Limonium Bicolor Research Articles

Salt gland distribution in limonium bicolor at the individual level

Limonium bicolor is a typical exo-recretohalophyte with multi-cellular salt glands. A differential interference contrast (DIC) microscope were applied to investigate the pattern of salt gland distribution in L. bicolor at the individual level. For a single mature leaf, more salt glands are distributed in the leaf central and apical regions than leaf base. For the leaves in different developmental stages, firstly, the density of salt glands linearly decreased at the beginning of leaf expansion and kept a relatively constant value in the later periods, which was mainly due to the rapid expansion of epidermal cells. Secondly, the total number of glands per leaf showed a reversed trend compared to the density of salt glands. These results suggested that the salt gland density was adapted to the leaf age and area as more and more salt accumulated in the saline soils.

Effect of salinity on ion homeostasis in three halophyte species, Limonium bicolor, Vitex trifolia Linn. var. simplicifolia Cham and Apocynaceae venetum

Limonium bicolor, Vitex trifolia Linn. var. simplicifolia Cham and Apocynaceae venetum are halophyte species with ecological restoration function of saline soil that are widely distributed in coastal areas around Bohai Sea in China. The mechanism of response to salt stress is different between them. The salt-tolerance mechanism, especially for ion homeostasis, of these halophytes varies and is not properly understood. The purpose of the work was to reveal the effect of salinity on ion homeostasis in these three halophyte species. Plant growth, leaf succulence, Na+, K+ and Cl− concentrations, as well as ion flux were examined at various concentrations (0–100%) of artificial sea water after 10 days. The results showed as follows: the growth of L. bicolor was obviously stimulated under salt stress, while V. trifolia Linn. var. simplicifolia Cham and A. venetum was strongly inhibited. The contents of Na+ and Cl− increased with salinity in the three halophyte species, in both the leaves and roots. The accumulation of K+ in roots was promoted by salinity except in L. bicolor subjected to 100% artificial sea water concentration. L. bicolor roots showed a lower net Na+ efflux compared with the control. By contrast, V. trifolia Linn. var. simplicifolia Cham and A. venetum roots retained a greater capacity for net Na+ efflux compared with the control. L. bicolor and V. trifolia Linn. var. simplicifolia Cham roots exhibited a lower and higher net Cl− influx, respectively, than the control, but the differences were not significant. By contrast, A. venetum roots exhibited a Cl− efflux compared with the control. L. bicolor and V. trifolia Linn. var. simplicifolia Cham roots reduced net K+ efflux, and L. bicolor even appeared to exhibit a K+ influx at certain points in time, whereas A. venetum strongly increased net K+ efflux compared with the control. The accumulation and partitioning of ions differed between the three halophyte species, and the salt-tolerance ranking from high to low was L. bicolor, V. trifolia Linn. var. simplicifolia Cham, and A. venetum.

Salt secretion in different leaf ages and leaf positions of Limonium bicolor

Recretohalophytes can excrete excessive salt from salt glands or salt bladders in order to avoid salt damage. Limonium bicolor, belonged to Plumbaginaceae, is a typical exo-recretohalophyte with salt glands. A differential interference contrast (DIC) microscope and leaf discs secretion model was used in the study to explore the salt secretion of different leaf ages in L. bicolor at the individual level. For a single mature leaf, more salt glands are distributed in the leaf abaxial than leaf adaxial. The total Na+ secretion amount was positively proportional to the leaf age and leaf area. And the Na+ secretion rate per salt gland obviously increased at the beginning and kept relatively stable at late period of leaf development. These results suggested that Na+ secretion rate of L. bicolor were adapted to the leaf age and area.

