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.

The NAC gene family in the halophyte Limonium bicolor: Identification, expression analysis, and regulation of abiotic stress tolerance.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Phytochemical profiling and anti-fibrotic activities of Plumbago indica L. and Plumbago auriculata Lam. in thioacetamide-induced liver fibrosis in rats.

This study aimed at investigating the chemical composition and the hepatoprotective activities of Plumbago indica L. and P. auriculata Lam. LC-MS/MS analyses for the hydroalcoholic extracts of the aerial parts of the two Plumbago species allowed the tentative identification of thirty and twenty-five compounds from P. indica and P. auriculata, respectively. The biochemical and histopathological alterations associated with thioacetamide (TAA)-induced liver fibrosis in rats were evaluated in vivo where rats received the two extracts at three different dose levels (100, 200 and 400 mg/kg p.o, daily) for 15 consecutive days with induction of hepatotoxicity by TAA (200 mg/kg/day, i.p.) at 14th and 15th days. Results of the present study showed a significant restoration in liver function biomarkers viz. alanine transaminase (ALT), aspartate transaminase (AST), gamma glutamyl transferase and total bilirubin. The liver homogenates exhibited increased levels of antioxidant biomarkers: reduced glutathione (GSH) and catalase (CAT), accompanied with decline in malondialdehyde (MDA). Furthermore, treated groups exhibited a significant suppression in liver inflammatory cytokines: tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6), and fibrotic biomarker: alpha smooth muscle relaxant. Histopathological examination of the liver showed normality of hepatocytes. Noteworthy, P. indica extract showed better hepatoprotective activity than P. auriculata, particularly at 200 mg/kg. To sum up, all these results indicated the hepatoprotective properties of both extracts, as well as their antifibrotic effect was evidenced by reduction in hepatic collagen deposition. However, additional experiments are required to isolate their individual secondary metabolites, assess the toxicity of the extracts and explore the involved mechanism of action.

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.

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.

Antimicrobial activity of Plumbago indica and ligand screening of plumbagin against methicillin-resistant Staphylococcus aureus.

In this study, the antimicrobial properties of Plumbago indica root bark against bacterial strains and a fungal strain were investigatedusing the disc diffusion and minimum inhibitory concentration assays. Gas chromatography/mass spectrometry, nuclear magnetic resonance spectrometry, and column chromatography analyses were conducted to identify and isolate the active compounds. A docking study was performed to identify possible interactions between the active compound and DNA gyrase using the Schrödinger Glide docking program. Both methanol extract and the ethyl acetate fraction of the root bark showed significant antimicrobial activity against the gram-positive bacteria than against the gram-negative bacteria and the fungal strain. The active compound was identified as plumbagin. A disc diffusion assay of plumbagin revealed potent antimicrobial activity against methicillin-resistant Staphylococcus aureus. Molecular docking of plumbagin revealed high specificity towards the DNA gyrase binding site with a high fitness score and a minimum energy barrier of -7.651 kcal/mol. These findings indicate that P. indica exhibits significant antimicrobial activity, primarily due to the presence of plumbagin. The specificity of plumbagin toward DNA gyrase in S. aureus indicates the feasibility of utilizing P. indica for developing new drug leads against drug resistant microbial strain. Communicated by Ramaswamy H. Sarma.

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.

Plumbagin: A Potential Candidate for Future Research and Development.

Plumbagin has gained a lot of attention in research due to its various therapeutic actions. It is a secondary metabolite obtained from different plant families, such as Plumbaginaceae, Droseraceae, and Ebenceae. Various studies on plumbagin have revealed that it is a natural gift for mankind in treating chronic diseases, like cancer, diabetes, malaria, bacterial infection, and controlling cardiovascular disease. However, there are several challenges in developing plumbagin as a therapeutic agent. The first and foremost is its limited solubility and oral bioavailability. The second limitation is its toxicity. Plumbagin has a narrow therapeutic window, and literature reveals that the compound has moderate toxicity in animals. However, data are insufficient to prove that plumbagin is unsafe for humans. Despite the many therapeutic benefits of plumbagin, it remains unexploited for mankind. Thus, a systematic review of its toxicity, pharmacology, and safety is required to be performed. This review work signifies the depth of therapeutic applications proven via research, its different modes of isolation and separation of chemical constituents, and its modification. A thorough review of promising therapeutic targets via docking studies is also presented. Different methods used to quantify plumbagin from the plant are reviewed. An overview of attempts to design novel formulations which could enhance its bioavailability is also presented. The review paper will help the scientist to exploit the drug to its optimum, which will help to overcome the challenges faced during its design and developmental stages.

