In exo-recretohalophytes, specialized structures known as salt glands secrete excess salt ions from plant tissues, thereby maintaining intracellular ion homeostasis and sustaining normal metabolism under salt stress. Based on their cellular composition, salt glands can be unicellular, bicellular, or multicellular, and they originate from undifferentiated precursor cells known as multipotent epidermal stem cells. A complex regulatory network drives the division and differentiation of these cells into functional salt-secreting structures. Three hypotheses have been proposed to explain the physiological mechanisms underlying salt secretion by salt glands, each supported by experimental evidence: The osmotic mechanism, the reverse pinocytosis mechanism, and the animal-like fluid transport mechanism. This review summarizes the structural characteristics, developmental processes, salt secretion mechanisms, and potential applications of salt glands in exo-recretohalophytes, providing a foundation for future studies on salt gland biology and their utilization in developing salt-tolerant crops.

Background and Aims Soil salinization severely limits crop productivity worldwide. Halophytes provide important biological resources for identifying genes that confer salinity tolerance. Salt glands, specialized epidermal structures in recretohalophytes like Limonium bicolor, excrete excess Na+, yet their developmental and functional regulation remains poorly understood.Methods We focused on LbSTICHEL (LbSTI), a homologue of the Arabidopsis trichome regulator STI, and investigated its function and upstream regulation in L. bicolor.Key Results LbSTI was found to be specifically expressed in the salt glands of L. bicolor, as determined by RNA in situ hybridization and GUS histochemical staining driven by the LbSTI promoter. The function of LbSTI was further investigated in L. bicolor. In L. bicolor, plants overexpressing LbSTI produced more salt glands, exhibited increased salt secretion and greater tolerance to salinity stress compared with non-transformed controls. Conversely, LbSTI-knockdown plants generated by virus-induced gene silencing showed opposite phenotypes. Meanwhile, LbSTI increased trichome development and salinity tolerance in wild-type plants and an sti mutant of Arabidopsis, which indicated conservation across species. Furthermore, the upstream regulatory transcription factor LbMUSTANG3 (LbMUG3) was identified to bind to the LbSTI promoter and inhibit the transcription of LbSTI using a yeast one-hybrid and dual-luciferase reporter assay. Further functional validation revealed that LbMUG3 delayed salt gland development in L. bicolor. Additionally, the expression of LbSTI was significantly upregulated in LbMUG3-silenced lines.Conclusions The current LbMUG3-LbSTI module may be a useful tool to cultivate plants with salinity tolerance.

The Casparian strip (CS) and suberin form the apoplastic barrier, which blocks solute entry into the vascular stele under high-salt conditions. Here, transcriptome analysis of Arabidopsis roots during three stages of apoplastic barrier development (undifferentiated, differentiating, and mature) revealed differential expression of the transcription factor gene WRKY71. We generated WRKY71-overexpression (OE) and WRKY71-complementation (COM) lines. Metabolomics analysis showed that WRKY71 promotes the biosynthesis of S-type lignin monomers. Compared to wild type and wrky71 plants, WRKY71-OE and COM plants showed enhanced apoplastic barrier formation and less Na+ accumulation under 100 mM NaCl treatment, exhibiting better salt-exclusion ability during germination and seedling growth. Yeast one-hybrid assays revealed that WRKY71 interacts with the lignin biosynthesis genes 4CL3 and CUTICLE DESTRUCTING FACTOR 1 (CDEF1). WRKY71 promotes 4CL3 expression and inhibits CDEF1 expression, as determined by dual-luciferase reporter assay. To further investigate the functions of 4CL3 and CDEF1, we generated the corresponding OE lines. Under 100 mM NaCl treatment, 4CL3-OE plants showed enhanced salt tolerance through increased CS formation, whereas CDEF1-OE plants showed increased salt sensitivity due to inhibited suberin formation. Our identification of the WRKY71–4CL3/CDEF1 module sheds light on the role of the apoplastic barrier in salt tolerance.

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.

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.