Physiological and proteomic response of Limonium bicolor to salinity

Medicinal halophyte Limonium bicolor (Bag.) Kuntze was treated with 200 mM NaCl, the physiological parameters including fresh weight, dry weight, salt gland number, MDA levels and root activity were investigated, comparative proteomic analyses of leaf and root were carried out using 2D-PAGE combined with MALDI-TOF/TOF-MS. The results showed that L. bicolor performed a positive effect on its growth under NaCl stress since increase in different extent of fr wt, dry wt, salt gland number, MDA level and SOD activity of leaves and roots, as well as root activity. More than 460 protein spots in leaves and 600 protein spots in roots were visualized and matched in 2D gels, the results of proteomic analyses showed that 45 proteins were identified in leaves and 49 proteins in roots to be differentially expressed. Based on their functions, these proteins were grouped into categories covering a wide range of molecular processes, including carbohydrate, energy, lipid, nucleotide and amino acid metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, biosynthesis of secondary metabolites, redox homeostasis, transcription, cytoskeleton and transport, as well as folding, sorting and degradation. The upregulating and downregulating of differentially- expressed proteins with different physiological function were also reflected in the physiological parameters. These results are helpful for further research work on salt response mechanisms and medicinal utilization of L. bicolor.

Flavonoids isolated from the flowers of Limonium bicolor and their in vitro antitumor evaluation

Background:Limonium bicolor, a halophytic species, can grow in saline or saline-alkali soil, is well known as a traditional Chinese medicine. Recently it attracted much attention for its treatment for cancer.Objective:The present study was performed to evaluate this species from the phytochemical standpoint and the possible relationship between the antitumor activity and its natural products.Materials and methods:The chemical constituents from the flowers of L. bicolor were investigated through bioassay-guided fractionation and isolation. All the individual compounds were characterized by spectroscopic analysis and their potential antitumor activity was tested against three different human tumor cell lines by MTT assays.Results:The EtOAc extract was proven as the most potent fraction and further fractionation led to the isolation of 15 natural flavonoids, which were characterized as luteolin (1), acacetin (2), quercetin (3), isorhamnetin (4), kaempferol (5), eriodictyol (6), kaempferol-3-O-α-L-rhamnoside (7), kaempferol-3-O-β-D-glucoside (8), quercetin-3-O-α-L-rhamnoside (9), quercetin-3-O-β-D-glucoside (10), quercetin-3-O-β-D-galactoside (11), myricetin-3-O-α-L-rhamnoside (12), kaempferol-3-O-(6″-O-galloyl)-β-D-glucoside (13), hesperidin (14) and rutin (15). The biotesting results demonstrated that both compounds 1 and 3 showed good cytotoxicity against human colon cancer cells (LOVO). Compound 5 exhibited relative greater growth inhibition against both human breast cancer cells (MCF-7) and osteosarcoma cell lines (U2-OS) at the concentration of 100 μg/mL.Conclusion:On the basis of these findings, the flavonoids were deduced to be potentially responsible for the antitumor activity of L. bicolor. The preliminary structure–activity relationship analysis suggests that the 3-O-glycosylation moiety in natural flavonoids was not essential for the antiproliferative activity on LOVO and U2-OS cells.SUMMARY The phytochemical investigation of Limonium bicolor led to the isolation of 15 flavonoids.The biotesting of the isolates against three different human tumor cell lines was evaluated.The structure-antitumor activity relationship between the isolated flavonoids was discussed. Abbreviation used: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, EtOAc: Ethyl acetate; LOVO: human colon cancer; MCF-7: human breast, cancer; U2-OS: human osteosarcoma; 5-FU: 5-Fluorouracil; DMSO: dimethyl sulfoxide, NMR: nuclear magnetic resonance; HR-ESI-MS: high resolution electrospray ionization mass chromatography, HPLC: high performance liquid chromatography, EtOH: ethanol; n-BuOH: n-butanol; CC: column chromatography, TLC: thin layer chromatography; PBS: phosphate-buffered saline.

