The transmembrane protein LbRSG from the recretohalophyte Limonium bicolor enhances salt gland development and salt tolerance.

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.

Global dynamics and cytokinin participation of salt gland development trajectory in recretohalophyte Limonium bicolor.

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.

Global analysis of key post-transcriptional regulation in early leaf development of Limonium bicolor identifies a long non-coding RNA that promotes salt gland development and salt resistance.

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.

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

The recretohalophyte Limonium bicolor thrives in high-salinity environments because salt glands on the above-ground parts of the plant help to expel excess salt. Here, we characterize a nucleus-localized C3HC4 (RING-HC)-type zinc finger protein of L. bicolor named  RING  ZINC  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.

Genome-wide identification of the mitogen-activated kinase gene family from Limonium bicolor and functional characterization of LbMAPK2 under salt stress.

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.

Root-Extracted lignanamides from Limonium gmelinii (Willd.) Kuntze with a potential PTP1B inhibitory activity by regulating PI3K/AKT signaling pathway.

The phytochemical study of Limonium gmelinii roots resulted in the isolation of five lignanamides (1-5). Among them, limoniumins J, K, and M (1, 2, and 4) are undescribed compounds, limoniumin L (3) is a new naturally occurring lignanamide, and limoniumin B (5) is a known compound which showed PTP1B inhibition activity with an IC50 value of 5.05 ± 2.44 µM in our previous work. Spectroscopic data analysis, including 1D and 2D NMR and HRESIMS experiments, established the chemical structures of limoniumins J - M (1-4). Compounds 1-4 showed PTP1B inhibition activity, among which compound 3 showed the most potent PTP1B inhibition with an IC50 value of 2.07 ± 0.05 µM. Compounds 3 and 5 could significantly increase cellular glucose consumption and glucose uptake in L6 muscle cells and could synergize with insulin to promote glucose consumption and glucose uptake in a concentration-dependent manner. The treatment of compound 3 also promoted glycogen synthesis in skeletal muscle cells. Western blot analysis demonstrated that the good hypoglycemic effect of compounds 3 and 5 was achieved by activating PI3K/AKT signaling pathway to promote glucose consumption, glucose uptake, and glycogen synthesis. Furthermore, studies on molecular docking revealed the potent interactions between these bioactive substances and the PTP1B protein.

Approaches for in vitro propagation and production of plumbagin in Plumbago spp.

The genus Plumbago (family Plumbaginaceae), commonly known as leadwort, is a sub-tropical shrub that produces secondary metabolite plumbagin, which is employed by pharmaceutical companies and in clinical research. Plumbagin is a potent pharmaceutical because of its anti-microbial, anti-malarial, antifungal, anti-inflammatory, anti-carcinogenic, anti-fertility, anti-plasmodium, antioxidant, anti-diabetic, and other effects. This review documents the biotechnological innovations used to produce plumbagin. The use of modern biotechnological techniques can lead to a variety of benefits, including better yield, increased extraction efficiency, mass production of plantlets, genetic stability, increased biomass, and more. Large-scale in vitro propagation is necessary to minimize over-exploitation of the natural population and allow the use of various biotechnological techniques to improve the plant species and secondary metabolite production. During in vitro culture, optimum conditions are requisites for explant inoculation and plant regeneration. In this review, we provide information on various aspects of plumbagin, depicting its structure, biosynthesis, and biotechnological aspects (both conventional and advanced) along with the future prospects. KEY POINTS: • Critical assessment on in vitro biotechnology in Plumbago species • In vitro propagation of Plumbago and elicitation of plumbagin • Biosynthesis and sustainable production of plumbagin.

Exogenous 6-BA enhances salt tolerance of Limonium bicolor by increasing the number of salt glands.

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.

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features.

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a ß-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.

Micro-Evolutionary Processes in Armeria maritima at Metalliferous Sites.

Tolerance to heavy metals in plants is a model process used to study adaptations to extremely unfavorable environments. One species capable of colonizing areas with high contents of heavy metals is Armeria maritima (Mill.) Wild. A. maritima plants growing in metalliferous areas differ in their morphological features and tolerance levels to heavy metals compared to individuals of the same species growing in non-metalliferous areas. The A. maritima adaptations to heavy metals occur at the organismal, tissue, and cellular levels (e.g., the retention of metals in roots, enrichment of the oldest leaves with metals, accumulation of metals in trichomes, and excretion of metals by salt glands of leaf epidermis). This species also undergoes physiological and biochemical adaptations (e.g., the accumulation of metals in vacuoles of the root's tannic cells and secretion of such compounds as glutathione, organic acids, or HSP17). This work reviews the current knowledge on A. maritima adaptations to heavy metals occurring in zinc-lead waste heaps and the species' genetic variation from exposure to such habitats. A. maritima is an excellent example of microevolution processes in plants inhabiting anthropogenically changed areas.