Plant pathogens use a diverse arsenal of effectors to suppress host immunity, though the precise mechanisms of their action are often not fully understood. In this study, we characterize FolCP1b, a cerato-platanin (CP) effector secreted by Fusarium oxysporum f. sp. lycopersici (Fol), as a key intracellular virulence factor that disrupts host defenses and protects other Fol effectors. FolCP1b interacts with the host apoplastic subtilase SlSBT1 within the plant cytoplasm, leading to its intracellular retention and preventing its secretion to the apoplast. As a result, SlSBT1-mediated degradation of key Fol effectors, such as FolEP1 and FolEP2, is impaired, thereby promoting Fol infection. Unlike canonical protease inhibitors, FolCP1b operates by altering host protein subcellular localization rather than inhibiting enzymatic activity. Our findings unveil a novel "effector hijacking" mechanism, through which one intracellular effector safeguards apoplastic effectors from host proteolytic degradation, thereby enhancing fungal pathogenicity.

IntroductionDeveloping salt-tolerant crops is critical for utilizing saline soils in agriculture. Limonium bicolor, a recretohalophyte with epidermal salt glands, represents a valuable genetic resource for salt tolerance engineering. Although WD40 proteins are known regulators of plant stress responses, their roles in L. bicolor remain unexplored.MethodsWe performed a genome-wide analysis of WD40 genes in L. bicolor, including phylogenetic classification, subcellular localization prediction, cis-element analysis, and expression profiling during salt stress. Functional validation was conducted using virus-induced gene silencing (VIGS).ResultsAmong 367 identified WD40 genes (distributed across all chromosomes), Subfamily 6 was the largest. Two key members (Lb1G05968 and Lb3G17197, localized in cytoplasm) showed significant involvement in salt gland development and stress tolerance, as demonstrated by VIGS-induced phenotypic defects.DiscussionOur findings reveal the WD40 family's expansion in L. bicolor and its functional specialization in salt adaptation. The identified genes (e.g., Lb1G05968, Lb3G17197) provide targets for engineering salt-tolerant crops. This study establishes a foundation for further research on halophyte developmental genetics.

Constructed wetlands (CWs) are widely used for the denitrification of wastewater because of their high efficiency and low pollutant consumption. However, insufficient internal dissolved oxygen (DO) or a lack of electron donors has resulted in a blocked electron supply and acceptance process for the nitrogen removal (N-removal) process, severely restricting the N-removal efficiency of CWs. In this study, the electron transfer mechanism of the N-removal process in CWs was reviewed, and the effects of plant action and substrate adsorption on the nitrogen cycle were discussed. To address the challenge of restricted nitrogen cycling in CWs, innovative strategies such as intermittent aeration to optimize the distribution of DO, introduction of metal oxide substrates to strengthen the electron transfer efficiency, and coupled bioelectrochemical systems (BES) have been proposed to induce system electron donors and acceptors to maintain the transfer balance. In the future, further research should explore the deep synergy between CWs and BES, development of new types of CWs fillers, and overcome the effects of low temperatures, as well as to implement further intelligent monitoring and management measures to address impeded nitrogen cycling in CWs and enhance the potential of the application of CWs in the field of wastewater denitrification.

The recretohalophyte Limonium bicolor produces salt glands on its leaf surface and can thrive in saline soils. YABBY (YAB) family transcription factors participate in plant development and stress responses; our previous single-cell transcriptome data revealed high expression of LbYAB1 (Lb2G09016) in salt gland subclusters. Here, we confirmed the LbYAB1 expression in salt glands and leaf primordia using RNA in situ hybridization and promoter-driven GUS histological staining; moreover, LbYAB1 expression was induced under 6-benzylaminopurine treatment. We explored LbYAB1 function by generating overexpression and knockdown plants in L. bicolor. Compared with the wild-type, LbYAB1-overexpressing lines showed suppressed salt gland development, diminished salt secretion, and lower salinity tolerance; LbYAB1 knockdown plants displayed the opposite phenotypes, demonstrating that LbYAB1 is a negative regulator of salt gland development. LbYAB1 conferred greater salinity sensitivity in L. bicolor and in Arabidopsis thaliana when overexpressed. Multiple assays (a yeast two-hybrid, split-luciferase complementation and GST pull-down) supported the interaction of LbYAB1 with LbKNAT7 (Homeobox protein knotted-1-like 7), recently shown to inhibit transcription of LbSAD2, a positive regulator of salt gland development and salt tolerance. The LbYAB1-LbKNAT7 interaction enhanced the transcriptional repression of LbSAD2, thus regulating salt gland development. Our work places LbYAB1 upstream of LbSAD2 in salt gland development, providing insights into salinity tolerance genetics and paving the way for engineering salt-resistant crops, even in species lacking salt glands.