The transcriptome of NaCl-treated Limonium bicolor leaves reveals the genes controlling salt secretion of salt gland

Limonium bicolor, a typical recretohalophyte that lives in saline environments, excretes excessive salt to the environment through epidermal salt glands to avoid salt stress. The aim of this study was to screen for L. bicolor genes involved in salt secretion by high-throughput RNA sequencing. We established the experimental procedure of salt secretion using detached mature leaves, in which the optimal salt concentration was determined as 200mM NaCl. The detached salt secretion system combined with Illumina deep sequencing were applied. In total, 27,311 genes were annotated using an L. bicolor database, and 2040 of these genes were differentially expressed, of which 744 were up-regulated and 1260 were down-regulated with the NaCl versus the control treatment. A gene ontology enrichment analysis indicated that genes related to ion transport, vesicles, reactive oxygen species scavenging, the abscisic acid-dependent signaling pathway and transcription factors were found to be highly expressed under NaCl treatment. We found that 102 of these genes were likely to be involved in salt secretion, which was confirmed using salt-secretion mutants. The present study identifies the candidate genes in the L. bicolor salt gland that are highly associated with salt secretion. In addition, a salt-transporting pathway is presented to explain how Na(+) is excreted by the salt gland in L. bicolor. These findings will shed light on the molecular mechanism of salt secretion from the salt glands of plants.

Salt stress changes chemical composition in Limonium bicolor (Bag.) Kuntze, a medicinal halophytic plant

In order to understand the response mode of the main chemical compounds to salt-stress, the chemical compositions in leaves and roots of NaCl-treated Limonium bicolor (Bag.) Kuntze, a medicinal halophytic plant with efficacy of blood enriching and hemostasis, were determined by UPLC-Q-TOF-MS2 and HPLC, and compared to the control, the expression analysis of chalcone synthase (CHS) gene under salt-stress was also carried out by quantitative real-time PCR. As a result, eleven chemical constituents including triethyl citrate (1), gallic acid (2), vanillic acid (3), vanillic acid derivative (4), vanillic acid-hexose (5), N-(2-carboxyethyl) iminodiacetic acid (6), epigallocatechin gallate (7), myricetin-3-O-glucoside/galactoside (8), myricetin-3-O-rhamnoside (9), quercetin-3-O-rhamnoside (10) and myricetin-3-O-(O-galloyl)-rhamnoside (11) were identified, and showed that NaCl-stress can induce accumulation changes of these compounds. Among them, compound 9 showed the highest abundance both in leaf and root of L. bicolor, which can be markedly enhanced in root by NaCl-stress. CHS gene was sensitive to NaCl-stress, its expression level was up-regulated in leaf while inhibited in root by NaCl-stress. These results will provide references for further research of the directional induction of medicinal active ingredients and the salt response mechanism of secondary metabolism in medicinal halophyte L. bicolor.

Identification and functional analysis of the autofluorescent substance in Limonium bicolor salt glands

Limonium bicolor is a typical recretohalophyte with salt glands for the secretion of excess salts into the environment. We observed that L. bicolor salt glands showed obvious blue autofluorescence under UV excitation (330–380 nm). The aim of the present study was to identify and clarify a role for this autofluorescent substance in salt secretion. Sudan IV staining showed that the autofluorescent substance was localized in the cuticle of the salt glands. Moreover, the primary autofluorescent substance was identified as ferulic acid after treatment with 0.1 M ammonium hydroxide solution, alkaline and enzymatic hydrolysis. Additional experiments using two mutants exhibiting increased (fii) and decreased (fid) salt gland fluorescence indicated that the fluorescence intensity of salt glands under UV excitation was positively correlated with the content of ferulic acid in the cuticle, strongly suggesting that the primary autofluorescent substance in the salt glands was ferulic acid. Salt gland secretion was determined using leaf discs, and the results showed that the Na+ secretion rate per single salt gland was also positively correlated with the content of ferulic acid in the cuticle, suggesting that ferulic acid in the cuticle was directly involved in salt secretion of salt gland.

