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Functional study of the AfRAP2 gene in Amorpha fruticosa L. tolerance to saline-alkali and drought stress.

BACKGROUND: Amorpha fruticosa L. is a leguminous shrub with high tolerance to drought, poor soil, and saline-alkali stress conditions. As a member of the family of transcription factors in higher plants, the ethylene response factor AP2/ERF plays a crucial role in both plant adaptation to abiotic stress and in growth and development. In this study, based on genes identified from the transcriptomic sequencing of Amorpha fruticosa L. under drought stress, the upregulated gene AfRAP2 was isolated from its seedlings, with the aim of elucidating its stress-response function using molecular biological techniques. RESULTS: In this study, the AfRAP2 gene was cloned from the leaves of Amorpha fruticosa L. using RT-PCR. Bioinformatics analysis revealed that AfRAP2 contains an AP2 domain and belongs to the DREB subfamily of the AP2/ERF transcription factor family, showing close phylogenetic relationships with LaEREBP from Lathyrus albus. Real-time quantitative PCR (RT-qPCR) results indicate that AfRAP2 is expressed in various tissues of Amorpha fruticosa L., with the highest expression in leaves and the lowest in stems, furthermore, its expression is significantly upregulated in roots and leaves upon induction by NaHCO3 and PEG6000. Subcellular localization experiments confirmed that the AfRAP2 protein is localized to the nucleus, and GUS histochemical staining assay revealed that its promoter drives GUS expression in anthers. Resistance analysis of overexpressing yeast strains showed that yeast transformed with the AfRAP2 gene exhibited significantly better growth under sorbitol, mannitol, and NaHCO3 stress conditions compared to the control, indicating that this gene enhances yeast tolerance to drought and saline-alkali stress. We screened transgenic tobacco and Populus davidiana × P. alba var. Pyramidalis. The results showed that under natural drought and saline-alkali stress treatments, the transgenic lines exhibited significantly improved growth and higher activities of the physiological indicators of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) compared with wild-type plants, indicating that the overexpression of the AfRAP2 gene plays a key role in the response to saline-alkali stress and drought stress. CONCLUSION: In summary, AfRAP2 contains an AP2 domain and belongs to the DREB subfamily of transcription factors, under abiotic stress induced by NaHCO₃ and mannitol, it can induce the expression of the AfRAP2 gene in tobacco and Populus davidiana×P. alba var. pyramidalis. AfRAP2 plays a vital role in the plant response to saline-alkali stress and drought stress and is a promising candidate gene for stress-tolerant breeding.

Plant Proteins

Physiological and transcriptomic responses of sunflower to combined saline-alkali stress.

BACKGROUND: Sunflower (Helianthus annuus L.), an important oilseed crop, is often used as a pioneer species for improving saline-alkali soils. However, the molecular mechanisms underlying sunflower seedling responses to combined saline-alkali stress remain unclear. This study aimed to elucidate the molecular basis of saline-alkali tolerance at the seedling stage by comparing physiological and transcriptomic responses between tolerant and sensitive sunflower hybrids. The saline-alkali tolerant hybrid K-27 and the sensitive hybrid K-7 were used as experimental materials. Root samples were collected at 0, 3, 12, 24, 48, and 96 h after exposure to combined saline-alkali stress (0.5% NaCl + Na2CO3, adjusted to pH 9.0). Physiological parameters, including antioxidant enzyme activities, osmolyte contents, ion concentrations, membrane damage levels, and cell wall components, were measured, followed by transcriptome sequencing analysis. RESULTS: Phenotypic analysis showed that the root length inhibition rate and fresh weight loss rate of K-27 were significantly lower than those of K-7, indicating stronger tolerance. Physiological analysis revealed that K-27 exhibited an inducible antioxidant enzyme response pattern. In addition, K-27 achieved osmotic adjustment through sustained proline accumulation (peaking at 12 h and remaining significantly higher than that of K-7 at 96 h) and exhibited higher basal levels of lignin and hemicellulose. Transcriptome analysis showed that the number of upregulated genes in K-27 was consistently higher than in K-7 at all time points, with 5,283 genes upregulated as early as 3 h after stress exposure. Venn analysis identified 44 core differentially expressed genes (cDEGs) shared between the two genotypes, which were mainly enriched in auxin biosynthesis regulation, phenylpropanoid biosynthesis, and glutathione metabolism. Among them, the benzoic acid carboxyl methyltransferase gene (BAMT) was continuously upregulated in K-27 but persistently downregulated in K-7. In addition, five other genes (encoding fatty aldehyde dehydrogenase, pectin methylesterase inhibitor, glutathione S-transferase, INPP5E, and HXXXD-type acyltransferase) exhibited significantly higher expression levels in K-27. CONCLUSION: K-27 tolerates combined saline-alkali stress through coordinated multi-layered response mechanisms, including inducible antioxidant defense, maintenance of ion homeostasis, sustained osmotic adjustment, and activation of the phenylpropanoid metabolic pathway. Candidate genes such as BAMT may provide potential targets for molecular breeding of saline-alkali tolerant sunflower, although their functions require further experimental validation.