The architecture, shape, size, and structure of leaves are regulated by endogenous and exogenous signals, including phytohormones, various stresses, and pathogens. Plants can sense these stimuli through various receptors, which trigger intracellular signaling cascades, modulate gene expression, and guide leaf development. In recent years, many receptors located at the plasma membrane have been reported to participate in leaf development in response to developmental signals and environmental factors. These membrane receptors, which perceive signals in epidermis cells, mesophyll cells, and vascular tissues, act as the "eyes" of plants to regulate leaf development, especially under various stresses. In this review, we briefly summarize the signaling mechanisms mediated by membrane receptors during leaf development, including the upstream factors influencing leaf development, their dedicated membrane receptors, and their downstream signaling pathways. Plants perceive external signals (such as salt, drought, pathogens, etc.) through receptors, which then initiate signal transduction cascades and ultimately form a regulatory network. Understanding how plant membrane receptors sense, decode, and transduce signals has important theoretical and practical implications for the study of plant responses to the environment and the genetic improvement of crop resistance to stress conditions imposed by the external milieu.

Limonium bicolor is a typical recretohalophyte with specialised salt glands that secrete excessive Na+ out of the plant. The detailed mechanisms of salt gland development and salt resistance are largely unclear. Here, we investigated the function of the importin-β protein LbSAD2 from L. bicolor. Lines with silenced LbSAD2 expression showed significantly lower salt gland density, salt-secretion ability and salt resistance, whereas LbSAD2 overexpression lines had a greater number of salt glands with an abnormal distribution in the abaxial and leaf adaxial surfaces. A previously uncharacterised hydrophobic protein, Lb2G12077, can bind to the LbSAD2 promoter to inhibit the transcription of LbSAD2 verified by a yeast one-hybrid, electrophoretic mobility shift and dual-luciferase reporter assay. Further functional validation revealed that Lb2G12077 demoted salt gland development and salt resistance of L. bicolor. Moreover, a yeast two-hybrid, bimolecular fluorescence complementation and GST pull-down assays indicated that the hypothetical protein Lb2G12567 interacts with LbSAD2, whose silenced lines also showed significantly reduced salt gland density, salt-secretion ability and salt resistance, suggesting Lb2G12567 played a similar positive role in salt resistance. The current LbSAD2 pathway sheds light on salt gland development and salt resistance in L. bicolor, laying the foundation for increasing salt tolerance in crops.

Halophytes can grow and reproduce normally in an environment containing more than 200 mM NaCl, offering untapped gene resources for improving crop salinity tolerance. As a recretohalophyte, Limonium bicolor can secrete excess Na+ through salt glands, specialized structures on the leaf and stem epidermis. Here, we identified a MYB transcription factor gene, LbMYB368, that is highly expressed during salt gland development. We confirmed its expression in salt glands using RNA in situ hybridization and a promoter reporter construct. To investigate in detail the roles of LbMYB368 in salinity tolerance, we overexpressed and knocked down the gene, via virus-induced gene silencing (VIGS), in L. bicolor. The transgenic L. bicolor overexpression lines developed more salt glands, while the VIGS plants had fewer salt glands. The salt secretion ability and salt tolerance of these plants were correlated with the changes in salt gland development, indicating that LbMYB368 plays an important role in the salt tolerance of L. bicolor by enhancing salt gland development and salt secretion. We also investigated the effect of LbMYB368 on enhanced salinity tolerance when heterologously expressed in Arabidopsis to assess its potential applications in non-halophytes for future conferring salinity tolerance in crops.

NAC transcription factors regulate plant growth, development, and stress responses. However, the number, types, and biological functions of Limonium bicolor LbNAC genes have remained elusive. L. bicolor secretes excessive salt ions through salt glands on its stems and leaves to reduce salt -induced damage. Here, we identified 63 NAC members (LbNAC1-63) in L. bicolor, which were unevenly distributed across eight chromosomes. Cis-elements in the LbNAC promoters were related to growth and development, stress responses, and phytohormone responses. We observed strong colinearity between LbNACs and GmNACs from soybean (Glycine max). Thus, LbNAC genes may share similar functions with GmNAC genes. Expression analysis indicated that 16 LbNAC genes are highly expressed in roots, stems, leaves, and flowers, whereas 17 LbNAC genes were highly expressed throughout salt gland development, suggesting that they may regulate this developmental stage. Silencing LbNAC54 in L. bicolor decreased salt gland density, salt secretion from leaves, and overall salt tolerance. In agreement, genes related to salt gland development were significantly downregulated in LbNAC54-silenced lines. Our findings shed light on LbNAC genes and help elucidate salt gland development and salt secretion in L. bicolor. Our data also provide insight into NAC functions in halophytes.

