Selection of salt-tolerant plants using tissue culture.
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The perennial herbaceous plant, Phyla nodiflora (Verbenaceae), which possesses natural resistance to multiple abiotic stresses, is widely used as a pioneer species in island ecological restoration. Due to the lack of information about its genome, the mechanism underlying its tolerance to environmental stresses, such as salinity, is almost entirely unknown. Here, we report on the high-quality genome of P. nodiflora that is 403.07 Mb in size, and which was assembled and anchored onto 18 pseudo-chromosomes. Genomic synteny revealed that P. nodiflora underwent two whole genome duplication events, which promoted the expansion of genes related to environmental adaptation and the biosynthesis of secondary metabolites. An integrated genomic and transcriptomic analysis suggested that salt stress tolerance in P. nodiflora is associated with the expansion and activated expression of genes related to abscisic acid (ABA) homeostasis and signaling. The expansion of ZEP family genes may contribute to the consistent increase in ABA levels under salt stress. Lysine acetylomic analysis revealed that exposure to salt led to widespread protein deacetylation, with these proteins primarily involved in signal transduction, carbohydrate transport and metabolism, and transcription regulation. Deacetylation of glutathione S-transferase increased enzymatic activities in response to salt-induced oxidative stress. Collectively, the genomic, transcriptomic, and lysine acetylomic analyses provide profound insight into the molecular basis of the adaptation of P. nodiflora to salt stress, and will be helpful to engineer salt-tolerant plants for ecological restoration.
Soil salinization severely threatens global food security, necessitating systematic investigations of halophytes like Portulaca oleracea to decode the molecular mechanisms of environmental resilience. Utilizing an integrated framework of deep learning-based genome annotation (58,817 predicted genes; 96.5% BUSCO completeness), multi-tissue RNA-Seq, phylogenomics, and gene regulatory network (GRN) inference, the synergistic orchestration of 78 aquaporins (AQPs), 525 heat shock proteins (HSPs), and 119 late embryogenesis abundant (LEA) proteins was elucidated. The active transcriptome, encompassing 39,065 expressed loci, revealed a systemic growth-defense trade-off. Tissues displayed distinct adaptive mechanisms: leaves modulated intracellular water balance via specialized AQPs, whereas adult roots maintained proteostasis through robust HSP20/HSP70 induction. Phylogenomic clustering across 154 species demonstrated that salinity tolerance constitutes an evolutionary mosaic, identifying 81 halophyte-exclusive orthogroups and 1129 species-specific clusters. Comparative topology across six independent GRNs (4.2M-5.3 M edges) unmasked a highly modular transcriptional reprogramming strategy governed by a core apparatus of 22 stress-exclusive regulators, with functional enrichment heavily prioritizing protein dimerization and chromatin remodeling. Theoretically, the distinct convergence of Trihelix transcription factors with guard cell differentiation pathways offers a candidate transcriptomic framework to explain the plant's characteristic C4-CAM photosynthetic plasticity under severe osmotic pressure. Practically, these evolutionary blueprints and specific master switches transcend single-gene transgenic limitations. Utilizing these root-sustained and stress-inducible targets under localized promoters provides a naturally optimized, network-level precision engineering roadmap to transfer robust, compartmentalized halotolerance to sensitive glycophytic crops.
Amid escalating water scarcity and groundwater depletion, halophytes such as Salicornia (Amaranthaceae) represent valuable models for extreme salt tolerance and hold promise for saltwater-based agriculture. Here, we show chromosome-scale genome assemblies for six Salicornia species, revealing four distinct subgenomes, reconciling our assemblies with two existing reference genomes (S. ramosissima UK and S. europaea China), correcting chromosome numbering and orientation. Comparative analyses across ploidy levels demonstrate genome expansion in North American lineages driven by Gypsy retrotransposons, and lineage-specific expansions of two gene families implicated in stress metabolism. Phylogenetic and population-structure analyses of a global resequencing panel of 318 accessions resolve interspecific relationships and establish curated germplasm collections for future crop breeding. Genetic analyses uncover a contrasting population-genetic signal on chromosome 6A between two species, highlighting an OSCA calcium-permeable channel gene as a candidate locus for osmotic adaptation. Together, these resources establish a genomic framework for Salicornia that supports evolutionary studies of halophyte adaptation and crop development.
