Salt Tolerance In Transgenic Plants Research Articles

MdWRKY71 as a positive regulator involved in 5-aminolevulinic acid-induced salt tolerance in apple

5-Aminolevulinic acid (ALA), is a novel plant growth regulator that can enhance plant tolerance against salt stress. However, the molecular mechanism of ALA is not well studied. In this study, ALA improved salt tolerance of apple (Malus × domestica 'Gala') when the detached leaves or cultured calli were used as the materials. The expression of MdWRKY71, a WRKY transcription factor (TF) gene was found to be responsive to NaCl as well as ALA treatment. Functional analysis showed that overexpressing (OE)-MdWRKY71 significantly improved the salt tolerance of the transgenic apple, while RNA interfering (RNAi)-MdWRKY71 reduced the salt tolerance. However, exogenous ALA alleviated the salt damage in the RNAi-MdWRKY71 apple. When MdWRKY71 was transferred into tobacco, the salt tolerance of transgenic plants was enhanced, which was further improved by exogenous ALA. Subsequently, MdWRKY71 bound to the W-box of promoters of MdSOS2, MdNHX1, MdCLC-g, MdSOD1, MdCAT1 and MdAPX1, transcriptionally activating the gene expressions. Since the genes are responsible for Na+ and Cl− transport and antioxidant enzyme activity respectively, it can be concluded that MdWRKY71, a new TF, is involved in ALA-improved salt tolerance by regulating ion homeostasis and redox homeostasis. These results provided new insights into the transcriptional regulatory mechanism of ALA in enhancing apple salt tolerance.

Verification of the peanut vitamin C synthesis-related gene AhPMM and its role in stress resistance

Vitamin C plays an important role in plant antioxidation, photosynthesis, growth and development, and metabolism. In this study, a gene AhPMM, which is involved in vitamin C synthesis and responds significantly to low temperature, NaCl, polyethylene glycol (PEG) and abscisic acid (ABA) treatments, was cloned from peanut. An AhPMM overexpression vector was constructed, and transferred to a peanut variety Junanxiaohong using the pollen tube injection method. PCR test on the T3 generation transgenic peanut plants showed a transgenics positive rate of 42.3%. HPLC was used to determine the content of reducing vitamin C (AsA) and total vitamin C in the leaves of transgenic plants. The results showed that the content of AsA in some lines increased significantly, up to 1.90 times higher than that of the control, and the total vitamin content increased by up to 1.63 times compared to that of the control. NaCl and ABA tolerance tests were carried out on transgenic seeds. The results showed that the salt tolerance of transgenic seeds was significantly enhanced and the sensitivity to ABA was weakened compared to that of the non-transgenic control. Moreover, the salt tolerance of the transgenic plants was also significantly enhanced compared to that of the non-transgenic control. The above results showed that AhPMM gene not only increased the vitamin C content of peanut, but also increased the salt tolerance of transgenic peanut seeds and plants. This study may provide a genetic source for the molecular breeding of peanut for enhanced salt tolerance.

Overexpression of high affinity K+ transporter from Nitraria sibirica enhanced salt tolerance of transgenic plants