Quercetin 3-O-Galactoside Isolated from Limonium tetragonum Inhibits Melanogenesis by Regulating PKA/MITF Signaling and ERK Activation.

Quercetin 3-O-galactoside (Q3G) is a common dietary flavanol that has been shown to possess several bioactivities, including anti-melanogenesis. However, how Q3G exerts its anti-melanogenic effect has not been studied. The current study, therefore aimed to investigate the anti-melanogenesis potential of Q3G and elucidate the underlying action mechanism in α-melanocyte-stimulating hormone (α-MSH)-induced hyperpigmentation model of B16F10 murine melanoma cells. Results showed that α-MSH stimulation significantly increased tyrosinase (TYR) and melanin production, which were significantly downregulated by Q3G treatment. The treatment with Q3G suppressed the transcriptional and protein expressions of melanogenesis-related enzymes TYR, tyrosinase related protein-1 (TRP-1), and TRP-2, along with the melanogenic transcription factor microphthalmia-associated transcription factor (MITF) in B16F10 cells. It was shown that Q3G downregulated MITF expression and suppressed its transcriptional activity by inhibiting the cAMP-dependent protein kinase A (PKA)-mediated activation of CREB and GSK3ß. In addition, MAPK-regulated MITF activation signaling was also involved in the inhibition of melanin production by Q3G. The results suggest that the anti-melanogenic properties of Q3G rationalize further studies in vivo to confirm its action mechanism and consequent utilization as a cosmetic ingredient against hyperpigmentation.

Study on the Alleviating Effect and Potential Mechanism of Ethanolic Extract of Limonium aureum (L.) Hill. on Lipopolysaccharide-Induced Inflammatory Responses in Macrophages.

Inflammation is the host response of immune cells during infection and traumatic tissue injury. An uncontrolled inflammatory response leads to inflammatory cascade, which in turn triggers a variety of diseases threatening human and animal health. The use of existing inflammatory therapeutic drugs is constrained by their high cost and susceptibility to systemic side effects, and therefore new therapeutic candidates for inflammatory diseases need to be urgently developed. Natural products are characterized by wide sources and rich pharmacological activities, which are valuable resources for the development of new drugs. This study aimed to uncover the alleviating effect and potential mechanism of natural product Limonium aureum (LAH) on LPS-induced inflammatory responses in macrophages. The experimental results showed that the optimized conditions for LAH ultrasound-assisted extraction via response surface methodology were an ethanol concentration of 72%, a material-to-solvent ratio of 1:37 g/mL, an extraction temperature of 73 °C, and an extraction power of 70 W, and the average extraction rate of LAH total flavonoids was 0.3776%. Then, data of 1666 components in LAH ethanol extracts were obtained through quasi-targeted metabolomics analysis. The ELISA showed that LAH significantly inhibited the production of pro-inflammatory cytokines while promoting the secretion of anti-inflammatory cytokines. Finally, combined with the results of network pharmacology analysis and protein expression validation of hub genes, it was speculated that LAH may alleviate LPS-induced inflammatory responses of macrophages through the AKT1/RELA/PTGS2 signaling pathway and the MAPK3/JUN signaling pathway. This study preliminarily revealed the anti-inflammatory activity of LAH and the molecular mechanism of its anti-inflammatory action, and provided a theoretical basis for the development of LAH as a new natural anti-inflammatory drug.

Integrative transcriptome and proteome analyses provide deep insights into the molecular mechanism of salt tolerance in Limonium bicolor.

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.

Melatonin increases growth and salt tolerance of Limonium bicolor by improving photosynthetic and antioxidant capacity.

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.

Functional genomics-enabled characterization of CYP81B140 and CYP81B141 from Plumbago zeylanica L. substantiates their involvement in plumbagin biosynthesis.

