Confers Salt Tolerance Research Articles

OsAAH confers salt tolerance in rice seedlings.

Soil salinization is becoming a great threat that reduces crop productivity worldwide. In this study, we found that rice allantoate amidohydrolase (OsAAH) expression was significantly upregulated by salt stress, and its overexpression conferred salt tolerance at the seedling stage. Compared to wild type (WT), the contents of ureides (allantoin and allantoate) were significantly increased in Osaah mutants and reduced in OsAAH overexpression lines both before and after salt treatments. Exogenous allantoin significantly promoted salt tolerance in OsAAH overexpression, but not in Osaah mutants. Subcellular localization showed that OsAAH was also localized to the peroxisomes in addition to the previously reported endoplasmic reticulum (ER). The differential expression of peroxisome-related genes was identified between Osaah mutants and WT. Furthermore, the contents of H2O2 and malondialdehyde (MDA) were significantly accumulated in Osaah mutants and reduced in OsAAH overexpression lines. The activities of antioxidant enzymes were significantly reduced in Osaah mutants and enhanced in OsAAH overexpression under NaCl treatment. The transcription factor OsABI5 could directly bind to OsAAH promoter and activate OsAAH expression. Our findings reveal that OsAAH could be induced by salt stress through the activation of OsABI5 and then confer salt tolerance by enhancing the scavenging capacity of reactive oxygen species (ROS), which contributes to rice breeding in salt tolerance.

Characterization of lycopene β-cyclase from Dunaliella bardawil for enhanced β-carotene production and salt tolerance

Dunaliella can accumulate more β-carotene (10 % or even more of the dry weight of cells) than any other species. Lycopene β-cyclase (LcyB) is the key enzyme in the catalysis of lycopene to β-carotene. In the present research, we used Escherichia coli BL21 (DE3) as host to construct two different types of engineering bacteria, one expressing the D. bardawil LcyB and the other expressing the orthologue Erwinia uredovora crtY. The catalytic ability of LcyB and CrtY were evaluated by comparing the β-carotene yields of the two E. coli BL21(DE3) strains, whose salt tolerance was simultaneously compared by cultivated them under different NaCl concentrations (1 %, 2 %, and 4 %). We also interfered with the LcyB gene to investigate the effect of LcyB in D. bardawil. Results displayed that the β-carotene yield of the LcyB-transformant significantly increased by about 48 % compared with the crtY-transformant. Additionally, LcyB was verified to be able to enhance the salt tolerance of E. coli BL21 (DE3). It is concluded that D. bardawil LcyB not only has better catalytic ability but also is able to confer salt tolerance to cells. Interfering D. bardawil LcyB induced the low expression of LcyB and the changes of growth and carotenoids metabolism in D. bardawil.

Biopriming of Solanum Lycopersicum seeds with novel root endophytic bacterial consortium retrieved from halotolerant Sundarban Mangroves to sustain growth and yield with salt resilience

Mangroves are often found in coastal areas and tropical wetlands that can withstand high salinity. We hypothesized that endophytes that are harbouring in the roots of mangrove plants may improve the innate immunity of host plants to survive naturally in saline environment. Retrieving these endophytes and sequential characterization may function as a novel bio-effector for non-host food crops as well. We focused on the integrated approach towards formulating a novel bacterial consortium. Thirty-one bacterial endophytes isolated from the roots of mangrove plants were screened for plant growth promoting potential by inoculating our model crop (Tomato). Seven most promising isolates impacting plant growth were identified. In-vitro plant growth promoting characters were also analysed. The root colonization by the isolates was confirmed by Scanning Electron Microscopy (SEM). Among the screened isolates, four of them were found to be compatible with each other when grown together and were selected to formulate a novel biostimulant consortia. The consortia treated Tomato plants exhibited superior phenological characters. In the pot experiment, plant height of the treated plants was about ≈43cm while the non-treated plants under salt stress could grow only up to a height of ≈26cm. Similarly, a total fruit yield of ≈6.8 kg was observed in case of treated plants under salt stress whereas the non-treated plants under salt stress could only produce ≈4.7 kg of fruit. This study demonstrated that the beneficial bacteria inhabiting in mangrove roots can increase the potential of conferring salt tolerance to non-host crops, thereby contributing to sustainable agriculture.