Nitraria sibirica Pall is a halophytic shrub growing in desert steppe zones. It exhibits extraordinary adaptability to saline–alkali soil, drought, and sand burial. In this study, the high-affinity K+ transporter NsHKT1 was identified and found to play a key role in salt tolerance in N. sibirica. NsHKT1 was used to improve salt tolerance in a poplar hybrid. The expression characteristics of NsHKT1 were analyzed by transforming Arabidopsis and poplar with the β-glucuronidase (GUS) gene driven by the NsHKT1 promoter. The results showed that NsHKT1 expression was induced by various abiotic stresses and phytohormones. GUS expression was also detected in the reproductive organs of transgenic Arabidopsis, indicating its function in regulating plant reproductive growth. Transgenic 84 K poplar plants overexpressing NsHKT1 exhibited less damage, higher antioxidant capacity, higher chlorophyll and proline levels, and lower malondialdehyde content compared with non-transgenic plants under salt stress. These results are consistent with the salt tolerance results for transgenic Arabidopsis overexpressing NsHKT1, indicating that NsHKT1 plays a key role in salt tolerance in herbaceous and ligneous plants. Inductively coupled plasma–optical emission spectrometry showed a significantly lower leaf Na+ content in transgenic poplar than in the non-transgenic line, revealing that NsHKT1, as a member of HKT family subclass 1, was highly selective to Na+ and prevented shoot Na+ accumulation. Transcriptome analysis indicated that differentially expressed genes in transgenic poplars under salt stress were associated mainly with the isoflavonoid, cutin, suberine, wax, anthocyanin, flavonoid, and cyanoamino biosynthesis pathways, as well as the MAPK signaling pathway, indicating that NsHKT1 not only regulates ion homeostasis but also influences secondary metabolism and signal transaction in transgenic plants.

Genome-wide identification and expression analysis of the NHX gene family under salt stress in wheat (Triticum aestivum L).

Salt stress affects plant growth and development, resulting in the loss of crop yield across the world, and sodium-proton antiporters (NHXs) are one of the genes known to promote salt tolerance in transgenic plants. In this study, we conducted a comprehensive genome-wide analysis and expression profile of NHX genes in wheat under salinity stress. We identified 30 TaNHX genes in wheat based on the Na+/H+ exchanger domain, with all genes containing an amiloride motif except one, a known for inhibiting Na+ ions in plants. Phylogenetic analysis classified these genes into three classes with subfamilies: 12 were localized in vacuoles, while 18 were in the endoplasmic reticulum and plasma membrane. Promoter analysis revealed stress-related cis-acting elements, indicating their potential role in abiotic stress tolerance. The non-synonymous (Ka)/synonymous (Ks) ratios highlighted that the majority of TaNHX genes experienced robust purifying selection throughout their evolutionary history. Transcriptomis data analysis and qRT-PCR demonstrated distinct expression patterns for TaNHX genes across various tissues when subjected to salt stress. Additionally, we predicted 20 different miRNA candidates targeting the identified TaNHX genes. Protein-protein interaction prediction revealed NHX6's involvement in the SOS1 pathway, while NHX1 gene exhibit proton antiporter activity. Molecular dynamics (MD) simulations were also conducted to examine the interactions of TaNHX1, TaNHX2, and TaNHX3. These results represent a significant advancement in our understanding of the molecular mechanisms governing Na+ transporters. This may also offer promising avenues for future studies aimed at unraveling the intricate details of their biological roles and applications.

Wheat WRKY transcription factor TaWRKY24 confers drought and salt tolerance in transgenic plants

Drought and salt stress are major environmental conditions that severely limit plant growth and productivity. WRKY transcription factors play a vital role in the responses against biotic or abiotic stress. In this study, TaWRKY24, a gene of the IIe WRKY family identified in wheat, was cloned and characterized. TaWRKY24 was mainly expressed in wheat leaf and stem and induced by treatment with PEG6000, salt, H2O2, ABA, MeJA, and ethrel. TaWRKY24 transient expression in onion epidermal cells suggested its nuclear localization and its transcriptional activation capability characteristics. Overexpression of TaWRKY24 in tobacco improved the seed germination rate and root growth of seedlings in transgenic lines when subjected to higher mannitol and NaCl concentrations. Further research showed that transgenic lines had higher proline and soluble sugars and lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Moreover, compared to normal and negative control plants, TaWRKY24 silenced wheat seedlings had reduced growth under salt and drought stress. This study shows that wheat TaWRKY24 is crucial to plant stress, providing an excellent candidate gene for wheat resistance breeding.

Overexpression of the Purple Perilla (Perilla frutescens (L.)) FAD3a Gene Enhances Salt Tolerance in Soybean.