Helianthus

Evidence for the presence of nasal salt glands in the roadrunner and the Coturnix quail.

The purpose of this investigation was to determine whether or not the nasal glands of the roadrunner and the Coturnix quail show cytological specializations for salt secretion. In addition, the Na-K ATPase content of the quail gland was determined before and after drinking of saline solutions, in an effort to evaluate the functional status of the gland. The ability to maintain weight while drinking salt water was also measured as a general index of tolerance to saline conditions. The ultrastructure of the nasal glands of the roadrunner injected with salt and of quail drinking 200 mM NaCl was similar to that of salt glands in reptiles and the fresh-water acclimated duck. Numerous lateral cell evaginations and abundant mitochondria were present in the principal cell types. There was a significant increase in quail nasal gland Na-K ATPase when young birds were offered only saline solutions to drink. The ability of Coturnix quail to maintain weight while drinking saline solutions improves with age and at adulthood is comparable to that of some North American desert quail. Roadrunners were previously known to possess functional salt glands whereas quail were not. However the characteristic fine structure and the high Na-KATPase content of the quail nasal gland suggest that it is a salt gland.

Adenosine Triphosphatases

Genomic exploration of Bacillus paralicheniformis TB197: an agrobiotechnological tool from the Sonoran Desert.

Climate change and the harmful effects of extensive agrochemical use for plant nutrition and pest control on soils, the environment, and human health are driving the search for sustainable alternatives that reduce their use while increasing plant resilience. In regenerative agriculture, microorganisms have become valuable tools, acting as biological control agents or biostimulants, such as plant growth-promoting rhizobacteria, and/or to enhance plant performance under abiotic stress. The genus Bacillus is well known for its versatile interactions with plants. Specifically, Bacillus paralicheniformis TB197 has demonstrated high efficacy in controlling phytopathogenic nematodes and adapting to diverse soil and crop conditions. Based on these traits, we explored the agricultural potential of this strain through genomic analysis and in vitro and in vivo assays. Gene analysis identified functions related to three main areas: (i) stress resistance and plant colonization, (ii) plant growth promotion, and (iii) phytopathogen control. The strain showed high tolerance to salinity and temperature, promoted plant growth, and exhibited strong antifungal activity. These findings highlight the potential of the TB197 strain as a promising candidate for developing next-generation bioinoculants.IMPORTANCEThe use of beneficial microorganisms is a pivotal strategy for mitigating the environmental impacts of intensive agriculture while preserving crop productivity. Bacillus paralicheniformis TB197 is a native desert soil bacterium with genetic traits associated with stress tolerance, plant growth promotion, and suppression of plant pathogens. In this study, we employed a multifaceted approach integrating genomic analysis and functional assays to demonstrate the strain's multifunctional potential as an agricultural bioinoculant. The results of the study demonstrate that a singular bacterial strain can integrate multiple beneficial functions relevant to sustainable agriculture. This work contributes to the field of applied microbiology by expanding the understanding of how environmentally adapted bacteria can serve as biological alternatives to chemical inputs in agroecosystems.