Zoysia macrostachya Franch. & Sav. is a halophytic perennial turfgrass in the Poaceae family, commonly found in the coastal regions of Korea, Japan, and East Asia. Z. macrostachya thrives in high-salinity environments, making it an excellent model for studying abiotic stress resilience. In this study, we present a chromosome-level genome assembly of Z. macrostachya, constructed using Oxford Nanopore long reads, Illumina short reads, and Omni-C sequencing data. The assembly spans 329.78 Mb across 20 chromosomes, with a scaffold N50 of 19.24 Mb, and includes complete telomeric sequences at both ends. The assembly showed 97.8% complete BUSCOs, indicating high genome completeness. Repeat element and gene annotation identified 44.03% of the genome as repetitive elements and 33,474 protein-coding genes. The gene annotation showed 97.1% complete BUSCOs and 86.92% functionally characterized genes. Macrosynteny analysis highlighted highly collinear relationships with related species, providing a foundational understanding of the Z. macrostachya genomic structure. This high-quality genome serves as a valuable resource for advancing salinity tolerance research and improving the genetic diversity of Zoysia species.
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.
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.
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.
Gene editing of three key genes, G PROTEIN GAMMA SUBUNIT, CYTOKININ OXIDASE, AND IDEAL PLANT ARCHITECTURE 1, improved the salt-tolerant japonica rice variety Lansheng increased yield while maintaining its original salt tolerance and quality.
Salinity is a major constraint to crop productivity. Beneficial plant-fungus interactions represent a promising strategy to enhance stress resilience. Here, we investigated fungal endophytes isolated from the roots of Oryza sativa cultivated in saline-prone marshlands of the Guadalquivir River, Spain. From a collection of 38 isolates, five salt-tolerant strains exhibiting plant growth-promoting activity were identified, including a previously uncharacterized Reticulascus sp. strain S5. Co-cultivation assays with the non-native host plant Arabidopsis thaliana demonstrated that S5 increased the root and shoot biomass under salt stress. To elucidate the underlying molecular mechanisms, a comprehensive RNA-Seq analysis of the roots and shoots under control and saline conditions was performed. Fungal colonization induced pronounced transcriptomic changes, particularly in the shoots, including rewiring of the auxin- and abscisic acid-related pathways and the induction of genes associated with cell wall remodeling. Concurrently, defense-related processes, including glucosinolate biosynthesis and ethylene signaling, were broadly repressed, suggesting attenuated stress perception in colonized plants. In the roots, S5 inoculation suppressed the expression of genes involved in root hair development and cell wall organization, indicating a fungus-driven reconfiguration of root development. Moreover, comparative analysis with Fusarium sp. K-23, a fungus that has previously been demonstrated to promote plant growth under salinity stress, revealed distinct root-associated mechanisms but convergence on a shared regulatory module in shoots involving ABA-responsive transcription factors and osmotic stress regulators. Collectively, our findings demonstrate that Reticulascus sp. S5 enhances plant salt stress tolerance through the coordinated transcriptional reprogramming of growth, hormone signaling, and stress responses, highlighting a possible potential of habitat-adapted endophytes for sustainable crop improvement.
To explore the effects of salt-tolerance gene accumulation on salt tolerance in transgenic plant, we used four types of plant expression vector (N27, N28, N29, and N30) carrying mtlD, mtlD + gutD, mtlD + gutD + BADH, mtlD + gutD + BADH + sacB genes respectively, to transform tobacco through Agrobacterium-mediated method. Transgenic lines were identified through polymerase chain reaction (PCR) detection. Transgenic lines and non-transgenic plant (CK) were subjected to 6‰ sodium chloride solution stress; then, fluorescence quantitative PCR (FQ-PCR) and salt tolerance indexes were used to assess characteristics. PCR showed the exogenous genes had been integrated into the tobacco genome. FQ-PCR showed under clean water treatment the target genes were expressed in all transgenic plants at the transcriptional level. The transcript abundances of target genes changed with the number of genes increased, and improved following salt stress. Comparative analyses of salt tolerance indexes showed height growth, biomass (except for N29), chlorophyll content, net photosynthetic rate, Fv/Fm, and PI of all transgenic plants and CK were lower under salt stress than under clean water treatment, to varying degrees. However, the descent ratio was smaller in transgenic plants. A comprehensive evaluation of multiple salt-tolerance indicators performed using the membership function method showed the average salt tolerance of each vector transgenic line was higher than that of CK, and salt tolerance was greater in transgenic polyvalent gene lines than in transgenic monovalent gene lines. The average salt tolerance was N29 > N28 > N30 > N27 > CK. This study provides a theoretical and practical reference for salt tolerance breeding in other plants.
