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AP2/ERF transcription factors (TFs) play important roles in plant growth and development, plant morphogenesis and response to environmental stresses. However, their biological roles in recretohalophytes are still not fully revealed. Limonium bicolor L. is a typical recretohalophyte, which secretes excessive salt ions through the salt glands on the epidermis. Here, 64 LbAP2/ERF genes were identified in L. bicolor genome, which were unevenly distributed on the eight chromosomes. Cis-elements related to growth and development, stress response and phytohormone response are distributed in multiple LbAP2/ERF promoters. Expression analysis indicated that LbAP2/ERF genes responsed to NaCl, PEG and ABA. And the salt gland density, salt secretion of leaves and overall salt tolerance of LbAP2/ERF32 silenced lines were significantly reduced. In agreement, the genes related to salt gland development and ion transport were significantly changed in LbAP2/ERF32-silenced lines. Our findings provided fundamental information on the structure and evolutionary relationship of LbAP2/ERF gene family in salt gland development and salt secretion of L. bicolor and gave theoretical guideline for further functional study of LbAP2/ERF genes in response to abiotic stress.

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KEY MESSAGE: 63 L. bicolor WRKY genes were identified and their informatics was analyzed. The results suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Salt stress, as a universal abiotic stress, severely inhibits the growth and development of plants. WRKY transcription factors play a vital role in plant growth and development, as well as in response to various stresses. Nevertheless, little is known of systematic genome-wide analysis of the WRKY genes in Limonium bicolor, a model recretohalophyte. In this study, 63 L. bicolor WRKY genes were identified (LbWRKY1-63), which were unevenly distributed across seven chromosomes and one scaffold. Based on the structural and phylogenetic characteristics, 63 LbWRKYs are divided into three main groups. Cis-elements in the LbWRKY promoters were related to growth and development, phytohormone responses, and stress responses. Colinearity analysis showed strong colinearity between LbWRKYs and GmWRKYs from soybean (Glycine max). Therefore, LbWRKY genes maybe have similar functions to GmWRKY genes. Expression analysis showed that 28 LbWRKY genes are highly expressed in roots, 9 in stems, 26 in leaves, and 12 in flowers and most LbWRKY genes responded to NaCl, ABA, and PEG6000. Silencing LbWRKY10 reduced salt gland density and salt secretion ability of leaves, and the salt tolerance of the species. Consistent with this, genes associated with salt gland development were markedly down-regulated in the LbWRKY10-silenced lines. Our findings suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Our research provides new insights into the functions of the WRKY family in halophytes.

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Ulcerative colitis (UC) is a kind of inflammatory bowel condition characterized by inflammation within the mucous membrane, rectal bleeding, diarrhea, and pain experienced in the abdominal region. Existing medications for UC have limited treatment efficacy and primarily focus on symptom relief. Limonium bicolor (LB), an aquatic traditional Chinese medicine (TCM), exerts multi-targeted therapeutic effects with few side effects and is used to treat anemia and hemostasis. Nevertheless, the impact of LB on UC and its mechanism of action remain unclear. Therefore, the objective of this study was to investigate the anti-inflammatory effects and mechanism of action of ethanol extract of LB (LBE) in lipopolysaccharide-induced RAW 264.7 macrophages and dextran sulfate sodium (DSS)-induced UC. The results showed that LBE suppressed the secretion of cytokines in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. LBE had protective effects against DSS-induced colitis in mice, decreased the disease activity index (DAI) score, alleviated symptoms, increased colon length, and improved histological characteristics, thus having protective effects against DSS-induced colitis in mice. In addition, it reversed disturbances in the abundance of proteobacteria and probiotics such as Lactobacillus and Blautia in mice with DSS-induced UC. Based on the results of network pharmacology analysis, we identified four main compounds in LBE that are associated with five inflammatory genes (Ptgs2, Plg, Ppar-γ, F2, and Gpr35). These results improve comprehension of the biological activity and functionality of LB and may facilitate the development of LB-based compounds for the treatment of UC.

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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.