The homeodomain (PHD) protein, PdbPHD3, confers salt tolerance by regulating squamosa promoter binding protein PdbSBP3 in Populus davidiana × P. bolleana

Plant homeodomain (PHD) proteins are a family of zinc finger transcription factors that play roles in abiotic stress tolerance. However, their mechanisms in conferring salt tolerance are largely unknown. In this study, we characterized a PHD gene, PdbPHD3, from Populus davidiana × P. bolleana (Shanxin poplar) in response to salt stress. PdbPHD3 is a nuclear protein that is strongly induced by salt and abscisic acid (ABA) treatments. Overexpression of PdbPHD3 conferred salt tolerance, while silencing of PdbPHD3 increased sensitivity to salt. PdbPHD3 could enhance the activities of superoxide dismutase and peroxidase to reduce the abundance of reactive oxygen species, and enhance the osmotic potential by increasing the proline content. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that PdbPHD3 could bind to various DNA motifs, including the G-box (“CACGTG”), PALBOXAPC (“GGACGG”), and POLLEN1LELAT52 (“TTTCTT”). ChIP-seq combined with RNA sequencing identified a transcription factor gene, squamosa promoter binding protein 3 (PdbSBP3), which is directly regulated by PdbPHD3. Overexpression and silencing of PdbSBP3 improved and decreased salt tolerance, respectively. PdbSBP3 could also regulate all the physiological changes associated with salt tolerance, similar to PdbPHD3. These results suggest that PdbPHD3 confers salt tolerance by regulating PdbSBP3 to reduce ROS accumulation and increase proline content. Therefore, the regulatory axis of PdbPHD3 and PdbSBP3 confers salt tolerance in Shanxin poplar.

Unravelling Morpho‐Physiological Mechanism and Candidate Genes Associated With Salinity Tolerance in Superior Haplotypes of Barley (Hordeum vulgare L.)

ABSTRACTThe lack of suitable genetic resources for saline regions and the complexity of the traits involved impede the progress in crop breeding for salt tolerance. The present investigation was carried out using 27 diverse barley genotypes chosen based on the assessment of salt tolerance potential within the barley mini‐core collection at the National Genebank of India. The genotypes were exposed to salinity stress (200 mM NaCl) and were examined for morpho‐agronomic, physiological traits and salt uptake parameters. Exposure to salt stress resulted in a significant decline in all parameters ranging from 5.94% in relative water content to 80.04% in shoot K+/Na+ ratio compared to the control in the evaluated accessions. Moreover, the grain yield and its key attribute hundred‐grain weight decreased substantially by 65.35% and 48.62%, respectively, under saline treatment. The majority of the resilient accessions managed to uphold a higher K+/Na+ ratio ranging from 0.51 in EC0578359 to 1.19 EC0578251 in contrast to vulnerable germplasm (<0.56) under saline circumstances. The analysis of haplotype variants disclosed allelic diversity linked with two promising candidate genes, HVA1 and HvHKT2, recognised for conferring salt tolerance. Examination of nucleotide sequences revealed that the HVA1 remained considerably conserved among the evaluated genotypes as the majority of the SNPs (single‐nucleotide polymorphisms) were synonymous. Conversely, for HvHKT2, a significant level of genetic variation led to the identification of two primary haplotypic clusters—Hap1 associated with sensitivity to salinity and Hap2 linked with tolerant traits. Hap2 predominantly consisted of In/Dels which caused a modification in the overall protein length alongside the phospho‐variant allelic form of the HKT2 gene, further enhancing the biological specificity and functional stress response in a spatiotemporal fashion. These haplotype clusters correlated with favourable traits could be utilised for trait integration into breeding populations, thereby expediting the enhancement of superior salt‐tolerant barley cultivars.