The increasingly serious trend of soil salinization inhibits the normal growth and development of soybeans, leading to reduced yields and a serious threat to global crop production. Microsomal ω-3 fatty acid desaturase encoded by the FAD3 gene is a plant enzyme that plays a significant role in α-linolenic acid synthesis via regulating the membrane fluidity to better accommodate various abiotic stresses. In this study, PfFAD3a was isolated from perilla and overexpressed in soybeans driven by CaMV P35S, and the salt tolerance of transgenic plants was then evaluated. The results showed that overexpression of PfFAD3a increased the expression of PfFAD3a in both the leaves and seeds of transgenic soybean plants, and α-linolenic acid content also significantly increased; hence, it was shown to significantly enhance the salt tolerance of transgenic plants. Physiological and biochemical analysis showed that overexpression of PfFAD3a increased the relative chlorophyll content and PSII maximum photochemical efficiency of transgenic soybean plants under salt stress; meanwhile, a decreased accumulation of MDA, H2O2, and O2•-, increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbic acid peroxidase (APX), as well as the production of proline and soluble sugar. In summary, the overexpression of PfFAD3a may enhance the salt tolerance in transgenic soybean plants through enhanced membrane fluidity and through the antioxidant capacity induced by C18:3.

EARLY FLOWERING3 Gene Confers Earlier Flowering and Enhancement of Salt Tolerance in Woody Plant Osmanthus fragrans

Osmanthus fragrans Lour. is popular in landscaping and gardening in Asia. In recent years, growing attention has been given to evergreen tree flowering and adaptation. EARLY FLOWERING3 (ELF3) plays an essential role in plant flowering regulation and abiotic stress tolerance. However, there is very little known about how the ELF3 gene affects flowering time and salt tolerance in O. fragrans. To elucidate the potential role of the flowering-related gene ELF3 in responding to salt tolerance, a significantly upregulated gene OfELF3 was obtained by RNA sequencing (RNA-seq) after salt treatment in O. fragrans. Our results showed that OfELF3 is a nuclear protein, which did not have a transcriptional activation ability. OfELF3 accumulation was determined in different tissues and the differentiation process of floral buds by qRT–PCR, and the gene was also significantly induced by salt stress treatment. In addition, overexpression of OfELF3 accelerated the flowering time of transgenic Arabidopsis lines, and an increase in the expression of flowering integrators such as AtFT, AtSOC1, and AtAP1 was investigated. Moreover, OfELF3 overexpression significantly improved the salt tolerance of transgenic plants, seed germination and root length of transgenic plants and was superior to those of the wild type (WT) under NaCl treatment at 4 days post-germination and the 5-day-old seedling stage, respectively. Similarly, phenotype and physiological indexes (REL, MDA and soluble protein) of 3-week-old transgenic plants were superior to the WT plants as well. Together, our results suggest that OfELF3 is not only a positive regulator in the regulation of flowering but is also involved in the salt tolerance response in O. fragrans.

The phenylcoumaran benzylic ether reductase gene PtPCBER improves the salt tolerance of transgenic poplar through lignan-mediated reactive oxygen species scavenging

Phenylcoumaran benzylic ether reductase (PCBER) in the phenylpropane metabolic pathway plays a crucial role in controlling plant growth and development. However, its biological function in response to abiotic stress in perennial trees is still largely obscure. In this work, a phenylcoumaran benzylic ether reductase gene, PtPCBER , isolated from poplar was overexpressed in poplar. The growth and salt resistance of transgenic plants were investigated, and the possible regulatory mechanism of PtPCBER in response to abiotic stresses was verified. PtPCBER was constitutively expressed in various tissues and organs with a predominant expression in xylems. Overexpression of PtPCBER augmented the salt and oxidative stress tolerance of transgenic poplar plants. The increased salt tolerance was associated with a relatively lower chlorphyll loss and Na + accumulation, and a higher antioxidant enzyme activity and stress gene expression, in the leaves of transgenic plants. Further metabolomic analyses revealed that overexpression of PtPCBER cut down the accumulation of coniferyl alcohols and lignans which function in the reactive oxygen species (ROS) scavenging pathway to alleviate the damage caused by oxidative stress in the leaves of transgenic plants. Taken together, our results suggest that PtPCBER has an important function in plant response to salt stress, and it could be used as an ideal candidate gene for the genetic engineering of woody plants with enhance resistance to multiple abiotic stresses through improving the scavenging capacity of ROS. • Overexpression of PtPCBER promoted salt tolerance in transgenic poplar. • The salt tolerance of transgenic plants was associated with ion homeostasis changes. • The reactive oxygen species scavenging capacity was increased in transgenic poplar.