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Salt glands are the unique epidermal structures present in recretohalophytes, plants that actively excrete excess Na+ by salt secretory structures to avoid salt damage. Here, we describe a transmembrane protein that localizes to the plasma membrane of the recretohalophyte Limonium bicolor. As virus-induced gene silencing of the corresponding gene LbRSG in L. bicolor decreased the number of salt glands, we named the gene Reduced Salt Gland. We detected LbRSG transcripts in salt glands by in situ hybridization and transient transformation. Overexpression and silencing of LbRSG in L. bicolor pointed to a positive role in salt gland development and salt secretion by interacting with Lb3G16832. Heterologous LbRSG expression in Arabidopsis enhanced salt tolerance during germination and the seedling stage by alleviating NaCl-induced ion stress and osmotic stress after replacing or deleting the (highly) negatively charged region of extramembranous loop. After screened by immunoprecipitation-mass spectrometry and verified using yeast two-hybrid, PGK1 and BGLU18 were proposed to interact with LbRSG to strengthen salt tolerance. Therefore, we identified (highly) negatively charged regions in the extramembrane loop that may play an essential role in salt tolerance, offering hints about LbRSG function and its potential to confer salt resistance.

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KEY MESSAGE: Exogenous 6-BA can increase endogenous hormone content, improve photosynthesis, decrease Na+ by increasing leaf salt gland density and salt secretion ability, and reduce ROS content so that it can promote L. bicolor growth. 6-benzyl adenine (6-BA) is an artificial cytokinin and has been widely applied to improving plant adaptation to stress. However, it is rarely reported that 6-BA alleviates salt damage of halophytes. In this paper, we treated Limonium bicolor seedlings, a recretohalophyte with high medicinal and ornamental values, with 300 mM NaCl and different concentrations of 6-BA (0.5, 1.0, and 1.5 mg/L) and measured plant growth, physiological index, the density of salt gland, and the salt secretion ability of leaves. The results showed that exogenous applications 1.0 mg/L 6-BA significantly improved plant growth and photosynthesis, increased cytokinin and auxins contents, K+ and organic soluble matter contents, the activities of SOD, CAT, APX, and POD, and decreased Na+, H2O2, and O2- contents compared to that treated with 300 mM NaCl. Further research showed that exogenous 6-BA significantly increased the density of salt gland and the salt secretion ability of leaves by upregulating the expression of the salt gland developmental genes, therefore, can secrete more excess Na+, and thus reduces the Na+ concentration in leaves, which can alleviate Na+ damage to the species. In all, exogenous 1.0 mg/L 6-BA can increase endogenous hormone, improve photosynthesis, decrease Na+ by increasing secretion ability, and reduce ROS content of L. bicolor so that it can improve the growth. These results above systematically prove the new role of 6-BA in salt tolerance of L. bicolor.

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BACKGROUND: Mitogen-activated protein kinases (MAPKs) are ubiquitous signal transduction components in eukaryotes. In plants, MAPKs play an essential role in growth and development, phytohormone regulation, and abiotic stress responses. The typical recretohalophyte Limonium bicolor (Bunge) Kuntze has multicellular salt glands on its stems and leaves; these glands secrete excess salt ions from its cells to mitigate salt damage. The number, type, and biological function of L. bicolor MAPK genes are unknown. RESULTS: We identified 20 candidate L. bicolor MAPK genes, which can be divided into four groups. Of these 20 genes, 17 were anchored to 7 chromosomes, while LbMAPK18, LbMAPK19, and LbMAPK20 mapped to distinct scaffolds. Structure analysis showed that the predicted protein LbMAPK19 contains the special structural motif TNY in its activation loop, whereas the other LbMAPK members harbor the conserved TEY or TDY motif. The promoters of most LbMAPK genes carry cis-acting elements related to growth and development, phytohormones, and abiotic stress. LbMAPK1, LbMAPK2, LbMAPK16, and LbMAPK20 are highly expressed in the early stages of salt gland development, whereas LbMAPK4, LbMAPK5, LbMAPK6, LbMAPK7, LbMAPK11, LbMAPK14, and LbMAPK15 are highly expressed during the late stages. These 20 LbMAPK genes all responded to salt, drought and ABA stress. We explored the function of LbMAPK2 via virus-induced gene silencing: knocking down LbMAPK2 transcript levels in L. bicolor resulted in fewer salt glands, lower salt secretion ability from leaves, and decreased salt tolerance. The expression of several genes [LbTTG1 (TRANSPARENT TESTA OF GL1), LbCPC (CAPRICE), and LbGL2 (GLABRA2)] related to salt gland development was significantly upregulated in LbMAPK2 knockdown lines, while the expression of LbEGL3 (ENHANCER OF GL3) was significantly downregulated. CONCLUSION: These findings increase our understanding of the LbMAPK gene family and will be useful for in-depth studies of the molecular mechanisms behind salt gland development and salt secretion in L. bicolor. In addition, our analysis lays the foundation for exploring the biological functions of MAPKs in an extreme halophyte.