Soil salinization is a growing environmental problem and a major factor limiting crop production. Growing salt-adapted crops may allow farmers to utilize farmland with high salt concentrations. For example, leaves of the halophyte Mesembryanthemum crystallinum (also known as ‘ice plant’) are consumed raw, cooked, or pickled. Here, we examine the mechanism by which salt improved the quality of this vegetable crop and provide theoretical support for planting M. crystallinum in saline soils or growing it in hydroponic facilities. Four-leaf stage M. crystallinum seedlings were treated with 0, 50, 100, 150, 200, 250 and 300 mM NaCl for 4 weeks and then plant growth and ion, nutrient and betacyanin contents were measured. Compared to the control (0 mM NaCl), treatment with 50 and 100 mM NaCl significantly improved M. crystallinum growth and increased the contents of nutrients (protein, dietary fiber, organic acids, and soluble sugars) and betacyanins. By contrast, treatment with 150–300 mM NaCl had the opposite effect. Genes encoding the betacyanin biosynthesis enzymes tyrosinase, DODA, 5-GT, and CYP76AD1 were upregulated in plants treated with 100 mM NaCl. Four betacyanin derivatives were also identified in this species. Treatment with 100 mM NaCl significantly improved the growth, nutrient contents and betacyanin contents of this species, thereby improving its commercial value. These findings thus provide guidance for hydroponic or field growth of this emerging vegetable crop.

Transcription factors with basic helix-loop-helix (bHLH) structures regulate plant growth, epidermal structure development, metabolic processes, and responses to stress extensively. Sea lavender (Limonium bicolor) is a recretohalophyte with unique salt glands in the epidermis that make it highly resistant to salt stress, contributing to the improvement of saline lands. However, the features of the bHLH transcription factor family in L. bicolor are largely unknown. Here, we systematically analyzed the characteristics, localization, and phylogenetic relationships of 187 identified bHLH family genes throughout the L. bicolor genome, as well as their cis-regulatory promoter elements, expression patterns, and key roles in salt gland development or salt tolerance by genetic analysis. Nine verified L. bicolor bHLH genes are expressed and the encoded proteins function in the nucleus, among which the proteins encoded by Lb2G14060 and Lb1G07934 also localize to salt glands. Analysis of CRISPR-Cas9-generated knockout mutants and overexpression lines indicated that the protein encoded by Lb1G07934 is involved in the formation of salt glands, salt secretion, and salt resistance, indicating that bHLH genes strongly influence epidermal structure development and stress responses. The current study lays the foundation for further investigation of the effects and functional mechanisms of bHLH genes in L. bicolor and paves the way for selecting salt-tolerance genes that will enhance salt resistance in crops and for the improvement of saline soils.

Limonium bicolor, known horticulturally as sea lavender, is a typical recretohalophyte with salt glands in its leaf epidermis that secrete excess Na+ out of the plant. Although many genes have been proposed to contribute to salt gland initiation and development, a detailed analysis of alternative splicing, alternative polyadenylation patterns, and long non-coding RNAs (lncRNAs) has been lacking. Here, we applied single-molecule long-read mRNA isoform sequencing (Iso-seq) to explore the complexity of the L. bicolor transcriptome in leaves during salt gland initiation (stage A) and salt gland differentiation (stage B) based on the reference genome. We identified alternative splicing events and the use of alternative poly(A) sites unique to stage A or stage B, leading to the hypothesis that they might contribute to the differentiation of salt glands. Based on the Iso-seq data and RNA in situ hybridization of candidate genes, we selected the lncRNA Btranscript_153392 for gene editing and virus-induced gene silencing to dissect its function. In the absence of this transcript, we observed fewer salt glands on the leaf epidermis, leading to diminished salt secretion and salt tolerance. Our data provide transcriptome resources for unraveling the mechanisms behind salt gland development and furthering crop transformation efforts towards enhanced survivability in saline soils.

