd2+胁迫的响应。结果表明,甘蓝型油菜CAX家族共包含17个成员,系统进化分析结果表明BnaCAXs与拟南芥进化相似,并且明显地分为5个亚家族。BnaCAXs家族所有基因成员的Ka/Ks值均小于0.3,受到强烈的纯化选择作用。大部分BnaCAXs均属于稳定的疏水蛋白,并包含8~11个跨膜结构域。基因结构差异明显,内含子数目从6~11不等,并且Dof、MYB以及W-BOX是其启动子上丰度较大的顺式作用元件,可能参与到BnaCAX抗逆过程的调控。荧光定量PCR结果表明,甘蓝型油菜CAX基因主要在地下部表达;BnaC4.CAX1-2和BnaC3.CAX2-2是该家族的核心基因,并受到Cd2+胁迫的显著诱导。