K+ accumulation in the cytoplasm and nucleus of the salt gland cells of Limonium bicolor accompanies increased rates of salt secretion under NaCl treatment using NanoSIMS

Recretohalophytes with specialized salt-secreting structures (salt glands) can secrete excess salts from plant, while discriminating between Na+ and K+. K+/Na+ ratio plays an important role in plant salt tolerance, but the distribution and role of K+ in the salt gland cells is poorly understood. In this article, the in situ subcellular localization of K and Na in the salt gland of the recretohalophyte Limonium bicolor Kuntze is described. Samples were prepared by high-pressure freezing (HPF), freeze substitution (FS) and analyzed using NanoSIMS. The salt gland of L. bicolor consists of sixteen cells. Higher signal strength of Na+ was located in the apoplast of salt gland cells. Compared with control, 200mM NaCl treatment led to higher signal strength of K+ and Na+ in both cytoplasm and nucleus of salt gland cells although K+/Na+ ratio in both cytoplasm and nucleus were slightly reduced by NaCl. Moreover, the rate of Na+ secretion per salt gland of L. bicolor treated with 200mM NaCl was five times that of controls. These results suggest that K+ accumulation both in the cytoplasm and nucleus of salt gland cells under salinity may play an important role in salt secretion, although the exact mechanism is unknown.

Increases of Unsaturated Fatty Acids in Membrane Lipids Protects Photosystem II from Photoinhibition under Salinity in Different Halophytes

For the purpose of testing the function of unsaturated fatty acids in different halophytes in the process of photosynthesis under salt stress, the impact of saline stress on plant development, content of chlorophyll, the PSII photochemistry efficiency, content of membrane lipid and composition of fatty acid were investigated in the three halophytes Thellungiella halophila, Limonium bicolor and Suaeda salsa and non-halophyte Arabidopsis thaliana . Salinity (200 mM NaCl) did not reduce the value of Fv/Fm, OPSII, and chlorophyll content in the halophytes. While all of them decreased by 200 mM NaCl treatment in A. thaliana . In the non-halophytic, A. thaliana , when treated with NaCl, the content of unsaturated fatty acid and the DBI of membrane lipids MGDG, SQDG, PG and PC decreased. While the unsaturated fatty acid content and the DBI of T. halophila , L. bicolor and S. salsa increased. The DBI of total lipids increased in all halophytes but decreased in the non-halophyte, A. thaliana . The proportion of PG increased in T. halophila and S. salsa . It decreased in L. bicolor and A. thaliana . The DGDG (digalactosyldiacylglycerols)/MGDG (monogalactosyldiacylglycerols) ratio of S. salsa increased from 1.20 to 1.35, while it decreased in T. halophila , L. bicolor and A. thaliana under salt stress. These results suggest that unsaturated fatty acid levels increase in the halophytes under salt stress relative to the non-halophyte A. thaliana . The proportion of membrane lipids and unsaturated fatty acids is related to different levels of salt tolerance among different halophytes.

Study on pathway and characteristics of ion secretion of salt glands of Limonium bicolor

Recretohalophytes with specialized salt-secreting structures, including salt glands and salt bladders, can secrete excess salts from plant tissues and enhance salinity tolerance of plants. However, the pathway and property of salt secretion by the salt gland has not been elucidated. In the article, Limonium bicolor Kuntze was used to investigate the pathway and characteristics of salt secretion of salt gland. Scanning electron microscope micrographs showed that each of the secretory cells had a pore in the center of the cuticle, and the rice grain-like secretions were observed above the pore. The chemical composition of secretions from secretory pores was mainly NaCl using environmental scanning electron microscope technique. Non-invasive micro-test technology was used to directly measure ion secretion rate of salt gland, and secretion rates of Na+ and Cl− were greatly enhanced by a 200-mmol/L NaCl treatment. However, epidermal cells and stoma showed little secretion of ions. In conclusion, our results provide evidence that the salt glands of L. bicolor have four secretory pores and that NaCl is secreted through these pores of salt gland.

Kinetics on the Ultrasonic-Assisted Extraction of Polysaccharides from <i>Limonium bicolor k</i><i>unze </i>(Bge.)