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Halophytes can grow and reproduce in high-salinity environments, making them an important reservoir of genes conferring salt tolerance. With the expansion of saline soils worldwide, exploring the mechanisms of salt tolerance in halophytes and improving the salt tolerance of crops have become increasingly urgent. Limonium bicolor is a halophyte with salt glands that secrete excess Na+ through leaves. Here, we identified an uncharacterized gene Lb1G04794, which showed increased expression after NaCl treatment and was high during salt gland development in L. bicolor. Overexpression of Lb1G04794 in L. bicolor showed promoted salt gland development, indicating that this gene may promote salt gland differentiation. Transgenic Arabidopsis strains overexpressing Lb1G04794 showed increased trichomes and decreased root hairs under normal conditions. Compared with wild type (WT), root growth in the transgenic lines was less inhibited by NaCl treatment. Transgenic seedlings accumulated less fresh/dry weight reductions under long-term salt treatment, accompanied by lower Na+ and malondialdehyde accumulation than WT, indicating that these transgenic lines behave better growth and undergo less cellular damage under NaCl stress. These results were consistent with the low expression levels of salt-tolerance marker genes in the transgenic lines upon salt stress. We conclude that the unknown gene Lb1G04794 positively regulated salt gland development, and promoted salt tolerance of Arabidopsis, offering a new direction for improving salt tolerance of non-halophytes and crops.

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Soil salinization is one of the major factors restricting crop growth and agricultural production worldwide. Recretohalophytes have developed unique epidermal structures in their aboveground tissues, such as salt glands or salt bladders, to secrete excess salt out of the plant body as a protective mechanism from ion damage. Three hypotheses were proposed to explain how salt glands secrete salts: the osmotic hypothesis, a hypothesis similar to animal fluid transport, and vesicle-mediated exocytosis. However, there is no direct evidence to show whether the salt gland-secreted liquid contains landmark proteins or peptides which would elucidate the salt secretion mechanism. In this study, we collected the secreted liquid of salt glands from Limonium bicolor, followed by extraction and identification of its constituent proteins and peptides by SDS-PAGE and mass spectrometry. We detected 214 proteins and 440 polypeptides in the salt gland-secreted droplets of plants grown under control conditions. Unexpectedly, the proportion of energy metabolism-related proteins increased significantly though only 16 proteins and 35 polypeptides in the droplets of salt-treated plants were detected. In addition, vesicle transport proteins such as the Golgi marker enzyme glycosyltransferase were present in the secreted sap of salt glands from both control and salt-treated plants. These results suggest that trans-Golgi network-mediated vesicular transport and energy production contributes to salt secretion in salt glands.

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Limonium bicolor is a dicotyledonous recretohalophyte with several multicellular salt glands on the leaves. The plant can directly secrete excess salt onto the leaf surface through the salt glands to maintain ion homeostasis under salt stress. Therefore, it is of great significance to study the functions of genes related to salt gland development and salt tolerance. In this study, an R1-type MYB transcription factor gene was screened from L. bicolor, named LbMYB48, and its expression was strongly induced by salt stress. Subcellular localization analysis showed that LbMYB48 was localized in the nucleus. LbMYB48 protein has transcriptional activation activity shown by transcriptional activation experiments. The density of salt glands in the leaves and the salt secretion capacity of LbMYB48-silenced lines were decremented, as demonstrated by the leaf disc method to detect sodium ion secretion. Furthermore, salt stress index experiments revealed that the ability of LbMYB48-silenced lines to resist salt stress was significantly reduced. LbMYB48 regulates salt gland development and salt tolerance in L. bicolor mainly by regulating the expression of epidermal cell development related genes such as LbCPC-like and LbDIS3 and salt stress-related genes (LbSOSs, LbRLKs, and LbGSTs) as demonstrated by RNA-seq analysis of LbMYB48-silenced lines. The heterologous over-expression of LbMYB48 in Arabidopsis thaliana improves salt tolerance of plants by stabilizing ion and osmotic balance and is likely to be involved in the abscisic acid signaling pathway. Therefore, LbMYB48, a transcriptional activator regulates the salt gland development of L. bicolor and salt tolerance of L. bicolor and A. thaliana.