The salt-tolerant genes (STGs) play important roles in protecting plants against salt stress. Although various types of STGs have been systematically characterized in plant species, the key genes (KGs) regulating salt stress tolerance in rice (Oryza sativa L.) remain elusive. This study focused on the identification and characterization of the members of STGs in rice through integrated bioinformatic and molecular approaches, including chromosomal location, physicochemical characteristics, protein-protein interaction, and expression profiles of the identified genes. A total of 164 differentially expressed genes (DEGs) were systematically identified as responsive to salt tolerance and sorted out potential 12 kg (OsHSP20.2, OsGFP2, OsBBTI2, OsEN20.6, OsUBC17, OsACD5, OsPEAB5, OsDP11, OsDFP5, OsWD40.7, OsEP11.1, and OsGRAM12) through the CytoHubba algorithms analysis. Physicochemical characterization indicated substantial variation among KGs, including genomic sequences (824-4051 bp), amino acid length (148-659 aa), molecular weight (16.39-71.35 kDa), and isoelectric point (4.66-10.37). Protein-protein interaction (PPI) network prediction indicated intricate functional associations among key STGs. Gene Ontology (GO) enrichment analysis revealed that the KGs are involved in numerous biological processes and molecular functions. Moreover, gene homology results revealed that KGs have multiple relationships with other plant species. Co-expression network analysis revealed that 12 kg are potentially involved in the regulatory mechanisms underlying the biological process. Relative gene expression through the comparative threshold (ΔΔCT) of qRT-PCR revealed that the KGs are salt-induced and may play crucial roles in rice responses to salt stress. Tissue-specific expression patterns revealed that the KGs significantly altered expression levels across different tissues and under stress. This systematic investigation demonstrated that the 12 identified genes may play roles in the development of salt-tolerant rice varieties.
The Ankyrin-repeat proteins (ANKs) play a key role in plant development and in response to abiotic stress. This research identified family members of the ANK genes in Sorghum bicolor at the whole-genome level, analyzed their sequence characteristics, evolutionary relationships, and expression patterns, and provided a scientific basis for elucidating the functionality of SbANK genes and for salt-tolerant breeding. Using bioinformatics methods, this study conducted a comprehensive identification of the SbANK gene family, analyzing its physicochemical properties, domain composition, chromosomal distribution, colinearity relationships, promoter cis-acting elements, and conserved protein motifs. Transcriptomic data and qRT-PCR were used to detect changes in their expression under salt stress. A total of 186 ANK family members were identified in the Sorghum bicolor genome, classified into 13 subfamilies and unevenly distributed across 10 chromosomes. Intra-species colinearity analysis revealed 7 pairs of duplicated genes, while inter-species colinearity analysis showed that S. bicolor and Oryza sativa share 88 pairs of orthologs, far exceeding the number found in Arabidopsis thaliana (11 pairs). Promoter analysis indicated that SbANK genes are enriched with cis-acting elements associated with hormone responses (particularly MeJA elements, accounting for 51.7%) and stress responses (particularly anaerobic-inducible elements, accounting for 60.9%). Transcriptomic expression analysis revealed that SbANK genes exhibit distinct tissue specificity, with the ANK-IQ subfamily highly expressed in leaves and the ANK-M subfamily showing the most widespread response under salt stress. Expression levels of the 10 candidate genes showing the most significant responses to salt stress were analyzed using qRT-PCR. The results indicated that SbANK91, SbANK135, and SbANK136 were significantly upregulated under 200 mmol/L NaCl treatment. The SbANK family is distinguished by a large number of member genes and structural diversity, with the ANK-M subfamily being the primary group responding to salt stress. SbANK91, SbANK135, and SbANK136 are identified as putative candidate genes for salt stress responses.
Mediterranean regions are among the most important areas for global grape production, characterized by dry climates and frequent challenges associated with soil salinity. In these environments, chloride toxicity is a major factor limiting vine growth and fruit quality. Despite the critical role of chloride exclusion in salinity tolerance, the genetic mechanisms underlying this trait remain poorly understood. In this study, we analyzed natural variation in chloride exclusion using a diverse panel of 335 accessions representing 18 wild and cultivated Vitis species. This panel, comprising accessions from the southwestern United States and Mexico, captures a broad range of evolutionary adaptations to abiotic stress and provides a valuable genetic resource for breeding efforts aimed at introducing novel traits. Using genome-wide association and quantitative trait loci (QTL) mapping, we identified a major QTL on chromosome 8, now designated qClEx8.1, containing candidate genes encoding cation/H⁺ exchangers (CHXs), which are involved in ion transport and homeostasis. To validate these findings, we analyzed a mapping population derived from Vitis acerifolia longii 9018 and the commercial rootstock GRN3, confirming the chromosome 8 locus as a major determinant of chloride exclusion. Structural variant analysis revealed nonsynonymous substitutions within CHX genes that may influence protein function and salinity tolerance. Additionally, we discovered a novel QTL on chromosome 19 enriched with G-type lectin S-receptor-like serine/threonine-protein kinases, known regulators of stress signaling. By integrating phenotypic and genomic data across a diverse Vitis collection, this study advances our understanding of the genetic architecture underlying chloride exclusion and highlights candidate genes for breeding salt-tolerant rootstocks.