Co-overexpression of AVP1, PP2A-C5, and AtCLCc in Arabidopsis thaliana greatly increases tolerance to salt and drought stresses

Abiotic stresses such as salinity and drought impose a severe constraint on global food production, posing a serious challenge in agriculture. Stacking beneficial genes in transgenic crops will likely improve crop yield under abiotic stress conditions. Previous studies showed that individually overexpressing the Arabidopsis vacuolar H + -pyrophosphatase gene AVP1 , the protein phosphatase 2 A catalytic subunit gene PP2A-C5, and the chloride channel protein gene AtCLCc contributed to enhanced salt tolerance and overexpression of AVP1 alone could also improve drought tolerance. We hypothesized that co-overexpressing AVP1 , PP2A-C5, and AtCLCc, would combine the benefits of these three genes, leading to a further increase in salt tolerance in transgenic plants due to the potential synergism of these genes. Indeed, co-overexpression of these three genes in Arabidopsis significantly improved salt and drought tolerance under single as well as under combined salt and drought stresses. The AVP1 / PP2A-C5/AtCLCc co-overexpressing plants displayed robust growth and produced greater amount of biomass as well as viable seeds than wild-type and single gene overexpression plants under saline and drought conditions. This study demonstrates that successful co-overexpression of several well-chosen genes is an effective strategy to achieve greater abiotic stress tolerance and could potentially lead to higher crop yield in regions of the world with saline soil and low precipitation. • Co-overexpression of genes that function synergistically in plant cells could greatly increase salt tolerance in transgenic plants. • This research provides an effective strategy to achieve greater abiotic stress tolerance in plants. • This approach could potentially lead to higher crop yield in semiarid and arid regions of the world

Populus euphratica Phospholipase Dδ Increases Salt Tolerance by Regulating K+/Na+ and ROS Homeostasis in Arabidopsis.

Phospholipase Dα (PLDα), which produces signaling molecules phosphatidic acid (PA), has been shown to play a critical role in plants adapting to salt environments. However, it is unclear whether phospholipase Dδ (PLDδ) can mediate the salt response in higher plants. PePLDδ was isolated from salt-resistant Populus euphratica and transferred to Arabidopsis thaliana to testify the salt tolerance of transgenic plants. The NaCl treatment (130 mM) reduced the root growth and whole-plant fresh weight of wild-type (WT) A. thaliana, vector controls (VC) and PePLDδ-overexpressed lines, although a less pronounced effect was observed in transgenic plants. Under salt treatment, PePLDδ-transgenic Arabidopsis exhibited lower electrolyte leakage, malondialdehyde content and H2O2 levels than WT and VC, resulting from the activated antioxidant enzymes and upregulated transcripts of genes encoding superoxide dismutase, ascorbic acid peroxidase and peroxidase. In addition, PePLDδ-overexpressed plants increased the transcription of genes encoding the plasma membrane Na+/H+ antiporter (AtSOS1) and H+-ATPase (AtAHA2), which enabled transgenic plants to proceed with Na+ extrusion and reduce K+ loss under salinity. The capacity to regulate reactive oxygen species (ROS) and K+/Na+ homeostasis was associated with the abundance of specific PA species in plants overexpressing PePLDδ. PePLDδ-transgenic plants retained a typically higher abundance of PA species, 34:2 (16:0–18:2), 34:3 (16:0–18:3), 36:4 (18:2–18:2), 36:5 (18:2–18:3) and 36:6 (18:3–18:3), under control and saline conditions. It is noteworthy that PA species 34:2 (16:0–18:2), 34:3 (16:0–18:3), 36:4 (18:2–18:2) and 36:5 (18:2–18:3) markedly increased in response to NaCl in transgenic plants. In conclusion, we suppose that PePLDδ-derived PA enhanced the salinity tolerance by regulating ROS and K+/Na+ homeostasis in Arabidopsis.