Comparative proteomic discovery of salt stress response in alfalfa roots and overexpression of MsANN2 confers salt tolerance

Soil salinity constrains growth, development and yield of alfalfa (Medicago sativa L.). To illustrate the molecular mechanisms responsible for salt tolerance, a comparative proteome analysis was explored to characterize protein profiles of alfalfa seedling roots exposed to 100 and 200 mM NaCl for three weeks. There were 52 differentially expressed proteins identified, among which the mRNA expressions of 12 were verified by Real-Time-PCR analysis. The results showed increase in abundance of ascorbate peroxidase, POD, CBS protein and PR-10 in salt-stressed alfalfa, suggesting an effectively antioxidant and defense systems. Alfalfa enhanced protein quality control system to refold or degrade abnormal proteins induced by salt stress through upregulation of unfolded protein response (UPR) marker PDIs and molecular chaperones (eg. HSP70, TCP-1, and GroES) as well as the ubiquitin‐proteasome system (UPS) including ubiquitin ligase enzyme (E3) and proteasome subunits. Upregulation of proteins responsible for calcium signal transduction including calmodulin and annexin helped alfalfa adapt to salt stress. Specifically, annexin (MsANN2), a key Ca2+-binding protein, was selected for further characterization. The heterologous of the MsANN2 in Arabidopsis conferred salt tolerance. These results provide detailed information for salt-responsive root proteins and highlight the importance of MsANN2 in adapting to salt stress in alfalfa.

Exogenous proline suppresses endogenous proline and proline-production genes but improves the salinity tolerance capacity of salt-sensitive rice by stimulating antioxidant mechanisms and photosynthesis

Salinity is a critical environmental stress factor that significantly reduces crop productivity and yield. A mutant B-type response regulator gene (hst1) has been shown to promote salinity tolerance in the YNU genotype. Previous studies on the hst1 gene showed a higher proline production capacity under salt stress. Using almost identical genetic backgrounded salt-tolerant (YNU) and salt-sensitive (Sister line) rice genotypes, we tested the function of proline in the hst1 gene salinity-tolerance mechanism by applying exogenous proline under control and salt-stress conditions. Morpho-physiological, biochemical, and molecular analysis of ST and SS plants was performed to clarify the salinity tolerance mechanism mediated by the exogenous proline. The ST and SS genotypes accumulated exogenous proline, and the ST genotype has higher proline levels than the SS genotype. However, exogenous proline improved salt tolerance only in the SS genotype. Exogenous proline promotes plant and root growth by stimulating photosynthetic pigments and photosynthesis. The exogenous proline has a reductive effect on MDA, and H2O2 protects plants against ROS. Interestingly, exogenous proline lowers Na+ and raises K+ accumulations under salt stress. In the SS genotype, exogenous proline increases the activity of antioxidant enzymes (SOD, CAT, and APX) to protect against salinity-induced damage. The exogenous proline application down-regulates proline-synthesis genes (OsP5CS1 and OsP5CR) and up-regulates proline-degradation genes. Also, exogenous proline increases the expression of the OsSalT and OsGRAS29 genes, improving salinity tolerance in the SS genotype. Our study has demonstrated that proline plays a significant role in conferring salt tolerance with the salinity-tolerance-related hst1 mechanisms.

Aerial signaling by plant-associated Streptomyces setonii WY228 regulates plant growth and enhances salt stress tolerance