Spontaneous mutants of Rhizobium leguminosarum biovar viciae strain C1204b were selected for their ability to tolerate 0.2 M NaCl, a growth-inhibiting level of salt for the parental strain. Transposon-mediated salt-sensitive mutants of strain C1204b were screened for their inability to grow in 0.08 M NaCl. Quantitation of the free-amino acid pools in the mutants grown in NaCl revealed a dramatic increase in glutamine, serine, glutamate and proline, and to a lesser extent alanine and glycine in the salt-tolerant mutants in comparison with the parental strain exposed to NaCl; but only glutamate and proline increased in the salt-sensitive mutants under NaCl stress. Extracellular polysaccharide levels were quantitated for the salt-tolerant mutants and determined to be approximately two-fold higher than for the parental strain. Although the mutations that occurred in the NaCl-tolerant and NaCl-sensitive strains did not interfere with nodule formation, no nitrogenase activity could be observed in the NaCl tolerant mutants as evaluated by acetylene reduction.
GmCXE31 negatively regulates salt tolerance and lipid synthesis in soybean, and the cxe31-edited lines improve soybean yield and seed quality. Carboxylesterases (CXEs), as essential lipid hydrolases of the α/β-hydrolase fold superfamily, are critical for plant stress responses, hormone signaling and secondary metabolism. The key candidate gene GmCXE31 was previously identified in our laboratory through a genome‑wide association study (GWAS) of soybean lipid‑related traits. In the present study, we further identified 60 GmCXE family genes in soybean. Phylogenetic analysis clustered them into 11 conserved subfamilies. Cis-acting element analysis showed their promoters are enriched with elements related to abiotic stress, growth and hormone signaling, suggesting potential roles in soybean development and stress adaptation. GmCXE31 is highly expressed in seedling roots and responsive to strigolactones (SLs) and salt stress. Functional assays revealed that GmCXE31 negatively regulates soybean salt tolerance: its overexpression reduced salt tolerance in Arabidopsis and soybean under 150 mM NaCl stress, while its knockout enhanced this trait. Lipid profiling revealed GmCXE31-edited lines had higher seed oil content, elevated oleic/linoleic acid ratio and lower saturated fatty acid proportion, which was achieved by regulating lipid synthesis-related genes like GmNFYA. Agronomic trait analysis showed GmCXE31-edited lines had increased nodule number, plant height and single-plant yield at maturity, with opposite phenotypes in overexpression lines. In conclusion, this study elucidates the multifaceted roles of GmCXE31 in coordinating soybean salt tolerance, lipid metabolism and agronomic traits, providing theoretical and genetic resources for salt-tolerant and high-quality soybean molecular breeding.
Endo-β-1,4-mannanases (MANs) are glycoside hydrolase family 5 (GH5) enzymes that degrade cell wall mannan polysaccharides and participate in plant growth and stress adaptation. This gene family has not been systematically characterized in common wheat (Triticum aestivum L.). Here, we identified 24 TaMAN genes (TaMAN1-TaMAN24) genome-wide and analyzed their phylogeny, gene structures, chromosomal distribution, synteny, and promoter cis-acting elements. Expression profiles under biotic and abiotic stresses were investigated using public databases, salt-stress RNA-seq, and RT-qPCR. TaMAN proteins (386-475 aa) were mainly predicted to localize in the extracellular space. Phylogenetic analysis divided them into three groups, with Groups II and III representing monocot-specific expansions. Family expansion was driven primarily by whole-genome duplication, supplemented by tandem duplication on homoeologous group 6. Promoters were enriched in hormone- and stress-responsive cis-acting elements (ABRE, as-1/CGTCA-motif, W box). TaMAN1, TaMAN5, TaMAN8, TaMAN9, TaMAN16 and TaMAN19 were significantly induced by powdery mildew, while TaMAN3, TaMAN4 and TaMAN19-TaMAN22 rapidly responded to salt stress. This study provides candidate genes for disease-resistant and salt-tolerant wheat breeding.