EuPIP1;2, a Plasma Membrane Aquaporin Gene from Eucommia ulmoides, Enhances Drought and Salt Tolerance in Transgenic Tobacco

Aquaporins are a specific type of membrane channel proteins that efficiently transport water molecules and other small molecular substrates in plants. In this study, we isolated the plasma membrane aquaporin gene EuPIP1;2 from Eucommia ulmoides, an important medicinal plant in China. The EuPIP1;2 protein was localized on the plasma membrane. Quantitative RT-PCR analysis revealed that EuPIP1;2 was constitutively expressed in all analyzed tissues, with the highest expression levels detected in the fruit and root. Overexpression of EuPIP1;2 in transgenic tobacco enhanced plant tolerance of drought and salinity. Under drought and salt stress, the transgenic lines exhibited higher percentage germination, longer roots, and enhanced percentage survival compared with wild-type plants. The contents of malonaldehyde and proline suggested that EuPIP1;2 improved drought and salt tolerance in transgenic lines by reducing damage to membranes and improving osmotic adjustment. We predict that EuPIP1;2 could be applied to improve drought and salt tolerance in transgenic plants.

NHX Gene Family in Camellia sinensis: In-silico Genome-Wide Identification, Expression Profiles, and Regulatory Network Analysis.

Salt stress affects the plant growth and productivity worldwide and NHX is one of those genes that are well known to improve salt tolerance in transgenic plants. It is well characterized in several plants, such as Arabidopsis thaliana and cotton; however, not much is known about NHXs in tea plant. In the present study, NHX genes of tea were obtained through a genome-wide search using A. thaliana as reference genome. Out of the 9 NHX genes in tea, 7 genes were localized in vacuole while the remaining 2 genes were localized in the endoplasmic reticulum (ER; CsNHX8) and plasma membrane (PM; CsNHX9), respectively. Furthermore, phylogenetic relationships along with structural analysis which includes gene structure, location, and protein-conserved motifs and domains were systematically examined and further, predictions were validated by the expression analysis. The dN/dS values show that the majority of tea NHX genes is subjected to strong purifying selection under the course of evolution. Also, functional interaction was carried out in Camellia sinensis based on the orthologous genes in A. thaliana. The expression profiles linked to various stress treatments revealed wide involvement of NHX genes from tea in response to various abiotic factors. This study provides the targets for further comprehensive identification, functional study, and also contributed for a better understanding of the NHX regulatory network in C. sinensis.

A PIP-mediated osmotic stress signaling cascade plays a positive role in the salt tolerance of sugarcane