Plant-associated streptomycetes play important roles in plant growth and development. However, knowledge of volatile-mediated crosstalk between Streptomyces spp. and plants remains limited. In this study, we investigated the impact of volatiles from nine endophytic Streptomyces strains on the growth and development of plants. One versatile strain, Streptomyces setonii WY228, was found to significantly promote the growth of Arabidopsis thaliana and tomato seedlings, confer salt tolerance, and induce early flowering and increased fruit yield following volatile treatment. Analysis of plant growth-promoting traits revealed that S. setonii WY228 could produce indole-3-acetic acid, siderophores, ACC deaminase, fix nitrogen, and solubilize inorganic phosphate. These capabilities were further confirmed through genome sequencing and analysis. Volatilome analysis indicated that the volatile organic compounds emitted from ISP-2 medium predominantly comprised sesquiterpenes and 2-ethyl-5-methylpyrazine. Further investigations showed that 2-ethyl-5-methylpyrazine and sesquiterpenoid volatiles were the primary regulators promoting growth, as confirmed by experiments using the terpene synthesis inhibitor phosphomycin, pure compounds, and comparisons of volatile components. Transcriptome analysis, combined with mutant and inhibitor studies, demonstrated that WY228 volatiles promoted root growth by activating Arabidopsis auxin signaling and polar transport, and enhanced root hair development through ethylene signaling activation. Additionally, it was confirmed that volatiles can stimulate plant abscisic acid signaling and activate the MYB75 transcription factor, thereby promoting anthocyanin synthesis and enhancing plant salt stress tolerance. Our findings suggest that aerial signaling-mediated plant growth promotion and abiotic stress tolerance represent potentially overlooked mechanisms of Streptomyces-plant interactions. This study also provides an exciting strategy for the regulation of plant growth and the improvement of horticultural crop yields within sustainable agricultural practices.

Overexpression of GhGSTF9 Enhances Salt Stress Tolerance in Transgenic Arabidopsis.

Soil salinization is a major abiotic stress factor that negatively impacts plant growth, development, and crop yield, severely limiting agricultural production and economic development. Cotton, a key cash crop, is commonly cultivated as a pioneer crop in regions with saline-alkali soil due to its relatively strong tolerance to salt. This characteristic renders it a valuable subject for investigating the molecular mechanisms underlying plant salt tolerance and for identifying genes that confer salt tolerance. In this study, focus was placed on examining a salt-tolerant variety, E991, and a salt-sensitive variety, ZM24. A combined analysis of transcriptomic data from these cotton varieties led to the identification of potential salt stress-responsive genes within the glutathione S-transferase (GST) family. These versatile enzyme proteins, prevalent in animals, plants, and microorganisms, were demonstrated to be involved in various abiotic stress responses. Our findings indicate that suppressing GhGSTF9 in cotton led to a notably salt-sensitive phenotype, whereas heterologous overexpression in Arabidopsis plants decreases the accumulation of reactive oxygen species under salt stress, thereby enhancing salt stress tolerance. This suggests that GhGSTF9 serves as a positive regulator in cotton's response to salt stress. These results offer new target genes for developing salt-tolerant cotton varieties.

A high-affinity potassium transporter (MeHKT1) from cassava (Manihot esculenta) negatively regulates the response of transgenic Arabidopsis to salt stress

BackgroundHigh-affinity potassium transporters (HKTs) are crucial in facilitating potassium uptake by plants. Many types of HKTs confer salt tolerance to plants through regulating K+ and Na+ homeostasis under salinity stress. However, their specific functions in cassava (Manihot esculenta) remain unclear.ResultsHerein, an HKT gene (MeHKT1) was cloned from cassava, and its expression is triggered by exposure to salt stress. The expression of a plasma membrane-bound protein functions as transporter to rescue a low potassium (K+) sensitivity of yeast mutant strain, but the complementation of MeHKT1 is inhibited by NaCl treatment. Under low K+ stress, transgenic Arabidopsis with MeHKT1 exhibits improved growth due to increasing shoot K+ content. In contrast, transgenic Arabidopsis accumulates more Na+ under salt stress than wild-type (WT) plants. Nevertheless, the differences in K+ content between transgenic and WT plants are not significant. Additionally, Arabidopsis expressing MeHKT1 displayed a stronger salt-sensitive phenotype.ConclusionThese results suggest that under low K+ condition, MeHKT1 functions as a potassium transporter. In contrast, MeHKT1 mainly transports Na+ into cells under salt stress condition and negatively regulates the response of transgenic Arabidopsis to salt stress. Our results provide a reference for further research on the function of MeHKT1, and provide a basis for further application of MeHKT1 in cassava by molecular biological means.