Plumbagin and Plumbago indica Differentially Modulated Cytochrome P450 and Transporter Profiles in BeWo and HepG2 Cells.

<b>Background and Objective:</b> The medicinal herb <i>Plumbago indica</i> (PI) and its major constituent plumbagin have reported pharmacological properties but there is a lack of information about their herb-drug interactions. The effects of methanolic (PI-MeOH) and ethanolic (PI-EtOH) crude extracts of PI and plumbagin on the expression of cytochrome P450s (<i>CYP1A2</i>, <i>CYP2E1</i> and <i>CYP3A4</i>) and transporters (<i>ABCC1</i>, <i>ABCG2</i> and <i>SLC22A11</i>) were investigated in BeWo and HepG2 cells. <b>Materials and Methods:</b> BeWo or HepG2 cells were treated with 0.5-5 µM plumbagin or 25-500 µg mL¹ of PI-MeOH or PI-EtOH for 24 hrs. Total RNA was extracted and mRNA expression of CYPs and transporters were determined using RT-qPCR. <b>Results:</b> PI and plumbagin affected mRNA expression differently in the two tested cell types. In BeWo cells, all concentrations of PI-MeOH induced <i>CYP2E1</i>, 100 and 500 µg Ml¹ PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i>, <i>CYP3A4 </i>and <i>ABCG2 </i>and 500 µg mL¹ PI-EtOH induced <i>ABCG2</i> expression. Plumbagin suppressed <i>CYP1A2</i> and induced <i>SLC22A11 </i>expression at the highest concentration, 5 µM. In HepG2 cells, 5 µM plumbagin and 500 µg Ml¹ PI-EtOH suppressed <i>CYP3A4 </i>expression and 500 µg mL¹ PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i> and <i>CYP2E1 </i>expression. <i>ABCC1</i> expression was induced by all treatments while <i>ABCG2</i> and <i>SLC22A11 </i>were induced only by 500 µg mL¹ PI-MeOH and PI-EtOH. <b>Conclusion:</b> The use of PI or plumbagin supplements in large quantities or for long periods should be carefully considered due to the risk of herbal drug interactions via modulated expression of CYPs and transporters.

Limoniastrum guyonianum behavior under seasonal conditions fluctuations of Sabkha Aïn Maïder (Tunisia).

In Sabkha biotope, several environmental factors (i.e., salinity, drought, temperature, etc.) especially during dry season affect halophytes developments. To cope with these harmful conditions, halophytes use multiple mechanisms of adaptations. In this study, we focused on the effect of environmental condition changes over a year in the Sabkha of Aïn Maïder (Medenine - Tunisia) on the physiological and biochemical behavior of Limoniastrum guyonianum using a modeling approach. Our study showed that the model depicted well (R2 > 0.75) the monthly fluctuations of the studied parameters in this habitat. During the dry period (June to September), the salinity of the soil increased remarkably (high level of EC and Na+ content), resulting in high Na+ content in the aerial parts followed by a nutrient deficiency in K+, Ca2+, and Mg2+. As a result of this disruption, L. guyonianum decreased its water potential to more negative values to maintain osmotic potential using inorganic osmolytes (i.e., Na+) and organic osmolytes (i.e., sugars: sucrose, fructose, glucose, and xylitol, and organic acids: citric and malic acids). In addition, CO2 assimilation rate, stomatal conductance, transpiration rate, and photosynthetic pigments decreased significantly with increasing salinity. The phenolic compounds contents and the antioxidant activity increased significantly in the dry period as a result of increased levels of H2O2 and lipid peroxidation. This increase was highly correlated with soil salinity and air temperature. The maintenance of tissue hydration (i.e., moderate decrease of relative water content), the accumulation of sugars and organic acids, the enhancement of phenolic compounds amounts, and the increase of antioxidant activity during the dry period suggest that L. guyonianum possesses an efficient tolerance mechanism that allows the plant to withstand the seasonal fluctuations of climatic conditions in its natural biotope.