Bacillus

The 13-lipoxygenase GmLOX6 is involved in JA biosynthesis and serves as a positive regulator of salt stress tolerance in soybean.

Salinity represents a major abiotic stressor that significantly impairs soybean growth and yield. Although jasmonic acid (JA) has been firmly established as a key regulator of plant defense against salt stress, the precise functions of lipoxygenase (LOX) genes responsible for initiating JA biosynthesis remain poorly defined. Here, a comprehensive genome-wide analysis of the soybean LOX gene family was performed, and a detailed functional characterization of GmLOX6 was carried out. Subcellular localization confirmed that GmLOX6 is targeted to chloroplasts, while enzymatic assays demonstrated that it acts as a 13-LOX enzyme with a strong preference for α-linolenic acid as substrate. To clarify its role under salt stress, we generated both overexpression and CRISPR/Cas9-mediated knockout lines of soybean. Phenotypic and molecular evaluations revealed that GmLOX6 facilitates JA production under salt stress, thereby contributing to enhanced JA accumulation. This elevation in JA levels was associated with improved salt tolerance through multiple physiological adaptations, including the activation of antioxidant enzymes for the detoxification of reactive oxygen species (ROS), enhanced Na+ extrusion to preserve ionic balance, and reinforced membrane stability. Moreover, GmRWP-RK11 was identified as a transcriptional repressor of GmLOX6. Functional disruption of GmRWP-RK11 via CRISPR/Cas9 conferred greater salt tolerance, further supporting its negative regulatory role. Collectively, these findings uncover a novel regulatory axis in which GmLOX6-mediated JA biosynthesis enhances soybean resistance to salinity through modulation of ROS homeostasis and Na+ transport. These insights provide an expanded understanding of the transcriptional and biochemical mechanisms underpinning JA-driven stress adaptation in soybean.

Glycine max

Teleost chloride cell. I. Response of pupfish Cyprinodon variegatus gill Na,K-ATPase and chloride cell fine structure to various high salinity environments.

Certain euryhaline teleosts can tolerate media of very high salinity, i.e. greater than that of seawater itself. The osmotic gradient across the integument of these fish is very high and the key to their survival appears to be the enhanced ability of the gill to excrete excess NaCl. These fish provide an opportunity to study morphological and biochemical aspects of transepithelial salt secretion under conditions of vastly different transport rates. Since the cellular site of gill salt excretion is believed to be the "chloride cell" of the branchial epithelium and since the enzyme Na,K-ATPase has been implicated in salt transport in this and other secretory tissues, we have focused our attention on the differences in chloride cell structure and gill ATPase activity in the variegated pupfish Cyprinodon variegatus adapted to half-strength seawater (50% SW), seawater (100% SW), or double-stregth seawater (200% SW). The Na,K-ATPase activity in gill homogenates was 1.6 times greater in 100% SW. When 50% SW gills were compared to 100% SW gills, differences in chloride cell morphology were minimal. However, chloride cells from 200% SW displayed a marked hypertrophy and a striking increase in basal-lateral cell surface area. These results suggest that there are correlations among higher levels of osmotic stress, basal-lateral extensions of the cell surface, and the activity of the enzyme Na,K-ATPase.

Adenosine Triphosphatases

Identification and analysis of HD-ZIP transcription factors that regulate salt gland development and salt tolerance in Limonium bicolor.

Soil salinity severely constrains agricultural production. Elucidating the salt-tolerance mechanisms of halophytes can provide innovative approaches for improving the salt tolerance of crop plants. In this study, we performed genome-wide identification and analysis of 36 LbHDZ genes encoding homeodomain-leucine zipper (HD-ZIP) transcription factors in Limonium bicolor, a typical recretohalophyte that excretes excess salt ions through specialized salt glands. Expression profiling across different stages of salt gland development, as well as in various tissues under salt stress, indicated that multiple LbHDZ genes are involved in regulating salt gland development and salt tolerance. Among these genes, LbHDZ14 (a member of the HD-ZIP II subfamily) exhibited sustained high expression during the critical period of salt gland formation, while its transcript levels were significantly downregulated in leaves and roots under salt stress. Subsequent experiments demonstrated that LbHDZ14 is localized in the nucleus and negatively regulates salt gland density and salt tolerance by directly binding to the promoter of LbGDSL, a positive regulator of salt gland development. In conclusion, this study reveals the expression patterns of LbHDZ genes in L. bicolor, characterizes the functional mechanism of LbHDZ14, further elucidates the regulatory network underlying salt gland development, and provides candidate genes for enhancing crop salt tolerance.