HgS2, a novel salt-responsive gene from the Halophyte Halogeton glomeratus, confers salt tolerance in transgenic Arabidopsis.

Halophyte Halogeton glomeratus mostly grows in saline desert areas in arid and semi-arid regions and is able to adapt to adverse conditions such as salinity and drought. Earlier transcriptomic studies revealed activation of the HgS2 gene in the leaf of H. glomeratus seedlings when exposed to saline conditions. To identify the properties of HgS2 in H. glomeratus, we used yeast transformation and overexpression in Arabidopsis. Yeast cells genetically transformed with HgS2 exhibited K+ uptake and Na+ efflux compared with control (empty vector). Stable overexpression of HgS2 in Arabidopsis improved its resistance to salt stress and led to a notable rise in seed germination in salinity conditions compared to the wild type (WT). Transgenic Arabidopsis regulated ion homeostasis in plant cells by increasing Na+ absorption and decreasing K+ efflux in leaves, while reducing Na+ absorption and K+ efflux in roots. In addition, overexpression of HgS2 altered transcription levels of stress response genes and regulated different metabolic pathways in roots and leaves of Arabidopsis. These results offer new insights into the role of HgS2 in plants' salt tolerance.

Identification of potential morpho-biochemical determinants conferring salt tolerance in wheat (Triticum aestivum L.) through seedling- and -reproductive stage phenotyping

To optimize a reproductive-stage-specific phenotyping protocol and isolate potential determinants conferring salinity tolerance in wheat, two consecutive experiments were conducted using salt-tolerant varieties (Binagom-1 and BARI Gom 25) and a sensitive variety (BARI Gom 20). In the first experiment, seedlings were grown hydroponically, and 14-day-old seedlings were subjected to two different levels of salt stress (EC=12 dS/m and 16 dS/m) for 7 days. Based on the results of tolerant and susceptible varieties, parameters such as root and shoot weight, shoot Na+/K+ ratio, chlorophyll content, proline content, methylglyoxal content, H2O2 content and lipid peroxidation content and the activities of enzymes such as ascorbate peroxidase, peroxidase, and glyoxalase I were considered as potential determinants of salt tolerance. The second experiment employed four leaf cutting treatments under both control and salinity stress conditions. Seedlings were grown in perforated pots filled with field soil, and at the heading stage, plants were subjected to salt stress (12 dS/m) after trimming as indicated. The combined analysis of control and salt stress data obtained from setup B reflected a significant decrease in yield and yield-attributing traits; however, a lesser decrease was observed in tolerant varieties. Correlation studies revealed that grain yield per spike exhibited a significant positive correlation with the number of seeds per spike, spike weight, plant height, and days to first flowering under both stress and control conditions. Additionally, different stress tolerance indices also supported the results of reproductive stage phenotyping. However, further studies will be required to tag the genes/QTLs controlling salinity tolerance in wheat at various growth phases.

The Spartina alterniflora genome sequence provides insights into the salt-tolerance mechanisms of exo-recretohalophytes.

Spartina alterniflora is an exo-recretohalophyte Poaceae species that is able to grow well in seashore, but the genomic basis underlying its adaptation to salt tolerance remains unknown. Here, we report a high-quality, chromosome-level genome assembly of S. alterniflora constructed through PacBio HiFi sequencing, combined with high-throughput chromosome conformation capture (Hi-C) technology and Illumina-based transcriptomic analyses. The final 1.58 Gb genome assembly has a contig N50 size of 46.74 Mb. Phylogenetic analysis suggests that S. alterniflora diverged from Zoysia japonica approximately 21.72 million years ago (MYA). Moreover, whole-genome duplication (WGD) events in S. alterniflora appear to have expanded gene families and transcription factors relevant to salt tolerance and adaptation to saline environments. Comparative genomics analyses identified numerous species-specific genes, significantly expanded genes and positively selected genes that are enriched for 'ion transport' and 'response to salt stress'. RNA-seq analysis identified several ion transporter genes including the high-affinity K+ transporters (HKTs), SaHKT1;2, SaHKT1;3 and SaHKT1;8, and high copy number of Salt Overly Sensitive (SOS) up-regulated under high salt conditions, and the overexpression of SaHKT2;4 in Arabidopsis thaliana conferred salt tolerance to the plant, suggesting specialized roles for S. alterniflora to adapt to saline environments. Integrated metabolomics and transcriptomics analyses revealed that salt stress activate glutathione metabolism, with differential expressions of several genes such as γ-ECS, GSH-S, GPX, GST and PCS in the glutathione metabolism. This study suggests several adaptive mechanisms that could contribute our understanding of evolutional basis of the halophyte.