To explore the kinetics parameters in the ultrasonic-assisted extraction from Limonium bicolor Kunze (Bge.) , the kinetic equation for polysaccharide ultrasonic extraction process was established with ball model. According to Fick’s second law of diffusion, extraction process was analysed. The results can provide the valuable theory basis for the technical design and further research of polysaccharide extraction process.

Decolorization of <i>Limonium bicolor kunze</i> (<i>Bge</i>.) Polysaccharides by Resins

To select a suitable decolorizing resin for Limonium bicolor Kunze (Bge.). polysaccharides (LBPs), the decolorization effect of crude LBPs by five resins are studied. And the adsorption equilibrium of LBPs pigment on the selected resin was investigated.The results show that the decolorization ratios of LX-46 is higher than those of other resins. Under optimized dynamicad sorption conditions (flow rate of 1.5BV/h, 2BV of 6mg/ml LBPS solution in 298K), the decoloration, deproteinization and polysaccharide recovery ratios of LX-46 resin were 92.08%, 92.79% and 86.53%, respectively. The above results show that macroporous anion exchange resins are suitable for the decolorization of crude LBPs.

A system for the transformation and regeneration of the recretohalophyte Limonium bicolor

Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD600) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.

An efficient autofluorescence method for screening Limonium bicolor mutants for abnormal salt gland density and salt secretion

With the spread of saline soils worldwide, it has become increasingly important to understand salt-tolerant mechanisms and to develop halophytes with increased salt tolerance. Limonium bicolor is a typical recretohalophyte and has a typical salt excretory structure in the epidermis called the salt gland. A method that can be used to screen a large population of L. bicolor mutants for altered salt gland density and altered salt secretion is needed but is currently unavailable. Leaves of 1-month-old L. bicolor seedlings were processed by three traditional methods [epidermal peel, nail impression, and clearing/differential interference contrast microscope (clearing/DIC) method] and a fluorescence method (fluorescence microscopic examination of cleared leaves). With the fluorescence method, the autofluorescence of salt glands under UV excitation (330–380nm) was easily distinguished with the least labor and time. The fluorescence method was used to screen ~10,000 seedlings (which grew from gamma-irradiated seeds). Four mutants with reduced salt gland density and 15 with increased salt gland density were obtained. Both kinds of mutants will be useful for the isolation of genes involved in salt gland development and salt secretion in L. bicolor and other halophytes. The fluorescence method was also successfully used to observe the salt glands of Aegialitis rotundifolia and the stomata and trichomes of Arabidopsis. The fluorescence method described here will be useful for examining plant epidermal structures that have autofluorescence under UV or other wavelengths.

Chitinase Genes LbCHI31 and LbCHI32 from Limonium bicolor Were Successfully Expressed in Escherichia coli and Exhibit Recombinant Chitinase Activities

The two chitinase genes, LbCHI31 and LbCHI32 from Limonium bicolor, were, respectively, expressed in Escherichia coli BL21 strain. The intracellular recombinant chitinases, inrCHI31 and inrCHI32, and the extracellular exrCHI31 and exrCHI32 could be produced into E. coli. The exrCHI31 and exrCHI32 can be secreted into extracellular medium. The optimal reaction condition for inrCHI31 was 5 mmol/L of Mn2+ at 40°C and pH 5.0 with an activity of 0.772 U using Alternaria alternata cell wall as substrate. The optimal condition of inrCHI32 was 5 mmol/L of Ba2+ at 45°C and pH 5.0 with an activity of 0.792 U using Valsa sordida cell wall as substrate. The optimal reaction condition of exrCHI31 was 5 mmol/L of Zn2+ at 40°C and pH 5.0, and the activity was 0.921 U using the A. alternata cell wall as substrate. Simultaneously, the optimal condition of exrCHI32 was 5 mmol/L of K+ at 45°C and pH 5.0, with V. sordida cell wall as the substrate, and the activity was 0.897 U. Furthermore, the activities of extracellular recombinant enzymes on fungal cell walls and compounds were generally higher than those of the intracellular recombinant enzymes. Recombinant exrCHI31 and exrCHI32 have better hydrolytic ability on cell walls of different fungi than synthetic chitins and obviously showed activity against A. alternata.