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Halophytes have evolved specialized strategies to cope with high salinity. The extreme halophyte sea lavender (Limonium bicolor) lacks trichomes but possesses salt glands on its epidermis that can excrete harmful ions, such as sodium, to avoid salt damage. Here, we report a high-quality, 2.92-Gb, chromosome-scale L. bicolor genome assembly based on a combination of Illumina short reads, single-molecule, real-time long reads, chromosome conformation capture (Hi-C) data, and Bionano genome maps, greatly enriching the genomic information on recretohalophytes with multicellular salt glands. Although the L. bicolor genome contains genes that show similarity to trichome fate genes from Arabidopsis thaliana, it lacks homologs of the decision fate genes GLABRA3, ENHANCER OF GLABRA3, GLABRA2, TRANSPARENT TESTA GLABRA2, and SIAMESE, providing a molecular explanation for the absence of trichomes in this species. We identified key genes (LbHLH and LbTTG1) controlling salt gland development among classical trichome homologous genes and confirmed their roles by showing that their mutations markedly disrupted salt gland initiation, salt secretion, and salt tolerance, thus offering genetic support for the long-standing hypothesis that salt glands and trichomes may share a common origin. In addition, a whole-genome duplication event occurred in the L. bicolor genome after its divergence from Tartary buckwheat and may have contributed to its adaptation to high salinity. The L. bicolor genome resource and genetic evidence reported in this study provide profound insights into plant salt tolerance mechanisms that may facilitate the engineering of salt-tolerant crops.

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With global increases in saline soil, it has become increasingly important to decipher salt-tolerance mechanisms and identify strategies to improve salt tolerance in crops. Halophytes complete their life cycles in environments containing ≥200 mM NaCl; these remarkable plants provide a potential source of genes for improving crop salt tolerance. Recretohalophytes such as Limonium bicolor have salt glands that secrete Na+ on their leaf epidermis. Here, we identified Lb1G04202, an uncharacterized gene with no conserved domains, from L. bicolor, which was highly expressed after NaCl treatment. We confirmed its expression in the salt gland by in situ hybridization, and then heterologously expressed Lb1G04202 in Arabidopsis thaliana. The transgenic lines had a higher germination rate, greater cotyledon growth percentage, and longer roots than the wild type (WT) under NaCl treatments (50, 100 and 150 mM). At the seedling stage, the transgenic lines grew better than the WT and had lower Na+ and malonyldialdehyde accumulation, and higher K+ and proline contents. This corresponded with the high expression of the key proline biosynthesis genes AtP5CS1 and AtP5CS2 under NaCl treatment. Isotonic mannitol treatment showed that Lb1G04202 overexpression significantly relieved osmotic stress. Therefore, this novel gene provides a potential target for improving salt tolerance.