BackgroundPlasma membrane intrinsic proteins (PIPs) are plant channel proteins involved in water deficit and salinity tolerance. PIPs play a major role in plant cell water balance and responses to salt stress. Although sugarcane is prone to high salt stress, there is no report on PIPs in sugarcane.ResultsIn the present study, eight PIP family genes, termed ScPIP1–1, ScPIP1–2, ScPIP1–3, ScPIP1–4, ScPIP2–1, ScPIP2–2, ScPIP2–4 and ScPIP2–5, were obtained based on the sugarcane transcriptome database. Then, ScPIP2–1 in sugarcane was cloned and characterized. Confocal microscopy observation indicated that ScPIP2–1 was located in the plasma membrane and cytoplasm. A yeast two-hybridization experiment revealed that ScPIP2–1 does not have transcriptional activity. Real time quantitative PCR (RT-qPCR) analysis showed that ScPIP2–1 was mainly expressed in the leaf, root and bud, and its expression levels in both below- and aboveground tissues of ROC22 were up-regulated by abscisic acid (ABA), polyethylene glycol (PEG) 6000 and sodium chloride (NaCl) stresses. The chlorophyll content and ion leakage measurement suggested that ScPIP2–1 played a significant role in salt stress resistance in Nicotiana benthamiana through the transient expression test. Overexpression of ScPIP2–1 in Arabidopsis thaliana proved that this gene enhanced the salt tolerance of transgenic plants at the phenotypic (healthier state, more stable relative water content and longer root length), physiologic (more stable ion leakage, lower malondialdehyde content, higher proline content and superoxide dismutase activity) and molecular levels (higher expression levels of AtKIN2, AtP5CS1, AtP5CS2, AtDREB2, AtRD29A, AtNHX1, AtSOS1 and AtHKT1 genes and a lower expression level of the AtTRX5 gene).ConclusionsThis study revealed that the ScPIP2–1-mediated osmotic stress signaling cascade played a positive role in plant response to salt stress.

RsSOS1 Responding to Salt Stress Might Be Involved in Regulating Salt Tolerance by Maintaining Na+ Homeostasis in Radish (Raphanus sativus L.)

Radish is a kind of moderately salt-sensitive vegetable. Salt stress seriously decreases the yield and quality of radish. The plasma membrane Na+/H+ antiporter protein Salt Overly Sensitive 1 (SOS1) plays a crucial role in protecting plant cells against salt stress, but the biological function of the RsSOS1 gene in radish remains to be elucidated. In this study, the RsSOS1 gene was isolated from radish genotype ‘NAU-TR17’, and contains an open reading frame of 3414 bp encoding 1137 amino acids. Phylogenetic analysis showed that RsSOS1 had a high homology with BnSOS1, and clustered together with Arabidopsis plasma membrane Na+/H+ antiporter (AtNHX7). The result of subcellular localization indicated that the RsSOS1 was localized in the plasma membrane. Furthermore, RsSOS1 was strongly induced in roots of radish under 150 mmol/L NaCl treatment, and its expression level in salt-tolerant genotypes was significantly higher than that in salt-sensitive ones. In addition, overexpression of RsSOS1 in Arabidopsis could significantly improve the salt tolerance of transgenic plants. Meanwhile, the transformation of RsSOS1△999 could rescue Na+ efflux function of AXT3 yeast. In summary, the plasma membrane Na+/H+ antiporter RsSOS1 plays a vital role in regulating salt-tolerance of radish by controlling Na+ homeostasis. These results provided useful information for further functional characterization of RsSOS1 and facilitate clarifying the molecular mechanism underlying salt stress response in radish.

The gene encoding lycopene epsilon cyclase of celery enhanced lutein and β-carotene contents and confers increased salt tolerance in Arabidopsis

Celery (Apium graveolens L.) is a leafy vegetable of Apiaceae, which is greatly popular because of its rich nutrients. Lutein and β-carotene are two important carotenoids. Lycopene epsilon cyclase (LCY-ε) is a key branch point enzyme in the carotenoid biosynthetic pathway. In this study, we cloned the AgLCY-ε gene from celery and overexpressed it in Arabidopsis. The results showed that both lutein and β-carotene accumulation increased significantly in transgenic Arabidopsis hosting AgLCY-ε gene, compared with wild type (WT) plants. The transcription levels of AtPSY and AtCRTISO genes involved in carotenoids biosynthesis also increased in transgenic lines. One-month-old transgenic Arabidopsis seedlings were treated with 200 mM NaCl. The malondialdehyde (MDA) content in transgenic Arabidopsis plants after salt treatment was significantly lower, and the activities of the two antioxidant enzymes, superoxide dismutase (SOD) and peroxidase (POD), were significantly increased than that of WT plants. Overexpression of AgLCY-ε gene showed increased lutein and β-carotene accumulations, and enhanced salt tolerance in transgenic plants.