CwJAZ4/9 negatively regulates jasmonate-mediated biosynthesis of terpenoids through interacting with CwMYC2 and confers salt tolerance in Curcuma wenyujin.

Plant JASMONATE ZIM-DOMAIN (JAZ) genes play crucial roles in regulating the biosynthesis of specialized metabolites and stressful responses. However, understanding of JAZs controlling these biological processes lags due to numerous JAZ copies. Here, we found that two leaf-specific CwJAZ4/9 genes from Curcuma wenyujin are strongly induced by methyl-jasmonate (MeJA) and negatively correlated with terpenoid biosynthesis. Yeast two-hybrid, luciferase complementation imaging and in vitro pull-down assays confirmed that CwJAZ4/9 proteins interact with CwMYC2 to form the CwJAZ4/9-CwMYC2 regulatory cascade. Furthermore, transgenic hairy roots showed that CwJAZ4/9 acts as repressors of MeJA-induced terpenoid biosynthesis by inhibiting the terpenoid pathway and jasmonate response, thus reducing terpenoid accumulation. In addition, we revealed that CwJAZ4/9 decreases salt sensitivity and sustains the growth of hairy roots under salt stress by suppressing the salt-mediated jasmonate responses. Transcriptome analysis for MeJA-mediated transgenic hairy root lines further confirmed that CwJAZ4/9 negatively regulates the terpenoid pathway genes and massively alters the expression of genes related to salt stress signaling and responses, and crosstalks of multiple phytohormones. Altogether, our results establish a genetic framework to understand how CwJAZ4/9 inhibits terpenoid biosynthesis and confers salt tolerance, which provides a potential strategy for producing high-value pharmaceutical terpenoids and improving resistantC. wenyujin varieties by a genetic approach.

The PbbHLH62/PbVHA-B1 module confers salt tolerance through modulating intracellular Na+/K+ homeostasis and reactive oxygen species removal in pear

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit membrane protein complex, which plays pivotal roles in building up an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this study, a B subunit of V-ATPase gene, PbVHA-B1 was discovered and isolated from stress-induced P. betulaefolia combining with RT-PCR method. The RT-qPCR analysis revealed that the expression level of PbVHA-B1 was upregulated by salt, drought, cold, and exogenous ABA treatment. Subcellular localization analyses showed that PbVHA-B1 was located in the cytoplasm and nucleus. Moreover, overexpression of PbVHA-B1 gene noticeably increased the ATPase activity and the tolerance to salt in transgenic Arabidopsis plants. In contrast, knockdown of PbVHA-B1 gene in P.betulaefolia by virus-induced gene silencing had reduced resistance to salt stress. In addition, using yeast one-hybride (Y1H) and yeast two-hybride (Y2H) screens, PbbHLH62, a bHLH transcription factor, was identified as a partner of the PbVHA-B1 promoter and protein. Then, we also found that PbbHLH62 positively regulate the expression of PbVHA-B1 and the ATPase activity after salt stress treatment. These findings provide evidence that PbbHLH62 played a critical role in the salt response. Collectively, our results demonstrate that a PbbHLH62/PbVHA-B1 module plays a positive role in salt tolerance by maintain intracellular ion and ROS homeostasis in pear.

Arabidopsis class II TPS controls root development and confers salt stress tolerance through enhanced hydrophobic barrier deposition.