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BACKGROUND: Soil salinization is becoming an increasingly serious problem worldwide, resulting in cultivated land loss and desertification, as well as having a serious impact on agriculture and the economy. The indoleamine melatonin (N-acetyl-5-methoxytryptamine) has a wide array of biological roles in plants, including acting as an auxin analog and an antioxidant. Previous studies have shown that exogenous melatonin application alleviates the salt-induced growth inhibition in non-halophyte plants; however, to our knowledge, melatonin effects have not been examined on halophytes, and it is unclear whether melatonin provides similar protection to salt-exposed halophytic plants. RESULTS: We exposed the halophyte Limonium bicolor to salt stress (300 mM) and concomitantly treated the plants with 5 µM melatonin to examine the effect of melatonin on salt tolerance. Exogenous melatonin treatment promoted the growth of L. bicolor under salt stress, as reflected by increasing its fresh weight and leaf area. This increased growth was caused by an increase in net photosynthetic rate and water use efficiency. Treatment of salt-stressed L. bicolor seedlings with 5 µM melatonin also enhanced the activities of antioxidants (superoxide dismutase [SOD], peroxidase [POD], catalase [CAT], and ascorbate peroxidase [APX]), while significantly decreasing the contents of hydrogen peroxide (H2O2), superoxide anion (O2•-), and malondialdehyde (MDA). To screen for L. bicolor genes involved in the above physiological processes, high-throughput RNA sequencing was conducted. A gene ontology enrichment analysis indicated that genes related to photosynthesis, reactive oxygen species scavenging, the auxin-dependent signaling pathway and mitogen-activated protein kinase (MAPK) were highly expressed under melatonin treatment. These data indicated that melatonin improved photosynthesis, decreased reactive oxygen species (ROS) and activated MAPK-mediated antioxidant responses, triggering a downstream MAPK cascade that upregulated the expression of antioxidant-related genes. Thus, melatonin improves the salt tolerance of L. bicolor by increasing photosynthesis and improving cellular redox homeostasis under salt stress. CONCLUSIONS: Our results showed that melatonin can upregulate the expression of genes related to photosynthesis, reactive oxygen species scavenging and mitogen-activated protein kinase (MAPK) of L. bicolor under salt stress, which can improve photosynthesis and antioxidant enzyme activities. Thus melatonin can promote the growth of the species and maintain the homeostasis of reactive oxygen species to alleviate salt stress.

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KEY MESSAGE: Integrative transcriptome and proteome analyses revealed many candidate members that may involve in salt secretion from salt glands in Limonium bicolor. Limonium bicolor, a typical recretohalophyte, protects itself from salt damage by excreting excess salt out of its cells through salt glands. Here, to provide an overview of the salt-tolerance mechanism of L. bicolor, we conducted integrative transcriptome and proteome analyses of this species under salt treatment. We identified numerous differentially expressed transcripts and proteins that may be related to the salt-tolerance mechanism of L. bicolor. By measuring the Na+ secretion rate, were found that this cation secretion rate of a single salt gland was significantly increased after high salinity treatment compared with that in control and then reached the maximum in a short time. Interestingly, transcripts and proteins involved in transmembrane transport of ions were differentially expressed in response to high salinity treatment, suggesting a number of genes and proteins they may play important roles in the salt-stress response. Correlation between differentially expressed transcript and protein profiles revealed several transcripts and proteins that may be responsible for salt tolerance, such as cellulose synthases and annexins. Our findings uncovered many candidate transcripts and proteins in response to the salt tolerance of L. bicolor, providing deep insights into the molecular mechanisms of this important process in recretohalophytes.

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Salt-resistant plants have different mechanisms to limit the deleterious effects of high salt in soil; for example, recretohalophytes secrete salt from unique structures called salt glands. Salt glands are the first differentiated epidermal structure of the recretohalophyte sea lavender (Limonium bicolor), followed by stomata and pavement cells. While salt glands and stomata develop prior to leaf expansion, it is not clear whether these steps are connected. Here, we explored the effects of the five phytohormones salicylic acid, brassinolide, methyl jasmonate, gibberellic acid, and abscisic acid on the development of the first expanded leaf of L. bicolor and its potential connection to salt gland, stomata, and pavement cell differentiation. We calculated the total number of salt glands, stomata, and pavement cells, as well as leaf area and pavement cell area, and assessed the correlations between these parameters. We detected strong and positive correlations between salt gland number and pavement cell area, between stomatal number and pavement cell area, and between salt gland number and stomatal number. We observed evidence of coupling between the development of salt glands, stomata, and pavement cells in L. bicolor, which lays the foundation for further investigation of the mechanism behind salt gland development.