Abscisic acid (ABA) plays an important role in regulating plant stress responses. However, there are currently no reports on ABA in regulating the salt tolerance of Limonium bicolor. Here, we analyzed the effects of exogenous ABA on growth, photosynthesis, salt secretion, and physiological and transcriptome changes of L. bicolor seedlings under 300 mM NaCl. Exogenous 10 mu M ABA treatment in L. bicolor seedlings treated with salt upregulated the expression of genes related to stomatal development, increased the density and number of stomata, and improved plant gas exchange capacity, thereby increasing the net photosynthesis rate. Exogenous ABA treatment in L. bicolor seedlings under salt stress also upregulated the expression of genes related to salt gland development, promoted salt glands development, increased salt glands density, increased the salt secretion capacity of leaves, and reduced the accumulation of Na+ in the plant. ABA application enhanced salt tolerance of L. bicolor seedlings by increasing content of soluble solutes, enhancing antioxidant enzymes activities, decreasing accumulation of reactive oxygen species, and increasing ABA and indole acetic acid contents. Transcriptome sequencing revealed that genes differentially expressed in L. bicolor seedlings during ABA -induced salt tolerance are involved in many metabolic pathways, including plant hormone signal transduction, proline metabolism, endocytosis and protein processing in the endoplasmic reticulum. Furthermore, some transcription factors, MYB, bHLH, and WRKY, might contribute to the improved salt stress tolerance of L. bicolor conferred by ABA. These results provide a basis for further clarifying the salt tolerance mechanism mediated by exogenous ABA in L. bicolor seedlings.

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 FINGER PROTEIN 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.

Limited organic carbon in drinking water constrains the removal of nitrate‑nitrogen (NO3--N) via aerobic denitrification. This paper reports the use of silica-coated nano zero-valent iron (nZVI@SiO2) as a stable and sustainable electron donor to enhance aerobic denitrification. The nZVI@SiO2, synthesized via a one-step method, was resistant to oxidation and exhibited excellent stability. In conjunction with aerobic denitrifying bacteria, nZVI@SiO2 achieved NO3--N and total nitrogen TN removal efficiencies of 90.64 % and 80.94 %, respectively. This represents an increase of 24.15 % in the efficiency of TN removal compared with that of the nZVI system. The activity of the nZVI system diminished gradually after just three cycles, whereas nZVI@SiO2 maintained NO3--N and TN removal efficiencies of 89.33 % and 78.08 %, respectively, after four cycles, respectively, indicating its sustainable ability to enhance aerobic denitrification. Cyclic voltammetry and electrochemical impedance spectroscopy demonstrated enhanced electron transfer efficiency of nZVI@SiO2. Furthermore, nZVI@SiO2 significantly promoted the activity of the electron transfer system, ATP levels, nitrate/nitrite reductase activity, contents of complexes I and III, and extracellular polymeric substances. nZVI@SiO2 significantly enhanced electron generation, transfer, and consumption during biological denitrification by functioning as both an electron donor and mediator. The findings implicate nZVI@SiO2 as a means to enhance nitrogen removal by aerobic denitrifying microorganisms in oligotrophic water via sustained donation of electrons.

Salt gland is an epidermal Na+ secretory structure that enhances salt resistance in the recretohalophyte sea lavender (Limonium bicolor). To elucidate the salt gland development trajectory and related molecular mechanisms, we performed single-cell RNA sequencing of L. bicolor protoplasts from young leaves at salt gland initiation and differentiation stages. Dimensionality reduction analyses defined 19 transcriptionally distinct cell clusters, which were assigned into 4 broad populations-promeristem, epidermis, mesophyll, and vascular tissue-verified by in situ hybridization. Cytokinin was further proposed to participate in salt gland development by the expression patterns of related genes and cytological evidence. By comparison analyses of Single-cell RNA sequencing with exogenous application of 6-benzylaminopurine, we delineated 5 salt gland development-associated subclusters and defined salt gland-specific differentiation trajectories from Subclusters 8, 4, and 6 to Subcluster 3 and 1. Additionally, we validated the participation of TRIPTYCHON and the interacting protein Lb7G34824 in salt gland development, which regulated the expression of cytokinin metabolism and signaling-related genes such as GLABROUS INFLORESCENCE STEMS 2 to maintain cytokinin homeostasis during salt gland development. Our results generated a gene expression map of young leaves at single-cell resolution for the comprehensive investigation of salt gland determinants and cytokinin participation that helps elucidate cell fate determination during epidermis formation and evolution in recretohalophytes.