Plumbaginaceae

Effects of the synthetic enkephalin analogue FK 33-824 on pain threshold and pain tolerance in man.

Natural enkephalins exert weak and transitory analgesic effects. The synthetic enkephalin, FK 33-824 (FK), is less susceptible to metabolic breakdown and produces long-lasting analgesia in animals. The present studies examined the effects of FK on threshold and tolerance of electrically evoked pain in man under double blind conditions. 1.0 mg FK given intramuscularly (saline control) increased tolerance significantly without affecting the pain threshold, but also produced vasodilatation and feelings of oppression and heaviness (study I). In study II, where 50 mg betazole was employed as "placebo" because of its vasodilatatory effects, 1.0 mg FK increased pain tolerance significantly more than 0.25 mg FK while the threshold remained unchanged. Self-ratings of activation and well-being decreased; those of oppression increased, as did reaction time, equally after 0.25 and 1.0 mg FK but were not altered by betazole. In conclusion, 1.0 mg FK i.m. increases tolerance but not perception of pain, thus mimicking the analgesic effects of morphine.

Adult

Transcription factor LbUBC positively regulates salt gland development and salt tolerance by directly binding to the LbTTG1 promoter and repressing its transcription.

KEY MESSAGE:: LbUBC enhances salt tolerance by promoting salt gland development via repressing LbTTG1, revealing a synergisticregulatory mechanism in Limonium bicolor. In the context of increasingly severe soil salinization, salt-tolerant genetic resources from halophytes show great application potential. In particular, the recretohalophyte Limonium bicolor, which possesses specialized salt gland structures, has become a key model for deciphering the molecular mechanisms underlying salt tolerance and salt gland development. In this study, using LbTTG1-overexpressing and -silenced lines, we demonstrate that LbTTG1 negatively regulates salt-gland development and salt tolerance. Through yeast one-hybrid, EMSA, and dual-luciferase assays, Lb7G33228 (LbUBC) was screened and verified as an upstream transcriptional regulator of LbTTG1. LbUBC enhances salt tolerance in L. bicolor by positively regulating salt-gland development, verified using LbUBC silence and overexpression strains. Interestingly, LbUBC represses the expression of its downstream target LbTTG1, thereby releasing the inhibitory effect of LbTTG1 on salt-gland development. In this manner, LbUBC positively regulates salt-gland development, achieving a dynamic balance in the regulation of salt-gland development and salt tolerance in L. bicolor. This study reveals a synergistic regulatory mechanism involving multiple genes, offering new insights for comprehensively dissecting the molecular regulatory network of salt-gland development.

Salt Tolerance

Plant-derived and microbial biostimulants in sustainable agriculture: mechanisms, applications, and challenges.

Plant biostimulants have emerged as transformative and sustainable tools for improving crop productivity, resource-use efficiency, and resilience under rapidly intensifying environmental stresses. Unlike conventional agrochemicals, biostimulants function by activating physiological, biochemical, and molecular processes that optimize plant performance without directly supplying nutrients or exerting pesticidal effects. This review comprehensively examines the integrated roles of plant-derived and microbial biostimulants in sustainable agriculture, with particular emphasis on microbial-mediated mechanisms underlying plant stress adaptation and rhizosphere functioning. Plant-derived biostimulants, including seaweed extracts, humic substances, protein hydrolysates, amino acids, and chitosan, enhance nutrient acquisition, root architecture, hormonal regulation, and antioxidant defense systems. More importantly, microbial biostimulants, such as plant growth-promoting rhizobacteria (PGPR), endophytic microorganisms, mycorrhizal fungi, actinomycetes, yeasts, and cyanobacteria, exert multifunctional effects through biological nitrogen fixation, mineral solubilization, phytohormone biosynthesis, volatile signaling, osmolyte accumulation, pathogen suppression, and modulation of stress-responsive genes. These beneficial microorganisms reshape rhizosphere microbial communities, improve nutrient cycling, and enhance plant tolerance to drought, salinity, heat, and heavy metal toxicity. Emerging evidence from genomics, transcriptomics, metabolomics, and microbiome-based investigations has further revealed the molecular networks and signaling pathways governing biostimulant-induced resilience and plant-microbe interactions. Despite their substantial promise, inconsistent field performance, formulation instability, regulatory limitations, and inadequate mechanistic understanding continue to restrict their large-scale adoption. This review highlights recent advances in microbial and plant-derived biostimulants while identifying critical knowledge gaps and future opportunities for precision biostimulant engineering, microbiome manipulation, and climate-resilient crop management. The integration of next generation biostimulant technologies into sustainable agricultural systems may significantly reduce dependence on agrochemicals while improving crop productivity, environmental sustainability, and global food security.