MAIN CONCLUSION: Novel cytochrome P450s, CYP81B140 and CYP81B141 from Plumbago zeylanica were functionally characterized to understand their involvement in polyketide plumbagin biosynthesis. Further, we propose 3-methyl-1-8-naphthalenediol and isoshinanolone as intermediates for plumbagin biosynthesis. Plumbago zeylanica L. (P. zeylanica) is a medicinally important plant belonging to the family Plumbaginaceae. It comprises the most abundant naphthoquinone plumbagin having anti-cancer activity. Only the polyketide synthase (PKS) enzyme has been identified from the biosynthetic pathway which catalyzes iterative condensation of acetyl-CoA and malonyl-CoA molecules. The plumbagin biosynthesis involves hydroxylation, oxidation, hydration and dehydration of intermediate compounds which are expected to be catalyzed by cytochrome P450s (CYPs). To identify the CYPs, co-expression analysis was carried out using PKS as a candidate gene. Out of the eight identified CYPs, CYP81B140 and CYP81B141 have similar expression with PKS and belong to the CYP81 family. Phylogenetic analysis suggested that CYP81B140 and CYP81B141 cluster with CYPs from CYP81B, CYP81D, CYP81E and CYP81AA subfamilies which are known to be involved in the hydroxylation and oxidation reactions. Moreover, artificial microRNA-mediated transient individual silencing and co-silencing of CYP81B140 and CYP81B141 significantly reduced plumbagin and increased the 3-methyl-1-8-naphthalenediol and isoshinanolone content. Based on metabolite analysis, we proposed that 3-methyl-1-8-naphthalenediol and isoshinanolone function as intermediates for plumbagin biosynthesis. Transient silencing, over-expression and docking analysis revealed that CYP81B140 is involved in C-1 oxidation, C-4 hydroxylation and [C2-C3] hydration of 3-methyl-1-8-naphthalenediol to form isoshinanolone, whereas CYP81B141 is catalyzing [C2-C3] dehydration and C-4 oxidation of isoshinanolone to form plumbagin. Our results indicated that both CYP81B140 and CYP81B141 are promiscuous and necessary for plumbagin biosynthesis. This is the first report of identification and functional characterization of P. zeylanica-specific CYPs involved in plumbagin biosynthetic pathway and in general hexaketide synthesis in plants.

Lb1G04202, an Uncharacterized Protein from Recretohalophyte Limonium bicolor, Is Important in Salt Tolerance.

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.

A novel gene LbHLH from the halophyte Limonium bicolor enhances salt tolerance via reducing root hair development and enhancing osmotic resistance.

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.

Molecular cloning and characterization of type III polyketide synthase from Plumbago zeylanica.

Two cDNAs encoding type Ш polyketide synthase (PKS1) and chalcone synthase (CHS, PKS2), were cloned from fresh leaves of Plumbago zeylanica L. (P. zeylanica). Their heterologous expression revealed that PKS1 catalyzed the formation of five α-pyrones from three to six acetate units by accepting acetyl-CoA and malonyl-CoA. In contrast, PKS2 catalyzed the formation of naringenin and bisnoryangonin by accepting p-coumaroyl-CoA and malonyl-CoA. Naringenin is thought to be involved in the biosynthesis of various bioactive flavonoids. PKS2 can be used to molecular breeding to enhance the production of these useful secondary metabolites via its overexpression.[Formula: see text].

Exogenous melatonin enhances salt secretion from salt glands by upregulating the expression of ion transporter and vesicle transport genes in Limonium bicolor.

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.

Salinity influences Cd accumulation and distribution characteristics in two contrasting halophytes, Suaeda glauca and Limonium aureum.

The potential for the phytoremediation of halophytes has been widely recognized. However, the effects of salt on Cd accumulation characteristics in different halophytic species, which may also be related to their salt tolerance, are still unclear. This study investigated the effects of salinity on Cd accumulation and distribution in two distinct halophytes, Suaeda glauca (euhalophyte) and Limonium aureum (recretohalophyte). Seedlings of the two species were treated with 0, 3, and 6 mg kg-1 soil Cd in combination with or without 0.3% NaCl in a pot experiment. The amount of Cd within the rhizosphere and plant tissues, plant biomass, and the subcellular distribution and chemical forms of Cd were examined. Results showed that the addition of NaCl significantly increased Cd bioavailability at high Cd levels due to the rhizosphere acidification effect. Meanwhile, salinity differently impacted plant biomass allocation, and enhanced Cd uptake and translocation in both studied halophytes. Excess Cd was excreted from the leaf surface, possibly by salt glands of L. aureum, with the salinity facilitating this process. Majority of the Cd was found within the cell walls and vacuolar compartments of two species. However, S. glauca plants had higher proportions of inactive Cd (extracted by 2% HAc and 0.6 M HCl) and lower proportions of active Cd (extracted by 80% ethanol and water), as opposed to L. aureum, which would better inform S. glauca's higher Cd accumulation. Based on these results, S. glauca seems more applicable for phytomanagement of Cd-contaminated saline soils due to its higher capacity for Cd enrichment and tolerance amplified by NaCl.