Improving drought-, salinity-, and heat-tolerance in transgenic plants by co-overexpressing Arabidopsis vacuolar pyrophosphatase gene AVP1 and Larrea Rubisco activase gene RCA

The severity and frequency of many abiotic stresses such as drought, salinity and heat, cause substantial crop losses worldwide, which poses a serious challenge in food security. To increase crop production, new approaches are needed. Previous research has shown that overexpression of the tonoplast H+ pyrophosphatase gene AVP1 leads to improved drought and salt tolerance in transgenic plants. Other research showed that overexpression of thermotolerant ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase gene could maintain photosynthesis at higher temperatures, which contributes to higher heat tolerance in transgenic plants. In nature, abiotic stresses rarely come alone, instead these stresses often occur in various combinations. Therefore, it is desirable to make crops more tolerant to multiple stresses, which will likely lead to higher crop yield under various stress conditions. It is shown here that co-overexpression of the Arabidopsis gene AVP1 and the Larrea Rubisco activase gene RCA significantly increases drought, salinity and heat tolerance, resulting in higher biomass and seed yield than wild-type plants. AVP1/RCA co-overexpressing plants are as more drought- and salt-tolerant as AVP1-overexpressing plants, and as more heat-tolerant as RCA-overexpressing plants. More importantly, they produce higher seed yields than AVP1-overexpressing, RCA-overexpressing, and wild-type plants under combined drought and heat conditions.

Populus euphratica remorin 6.5 activates plasma membrane H+-ATPases to mediate salt tolerance.

Remorins (REMs) play an important role in the ability of plants to adapt to adverse environments. PeREM6.5, a protein of the REM family in Populus euphratica (salt-resistant poplar), was induced by NaCl stress in callus, roots and leaves. We cloned the full-length PeREM6.5 from P. euphratica and transformed it into Escherichia coli and Arabidopsis thaliana. PeREM6.5 recombinant protein significantly increased the H+-ATPase hydrolytic activity and H+ transport activity in P. euphratica plasma membrane (PM) vesicles. Yeast two-hybrid assay showed that P. euphratica REM6.5 interacted with RPM1-interacting protein 4 (PeRIN4). Notably, the PeREM6.5-induced increase in PM H+-ATPase activity was enhanced by PeRIN4 recombinant protein. Overexpression of PeREM6.5 in Arabidopsis significantly improved salt tolerance in transgenic plants in terms of survival rate, root growth, electrolyte leakage and malondialdehyde content. Arabidopsis plants overexpressing PeREM6.5 retained high PM H+-ATPase activity in both in vivo and in vitro assays. PeREM6.5-transgenic plants had reduced accumulation of Na+ due to the Na+ extrusion promoted by the H+-ATPases. Moreover, the H+ pumps caused hyperpolarization of the PM, which reduced the K+ loss mediated by the depolarization-activated channels in the PM of salinized roots. Therefore, we conclude that PeREM6.5 regulated H+-ATPase activity in the PM, thus enhancing the plant capacity to maintain ionic homeostasis under salinity.

Overexpression of the Cytokinin Oxidase/dehydrogenase (CKX) from Medicago sativa Enhanced Salt Stress Tolerance of Arabidopsis