The mechanism of conferring salt tolerance by AtTPS9 involves enhanced deposition of suberin lamellae in the Arabidopsis root endodermis, resulting in reduction of Na+ transported to the leaves. Members of the class I trehalose-6-phosphate synthase (TPS) enzymes are known to play an important role in plant growth and development in Arabidopsis. However, class II TPSs and their functions in salinity stress tolerance are not well studied. We characterized the function of a class II TPS gene, AtTPS9, to understand its role in salt stress response and root development in Arabidopsis. The attps9 mutant exhibited significant reduction of soluble sugar levels in the leaves and formation of suberin lamellae (SL) in the endodermis of roots compared to the wild type (WT). The reduction in SL deposition (hydrophobic barriers) leads to increased apoplastic xylem loading, resulting in enhanced Na+ content in the plants, which explains salt sensitivity of the mutant plants. Conversely, AtTPS9 overexpression lines exhibited increased SL deposition in the root endodermis along with increased salt tolerance, showing that regulation of SL deposition is one of the mechanisms of action of AtTPS9 in conferring salt tolerance to Arabidopsis plants. Our data showed that besides salt tolerance, AtTPS9 also regulates seed germination and root development. qRT-PCR analyses showed significant downregulation of selected SNF1-RELATED PROTEIN KINASE2 genes (SnRK2s) and ABA-responsive genes in the mutant, suggesting that AtTPS9 may regulate the ABA-signaling intermediates as part of the mechanism conferring salinity tolerance.

Birch WRKY transcription factor, BpWRKY32, confers salt tolerance by mediating stomatal closing, proline accumulation, and reactive oxygen species scavenging

Plant WRKY transcription factors (TFs) play important roles in abiotic stress responses. However, how WRKY facilitate physiological changes to confer salt tolerance still needs to be studied. Here, we identified a WRKY TF from birch (Betula platyphylla Suk), BpWRKY32, which is significantly (P < 0.05) induced by salt stress. BpWRKY32 binds to W-box motif and is located in the nucleus. Under salt stress conditions, fresh weights (FW) of OE lines (BpWRKY32 overexpression lines) are increased by 66.36% than that of WT, while FW of knockout of BpWRKY32 (bpwrky32) lines are reduced by 39.49% compared with WT. BpWRKY32 regulates the expression of BpRHC1, BpNRT1, and BpMYB61 to reduce stomatal, and width-length ratio of the stomatal aperture in OE lines are reduced by 46.23% and 64.72% compared with in WT and bpwrky32 lines. BpWRKY32 induces P5CS expression, but inhibits P5CDH expression, leading to the proline content in OE lines are increased by 33.41% and 97.58% compared with WT and bpwrky32 lines. Additionally, BpWRKY32 regulates genes encoding SOD and POD family members, which correspondingly increases the activities of SOD and POD. These results suggested that BpWRKY32 regulates target genes to reduce the water loss rate, enhance the osmotic potential, and reduce the ROS accumulation, leading to improved salt tolerance.

Acetylation of transcription factor BpTCP20 by acetyltransferase BpPDCE23 modulates salt tolerance in birch.

Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) transcription factors function in abiotic stress responses. However, how TCPs confer salt tolerance is unclear. Here, we characterized a TCP transcription factor, BpTCP20, that responds to salt stress in birch (Betula platyphylla Suk). Plants overexpressing BpTCP20 displayed increased salt tolerance, and Bptcp20 knockout mutants displayed reduced salt tolerance relative to the wild-type (WT) birch. BpTCP20 conferred salt tolerance by mediating stomatal closure and reducing reactive oxygen species (ROS) accumulation. Chromatin immunoprecipitation sequencing showed that BpTCP20 binds to NeuroD1, T-box, and two unknown elements (termed TBS1 and TBS2) to regulate target genes. In birch, salt stress led to acetylation of BpTCP20 acetylation at lysine 259. A mutated BpTCP20 variant (abolished for acetylation, termed BpTCP20259) was overexpressed in birch, which led to decreased salt tolerance compared with plants overexpressing BpTCP20. However, BpTCP20259-overexpressing plants still displayed increased salt tolerance relative to untransformed WT plants. BpTCP20259 showed reduced binding to the promoters of target genes and decreased target gene activation, leading to decreased salt tolerance. In addition, we identified dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (BpPDCE23), an acetyltransferase that interacts with and acetylates BpTCP20 to enhance its binding to DNA motifs. Together, these results suggest that BpTCP20 is a transcriptional regulator of salt tolerance, whose activity is modulated by BpPDCE23-mediated acetylation.