Agriculture

Advances in Understanding Salt Stress Effects on Growth and Productivity in Sorghum (Sorghum bicolor L. Moench).

Salinity is a growing problem for cereal cultivation because it imposes multiple stresses, including osmotic, ionic, nutritional, and oxidative constraints, on the crop. Sorghum (Sorghum bicolor L. Moench) is considered a climate-smart C4 cereal for food, feed, fodder, forage, and bioenergy, but recent studies indicate that salinity continues to hinder establishment, biomass formation, reproductive growth, and yield. This review compiles the literature on the impacts of salinity on sorghum from 2021 to 2026, with a focus on germination, vegetative growth, physiological and biochemical responses, ion homeostasis, genetic control, productivity, mitigation, and future breeding priorities. In total, 160 records were identified, 118 records were screened after duplicate removal, and 44 recent sources were included in the synthesis. Across comparable sorghum studies, saline/NaCl treatments of approximately 60-200 mM commonly reduced germination by about 20-40%, root and shoot elongation by 25-50%, and biomass by 20-55%, while tolerant genotypes generally maintained higher K+/Na+ balance, 40-60% greater biomass retention, or two- to five-fold stronger ion homeostasis indicators than sensitive lines under similar conditions. Salt stress also lowers leaf expansion, chlorophyll stability, gas exchange, dry matter accumulation, panicle fertility, and grain filling. Tolerant genotypes show greater antioxidant potential, osmotic adjustment, photosynthetic stability, and root system resilience. Recent omics and genome-wide association studies suggest that salinity tolerance in sorghum is polygenic and involves genes related to ion transport, stress signalling, antioxidant regulation, osmolyte metabolism, and growth maintenance. This review recommends a shift from descriptive trait lists to full-cycle field validation, multi-trait selection indices, and integrated packages combining breeding with seed priming, soil water management, amendments, and beneficial microorganisms.

PRISMA

Insights into salt adaptation from comparative genomics of Scirpus mariqueter and a related freshwater species.

The evolutionary mechanisms underlying ecological divergence between closely related species remain a central question in biology. Scirpus mariqueter is a coastal halophyte thriving in the saline intertidal zone and exhibits marked adaptive differences compared to its freshwater relative Bolboschoenus planiculmis. However, the genomic and physiological bases of its salt tolerance remain poorly understood. We generated high-quality genome assemblies for both species and investigated the anatomical and physiological innovations underpinning S. mariqueter's adaptation to extreme environments. Morphological analyses revealed that S. mariqueter evolved specialized traits-including denser leaf palisade tissues, enhanced stem aerenchyma, and compact root cortices-synergistically limiting salt intrusion. Using chromosome-level genomes, we identified lineage-specific expansions in S. mariqueter of gene families critical for salinity tolerance, including those regulating carbohydrate metabolism, photosynthetic fidelity, and reactive oxygen species (ROS) detoxification. Strikingly, germin-like protein (GLP) and wound-induced protein (WIP) families contain tandem repeats mediating ROS scavenging and cell wall integrity, underwent adaptive expansion, paralleling anatomical innovations. Physiological profiling under salt stress confirmed S. mariqueter's unique capacity to maintain photosynthetic activity and carbohydrate production, directly linking genomic adaptations to functional resilience. This study reveals an adaptive strategy whereby structural modifications, diversification of stress-responsive gene families, and metabolic stability collectively enable S. mariqueter to thrive in saline ecosystems.