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BACKGROUND: Identifying genes involved in salt tolerance in the recretohalophyte Limonium bicolor could facilitate the breeding of crops with enhanced salt tolerance. Here we cloned the previously uncharacterized gene LbHLH and explored its role in salt tolerance. RESULTS: The 2,067-bp open reading frame of LbHLH encodes a 688-amino-acid protein with a typical helix-loop-helix (HLH) domain. In situ hybridization showed that LbHLH is expressed in salt glands of L. bicolor. LbHLH localizes to the nucleus, and LbHLH is highly expressed during salt gland development and in response to NaCl treatment. To further explore its function, we heterologously expressed LbHLH in Arabidopsis thaliana under the 35S promoter. The overexpression lines showed significantly increased trichome number and reduced root hair number. LbHLH might interact with GLABRA1 to influence trichome and root hair development, as revealed by yeast two-hybrid analysis. The transgenic lines showed higher germination percentages and longer roots than the wild type under NaCl treatment. Analysis of seedlings grown on medium containing sorbitol with the same osmotic pressure as 100 mM NaCl demonstrated that overexpressing LbHLH enhanced osmotic resistance. CONCLUSION: These results indicate that LbHLH enhances salt tolerance by reducing root hair development and enhancing osmotic resistance under NaCl stress.

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Arabidopsis thaliana TRY is a negative regulator of trichome differentiation that promotes root hair differentiation. Here, we established that LbTRY, from the recretohalophyte Limonium bicolor, is a typical MYB transcription factor that exhibits transcriptional activation activity and locates in nucleus. By in situ hybridization in L. bicolor, LbTRY may be specifically positioned in salt gland of the expanded leaves. LbTRY expression was the highest in mature leaves and lowest under NaCl treatment. For functional assessment, we heterologously expressed LbTRY in wild-type and try29760 mutant Arabidopsis plants. Epidermal differentiation was remarkably affected in the transgenic wild-type line, as was increased root hair development. Complementation of try29760 with LbTRY under both 35S and LbTRY specific promoter restored the wild-type phenotype. qRT-PCR analysis suggested that AtGL3 and AtZFP5 promote root hair cell fate in lines heterologously producing LbTRY. In addition, four genes (AtRHD6, AtRSL1, AtLRL2, and AtLRL3) involved in root hair initiation and elongation were upregulated in the transgenic lines. Furthermore, LbTRY specifically increased the salt sensitivity of the transgenic lines. The transgenic and complementation lines showed poor germination rates and reduced root lengths, whereas the mutant unexpectedly fared the best under a range of NaCl treatments. Under salt stress, the transgenic seedlings accumulated more MDA and Na+ and less proline and soluble sugar than try29760. Thus, when heterologously expressed in Arabidopsis, LbTRY participates in hair development, similar to other MYB proteins, and specifically reduces salt tolerance by increasing ion accumulation and reducing osmolytes. The expression of salt-tolerance marker genes (SOS1, SOS2, SOS3 and P5CS1) was significant reduced in the transgenic lines. More will be carried by downregulating expression of TRY homologs in crops to improve salt tolerance.

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BACKGROUND: Salt, a common environmental stress factor, inhibits plant growth and reduces yields. Melatonin is a pleiotropic molecule that regulates plant growth and can alleviate environmental stress in plants. All previous research on this topic has focused on the use of melatonin to improve the relatively low salt tolerance of glycophytes by promoting growth and enhancing antioxidant ability. It is unclear whether exogenous melatonin can increase the salt tolerance of halophytes, particularly recretohalophytes, by enhancing salt secretion from the salt glands. RESULTS: To examine the mechanisms of melatonin-mediated salt tolerance, we explored the effects of exogenous applications of melatonin on the secretion of salt from the salt glands of Limonium bicolor (a kind of recretohalophyte) seedlings and on the expression of associated genes. A pretreatment with 5 µM melatonin significantly improved the growth of L. bicolor seedlings under 300 mM NaCl. Furthermore, exogenous melatonin significantly increased the dry weight and endogenous melatonin content of L. bicolor. In addition, this treatment reduced the content of Na+ and Cl- in leaves, but increased the K+ content. Both the salt secretion rate of the salt glands and the expression level of genes encoding ion transporters (LbHTK1, LbSOS1, LbPMA, and LbNHX1) and vesicular transport proteins (LbVAMP721, LbVAP27, and LbVAMP12) were significantly increased by exogenous melatonin treatment. These results indicate that melatonin improves the salt tolerance of the recretohalophyte L. bicolor via the upregulation of salt secretion by the salt glands. CONCLUSIONS: Our results showed that melatonin can upregulate the expression of genes encoding ion transporters and vesicle transport proteins to enhance salt secretion from the salt glands. Combining the results of the current study with previous research, we formulated a novel mechanism by which melatonin increases salt secretion in L. bicolor. Ions in mesophyll cells are transported to the salt glands through ion transporters located at the plasma membrane. After the ions enter the salt glands, they are transported to the collecting chamber adjacent to the secretory pore through vesicle transport and ions transporter and then are secreted from the secretory pore of salt glands, which maintain ionic homeostasis in the cells and alleviate NaCl-induced growth inhibition.