Cytokinin oxidase/dehydrogenase (CKXs) are involved in various physiological processes, including cytokinins (CKs) catabolism, root system architecture and response to abiotic stresses in plants. Alfalfa (Medicago sativa) is a widely cultivated forage which is frequently threatened by high salinity, and the potential role of CKXs in alleviating the salt stress in alfalfa lacked attention. In this study, we isolated a CKX gene from alfalfa, MsCKX (MK177192), and identified its biological functions by overexpressing it in Arabidopsis. MsCKX shares high sequence identity with CKX from other legume plants, especially Medicago truncatula (98%). MsCKX was clearly tissue-specific, and it was mainly expressed in roots. In addition, the expression of MsCKX increased under salt stress and abscisic acid (ABA) treatment. Overexpression of MsCKX gene increased the activity of CKX, which led to an enlarged root system in transgenic Arabidopsis plants. Overexpression of MsCKX gene enhanced salt tolerance of transgenic plants by maintaining a higher K+/Na+ ratio, enhancing the activities of antioxidant enzymes to scavenge ROS and increasing the expression levels of stress-related genes (P5CS1, DREB2, ion transporters and H+ pumps). Taken together, these results shed light on the roles of MsCKX involved in salt tolerance and may have applications in salt-resistant breeding of alfalfa.

MiR319 mediated salt tolerance by ethylene.

SummarySalinity‐induced accumulation of certain microRNAs accompanied by gaseous phytohormone ethylene production has been recognized as a mechanism of plant salt tolerance. MicroRNA319 (miR319) has been characterized as an important player in abiotic stress resistance in some C3 plants, such as Arabidopsis thaliana and rice. However, its role in the dedicated biomass plant switchgrass (Panicum virgatum L.), a C4 plant, has not been reported. Here, we show crosstalk between miR319 and ethylene (ET) for increasing salt tolerance. By overexpressing Osa‐MIR319b and a target mimicry form of miR319 (MIM319), we showed that miR319 positively regulated ET synthesis and salt tolerance in switchgrass. By experimental treatments, we demonstrated that ET‐mediated salt tolerance in switchgrass was dose‐dependent, and miR319 regulated the switchgrass salt response by fine‐tuning ET synthesis. Further experiments showed that the repression of a miR319 target, PvPCF5, in switchgrass also led to enhanced ethylene accumulation and salt tolerance in transgenic plants. Genome‐wide transcriptome analysis demonstrated that overexpression of miR319 (OE‐miR319) down‐regulated the expression of key genes in the methionine (Met) cycle but promoted the expression of genes in ethylene synthesis. The results enrich our understanding of the synergistic effects of the miR319‐PvPCF5 module and ethylene synthesis in the salt tolerance of switchgrass, a C4 bioenergy plant.

Populus euphratica JRL Mediates ABA Response, Ionic and ROS Homeostasis in Arabidopsis under Salt Stress.

Sodium chloride (NaCl) induced expression of a jacalin-related mannose-binding lectin (JRL) gene in leaves, roots, and callus cultures of Populus euphratica (salt-resistant poplar). To explore the mechanism of the PeJRL in salinity tolerance, the full length of PeJRL was cloned from P. euphratica and was transformed into Arabidopsis. PeJRL was localized to the cytoplasm in mesophyll cells. Overexpression of PeJRL in Arabidopsis significantly improved the salt tolerance of transgenic plants, in terms of seed germination, root growth, and electrolyte leakage during seedling establishment. Under NaCl stress, transgenic plants retained K+ and limited the accumulation of Na+. PeJRL-transgenic lines increased Na+ extrusion, which was associated with the upward regulation of SOS1, AHA1, and AHA2 genes encoding plasma membrane Na+/proton (H+) antiporter and H+-pumps. The activated H+-ATPases in PeJRL-overexpressed plants restricted the channel-mediated loss of K+ that was activated by NaCl-induced depolarization. Under salt stress, PeJRL–transgenic Arabidopsis maintained reactive oxygen species (ROS) homeostasis by activating the antioxidant enzymes and reducing the production of O2− through downregulation of NADPH oxidases. Of note, the PeJRL-transgenic Arabidopsis repressed abscisic acid (ABA) biosynthesis, thus reducing the ABA-elicited ROS production and the oxidative damage during the period of salt stress. A schematic model was proposed to show the mediation of PeJRL on ABA response, and ionic and ROS homeostasis under NaCl stress.