Salt Tolerance

Phenotypic and phylogenomic characterization of Lactococcus garvieae isolates from rainbow trout (Oncorhynchus mykiss) in Türkiye.

Lactococcosis is an important bacterial disease of farmed fish and causes substantial economic losses in rainbow trout (Oncorhynchus mykiss) aquaculture. In this study, Lactococcus garvieae isolates recovered from rainbow trout farms in Türkiye were characterized using phenotypic, molecular, and phylogenomic methods. Among 32 presumptive Lactococcus isolates recovered from 127 dead rainbow trout, four were confirmed as L. garvieae and exhibited identical biochemical characteristics, Pulsed Field Gel Electrophoresis (PFGE) profiles, and broad growth tolerance across different pH, salinity, and temperature conditions. All isolates were presumptively classified as resistant to ciprofloxacin and florfenicol, while remaining susceptible to tetracycline and penicillin. Based on the AMR profiles, strain LG2, which exhibited the most susceptible antimicrobial profile among the isolates, was selected for whole-genome sequencing (WGS). WGS of the representative isolate LG2 generated a single 2,214,687-bp chromosomal contig with 38.5% GC content and 99.0% BUSCO completeness. In silico PCR assigned LG2 to serotype I, and the genome contained an intact capsule-associated cps/kps locus. The chromosomal lsa(D) determinant and an mdt(A)-like efflux-associated gene were detected, whereas no plasmid replicons or acquired quinolone or florfenicol resistance genes were identified, indicating discordance between the phenotypic and genomic AMR results. Taxonomic verification of 236 publicly available Lactococcus assemblies yielded 41 verified public L. garvieae genomes, which, together with LG2, formed a 42-genome within-species dataset. LG2 was most closely related to the Turkish isolate OS-37, sharing 99.96% ANI and differing by three core SNPs; both belonged to ST109, whereas the other Turkish isolates belonged to ST139. cgMLST identified a conserved genomic backbone, while pan-genome analysis identified 5,655 gene clusters and an open pan-genome characterized by a large cloud-gene fraction. These findings demonstrate the importance of species verification in Lactococcus population genomics and reveal substantial accessory-genome diversity within L. garvieae. The genomic features of LG2 provide a basis for future pathogenicity and immunogenicity studies, although experimental validation is required. Overall, these findings highlight the importance of local genomic surveillance for understanding L. garvieae population structure and provide a genomic framework for future region-specific vaccine research.

Animals

MdWRKY75 interacts with MdWOX11 to modulate root growth under salt stress in apple.

The root system is pivotal for plant development, enabling both vegetative growth and tolerance to abiotic stresses like salinity. However, the molecular mechanisms governing root adaptive development in response to salt stress remain poorly understood in apple (Malus domestica Borkh.). In this study, we identified the salt stress-responsive WRKY transcription factor MdWRKY75. Overexpression of MdWRKY75 in transgenic apple negatively regulates adventitious root (AR) formation and salt stress tolerance, whereas reducing MdWRKY75 expression yields the opposite phenotype. Moreover, MdWRKY75 directly binds to the promoter of MdSAUR15 (SMALL AUXIN UP RNA15) and transcriptionally represses the expression of MdSAUR15, which, when overexpressed, promotes AR formation and enhances salt stress tolerance. We further demonstrated that MdWRKY75 interacts with MdWOX11, a WUSCHEL-related homeobox (WOX) transcription factor, both in vitro and in vivo. MdWOX11 expression is upregulated and enhances AR formation under salt stress. Additionally, MdWOX11 reduces the binding of MdWRKY75 to the MdSAUR15 promoter, and alleviates the MdWRKY75-mediated inhibitory effect on MdSAUR15 expression. Collectively, our study provides a MdWOX11-MdWRKY75-MdSAUR15 module regulating root adaptation in response to salt stress in apple.

Malus