Study on the α-Glucosidase Inhibitory Activity of <i>Limonium bicolor</i> Kuntze

The antidiabetic activities of the extracts with different solvents and methods from Limonium bicolor Kuntze were investigated. It was found that all the extracts with ethanol, petroleum ether, EtOAc, n-BuOH and H2O from the underground part of the plant possessed the α-Glucosidase inhibitory activity, and the fractions from the 80% ethanol extracts possessed strong activity. The extracts eluted with water from the 80% ethanol crude extract of the aerial part of the plant were inactive to α-Glucosidase inhibition, the other extracts from the aerial part all give potent α-Glucosidase inhibition activity.

Effects of salinity and nitric oxide donor sodium nitroprusside (SNP) on development and salt secretion of salt glands of Limonium bicolor

NO, as a signaling molecule, is involved in abiotic stresses. Limonium bicolor seedlings were treated with 200 mM NaCl combined with 0.05 mM SNP for 20 days to study the effects of NO on development and salt-secretion rates of salt glands. It was shown that the total number of salt glands on adaxial surfaces under condition of 200 mM NaCl containing 0.05 mM SNP treatment increased significantly compared with that under 200 mM NaCl treatment. Na+ secretion rate per leaf under 200 mM NaCl containing 0.05 mM SNP was significantly higher than that under 200 mM NaCl without SNP. However, there was no significant difference in salt-secretion rate of individual salt glands between 200 mM NaCl containing 0.05 mM SNP treatment and 200 mM NaCl treatment. Although there was no significant difference in salt-secretion rate of individual glands, Na+ concentration in the leaves treated with 200 mM NaCl solution containing SNP was significantly lower than that treated with 200 mM NaCl solution. Treatment with 200 mM NaCl solution containing SNP caused a remarkable increase in Na+ concentration in salt glands. Obviously, the efficiency of the secretion process per gland was enhanced by adding SNP to NaCl. The results showed NO may enhance the salt secretion by inducing more dermatogen cells to develop into salt glands and by enhancing the efficiency of the secretion process per gland.

Optimization of Ultrasonic Extraction Process by Response Surface Methodology of Polysaccharides from <i>Limonium bicolor Kunze</i> (<i>Bge.</i>)

Polysaccharide from Limonium bicolor Kunze (Bge.)(LBPs) was extracted with the assistance of ultrasonic.Box-Behnken design(BBD) was employed to optimize extraction time; extraction temperature;ultrasonic extraction power and ratio of water to raw material to obtain a high LBPs yield.The optimum extraction conditions were as follows: extraction temperature 81.23°C, ultrasonic power 190.86W, extraction time 42.39 min and ratio of water to raw material 15.4ml/g.The yield of polysaccharide was 7.11% based on the above mentioned conditions. Close agreement between experimetal and predicted values was found.

A glycine-rich RNA-binding protein can mediate physiological responses in transgenic plants under salt stress

Glycine-rich RNA-binding proteins (GRPs) are involved in post-transcriptional regulation of genes, which have been found to play a role in stress response. However, whether GRPs can mediate some physiological responses related to salt stress tolerance is still not known. In the present study, we investigated the role of GRPs in salt stress-induced physiological responses by generating transgenic tobacco lines overexpressing a GRP (LbGRP1) gene from Limonium bicolor (Bunge) Kuntze. Compared with wild type (WT) tobacco, the transgenic plants showed significantly improved superoxide dismutase and catalase activities under salt stress conditions. Levels of proline in the transgenic plants were significantly higher than those in the WT plants grown under NaCl stress conditions. Furthermore, Na(+) content and Na(+)/K(+) ratio in the transgenic plants were lower than those in the WT plants under both normal growth and stress conditions. These results suggested that overexpression of the LbGRP1 gene can affect some physiological processes associated with salt tolerance of plants. Therefore, we hypothesize that LbGST1 can enhance stress resistance by mediating some physiological pathways.