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AIMS: To elucidate the genetics underlying salt tolerance in recretohalophytes and assess its relevance to non-halophytes, we cloned the Limonium bicolor homolog of Arabidopsis thaliana (Arabidopsis) SUPER SENSITIVE TO ABA AND DROUGHT2 (AtSAD2) and named it LbSAD2, an importin-ß gene associated with trichome initiation and reduced abscisic acid (ABA) sensitivity, and then we assessed the heterologously expressed LbSAD2 in Arabidopsis. METHODS: We examined LbSAD2 expression and assessed the effect of heterologous LbSAD2 expression in Arabidopsis on root hair/trichome induction; the expression levels of possible related genes in trichome/root hair development; some physiological parameters involved in salt tolerance including germination rate, root length, and contents of Na+, proline, and malondialdehyde; and the response of ABA at the germination stage. RESULTS: The LbSAD2 gene is highly expressed in the salt gland development stage and salt treatment, especially located in the salt gland by in situ hybridization, and the LbSAD2 protein contains some special domains compared with AtSAD2, which may suggest the involvement of LbSAD2 in salt tolerance. Compared with the SAD2/GL1 mutant CS65878, which lacks trichomes, CS65878-35S:LbSAD2 had higher trichome abundance but lower root hair abundance. Under 100 mM NaCl treatment, CS65878-35S:LbSAD2 showed enhanced germination and root lengths; improved physiological parameters, including high proline and low contents of Na+ and malondialdehyde; higher expression of the salt-tolerance genes Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 (P5CS1) and GST CLASS TAU 5 (GSTU5); reduced ABA sensitivity; and increased expression of the ABA signaling genes RESPONSIVE TO ABA 18 (RAB18) and SNF1-RELATED PROTEIN KINASE 2 (SRK2E), but not of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3). CONCLUSION: LbSAD2 enhances salt tolerance in Arabidopsis by specifically reducing root hair development, Na+ accumulation, and ABA sensitivity.

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The Arabidopsis thaliana WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) controls epidermis development, playing opposite roles in trichome differentiation and root hair formation. We isolated and characterized LbTTG1 (encoding a WD40-repeat protein with high sequence similarity to TTG1) from the recretohalophyte Limonium bicolor, which actively excretes absorbed salt via a salt gland. The complete open reading frame of LbTTG1 was 1,095 bp, encoding a protein of 364 amino acids, and showed highest expression during the salt gland initiation stage. We heterologously expressed LbTTG1 in wild type and ttg1-13 Arabidopsis plants to verify the protein's function, and the copies of LbTTG1 were identified in transgenic strains using southern blotting. Trichomes were extremely induced on the first true leaves of plants heterologously expressing LbTTG1, whereas no trichomes were produced by ttg1-13 plants. Conversely, plants heterologously expressing LbTTG1 produced fewer root hairs than ttg1-13 plants. In plants heterologously expressing LbTTG1 compared to controls, epidermis differentiation genes (GLABRA1 and GLABRA3) were up-regulated while genes encoding negative regulators of trichome development (TRIPTYCHON and CAPRICE) were down-regulated. Under increased NaCl concentrations, both of the transgenic lines showed enhanced germination and root length, and accumulated less malondialdehyde (MDA) and Na+ and produced more proline, soluble sugar, and higher glutathione S-transferase activity, compared with the ttg1-13 mutant. These results indicate that LbTTG1 participates in epidermis development in Arabidopsis, similarly to other WD40-repeat proteins, and specifically increases salt tolerance of transgenic Arabidopsis by reducing ion accumulation and increasing osmolyte levels.