The RING zinc finger protein LbRZF1 promotes salt gland development and salt tolerance in Limonium bicolor.

The recretohalophyte Limonium bicolor thrives in high-salinity environments because salt glands on the above-ground parts of the plant help to expel excess salt. Here, we characterize a nucleus-localized C3HC4 (RING-HC)-type zinc finger protein of L. bicolor named RING ZINC FINGERPROTEIN 1 (LbRZF1). LbRZF1 was expressed in salt glands and in response to NaCl treatment. LbRZF1 showed no E3 ubiquitin ligase activity. The phenotypes of overexpression and knockout lines for LbRZF1 indicated that LbRZF1 positively regulated salt gland development and salt tolerance in L. bicolor. lbrzf1 mutants had fewer salt glands and secreted less salt than did the wild-type, whereas LbRZF1-overexpressing lines had opposite phenotypes, in keeping with the overall salt tolerance of these plants. A yeast two-hybrid screen revealed that LbRZF1 interacted with LbCATALASE2 (LbCAT2) and the transcription factor LbMYB113, leading to their stabilization. Silencing of LbCAT2 or LbMYB113 decreased salt gland density and salt tolerance. The heterologous expression of LbRZF1 in Arabidopsis thaliana conferred salt tolerance to this non-halophyte. We also identified the transcription factor LbMYB48 as an upstream regulator of LbRZF1 transcription. The study of LbRZF1 in the regulation network of salt gland development also provides a good foundation for transforming crops and improving their salt resistance.

Overexpression of a plasmalemma Na+/H+ antiporter from the halophyte Nitraria sibirica enhances the salt tolerance of transgenic poplar

The plasmalemma Na+/H+ antiporter Salt Overly Sensitive 1 (SOS1) is responsible for the efflux of Na+ from the cytoplasm, an important determinant of salt resistance in plants. In this study, an ortholog of SOS1, referred to as NsSOS1, was cloned from Nitraria sibirica, a typical halophyte that grows in deserts and saline-alkaline land, and its expression and function in regulating the salt tolerance of forest trees were evaluated. The expression level of NsSOS1 was higher in leaves than in roots and stems of N. sibirica, and its expression was upregulated under salt stress. Histochemical staining showed that β-glucuronidase (GUS) driven by the NsSOS1 promoter was strongly induced by abiotic stresses and phytohormones including salt, drought, low temperature, gibberellin, and methyl jasmonate, suggesting that NsSOS1 is involved in the regulation of multiple signaling pathways. Transgenic 84 K poplar (Populus alba × P. glandulosa) overexpressing NsSOS1 showed improvements in survival rate, root biomass, plant height, relative water levels, chlorophyll and proline levels, and antioxidant enzyme activities versus non-transgenic poplar (NT) under salt stress. Transgenic poplars accumulated less Na+ and more K+ in roots, stems, and leaves, which had a lower Na+/K+ ratio compared to NT under salt stress. These results indicate that NsSOS1-mediated Na+ efflux confers salt tolerance to transgenic poplars, which show more efficient photosynthesis, better scavenging of reactive oxygen species, and improved osmotic adjustment under salt stress. Transcriptome analysis of transgenic poplars confirmed that NsSOS1 not only mediates Na+ efflux but is also involved in the regulation of multiple metabolic pathways. The results provide insight into the regulatory mechanisms of NsSOS1 and suggest that it could be used to improve the salt tolerance of forest trees.