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.

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.

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 200 mM 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.

Cloning and characterization of a chitinase gene Lbchi31 from Limonium bicolor and identification of its biological activity.

Chitinases are digestive enzymes that break down glycosidic bonds in chitin. In the current study, an endochitinase gene Lbchi31 was cloned from Limonium bicolor. The cDNA sequence of Lbchi31 was 1,107 bp in length, encoding 322 amino acid residues with a calculated molecular mass of 31.7 kDa. Clustal analysis showed that there was a highly conserved chitin-binding domains in Lbchi31 protein, containing four sulfide bridges. The Lbchi31 gene was inserted into the pPIC9 vector and transferred into yeast Pichia pastoris GS115 and KM71 for heterologous expression. The transformant harboring the Lbchi31 gene showed a clearly visible protein band with a molecular mass of more than 31 kDa in the SDS-PAGE gel, indicating that it had been translated in P. pastoris. Enzyme characterization showed that the optimal reaction condition for chitinase LbCHI31 activity was: 40 degrees C, pH of 5.0 and 5 mmol l(-1) of Mn(2+). The maximum enzyme activity was 0.88 U ml(-1) following exposure to the cell wall chitin of Valsa sordida. The LbCHI31 enzyme can efficiently degrade cell wall chitin of the phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, V. sordida, Septoria tritici and Phytophthora sojae, suggesting that it has the biocontrol function to fungal phytopathogen.

Cloning, heterologous expression, and functional characterization of a chitinase gene, Lbchi32, from Limonium bicolor.

In the present study, an endochitinase gene, Lbchi32, was cloned from Limonium bicolor. The cDNA sequence of Lbchi32 was 1,443 bp in length and encoded 319 amino acid residues. The DNA sequence of Lbchi32 was 2,512 bp in length and contained three exons and two introns. The Lbchi32 gene was inserted into a pPIC9 vector and transferred into Pichia pastoris strains GS115 and KM71 for heterologous expression. SDS-PAGE analyses indicated that LbCHI32 was expressed in both GS115 and KM71 and that it was secreted extracellularly. The optimal reaction conditions for LbCHI32 activity are 45 degrees C, pH 5.0, and 5 mM Ba(2+). The LbCHI32 enzyme can efficiently degrade chitin, chitin derivatives, and the cell walls of different pathogenic fungi, including phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Valsa sordida, Septoria tritici, and Phytophthora sojae. These findings suggest that Lbchi32 has potential use in the degradation of chitin and chitin derivatives.

Multiple stressors in multiple species: Effects of different RDX soil concentrations and differential water-resourcing on RDX fate, plant health, and plant survival.

Response to simultaneous stressors is an important facet of plant ecology and land management. In a greenhouse trial, we studied how eight plant species responded to single and combined effects of three soil concentrations of the phytotoxic munitions constituent RDX and two levels of water-resourcing. In an outdoor trial, we studied the effects of high RDX soil concentration and two levels of water-resourcing in three plant species. Multiple endpoints related to RDX fate, plant health, and plant survival were evaluated in both trials. Starting RDX concentration was the most frequent factor influencing all endpoints. Water-resourcing also had significant impacts, but in fewer cases. For most endpoints, significant interaction effects between RDX concentration and water-resourcing were observed for some species and treatments. Main and interaction effects were typically variable (significant in one treatment, but not in another; associated with increasing endpoint values for one treatment and/or with decreasing endpoint values in another). This complexity has implications for understanding how RDX and water-availability combine to impact plants, as well as for applications like phytoremediation. As an additional product of these greenhouse and outdoor trials, three plants native or naturalized within the southeastern United States were identified as promising species for further study as in situ phytoremediation resources. Plumbago auriculata exhibited relatively strong and markedly consistent among-treatment mean proportional reductions in soil RDX concentrations (112% and 2.5% of the means of corresponding values observed within other species). Likewise, across all treatments, Salvia coccinea exhibited distinctively low variance in mean leaf chlorophyll content index levels (6.5% of the means of corresponding values observed within other species). Both species also exhibited mean wilting and chlorosis levels that were 66% and 35%, and 67% and 84%, of corresponding values observed in all other plants, respectively. Ruellia caroliniensis exhibited at least 43% higher mean survival across all treatments than any other test species in outdoor trials, despite exhibiting similar RDX uptake and bioconcentration levels.

Exogenous melatonin improves seed germination in Limonium bicolor under salt stress.

Melatonin involves in improving tolerance to abiotic and biotic stresses by regulating various biological processes. However, little is known about the underlying mechanism. Here, we investigated the effects of exogenous melatonin on seed germination in the halophyte Limonium bicolor under salt stress. Specifically, we examined the effect of salt stress on seed germination, melatonin concentration, and changes in the concentrations of nutrients, amylase activity, and hormones in L. bicolor seeds with and without pre-treatment with melatonin. Seed germination was significantly suppressed under a 200 mM NaCl treatment, but pre-treatment with melatonin significantly improved seed germination under salt stress. During seed germination, seeds pre-treated with melatonin contained high levels of melatonin and gibberellic acid (GA), low levels of abscisic acid (ABA), and high levels of amylase and alpha-amylase activity. Melatonin treatment upregulated the expression of key genes involved in GA biosynthesis (GA20ox and GA3ox), downregulated key genes involved in ABA biosynthesis (LbNCED1 and LbNCED3), and upregulated ABA 8'-hydroxylase genes (LbCYP707A1 and LbCYP707A2), which mediate the changes in GA and ABA levels in seeds during germination. A high melatonin concentration in seeds promotes the utilization of nutrients and the synthesis of new proteins to enhance seed germination.

Exogenous salicylic acid improves the germination of Limonium bicolor seeds under salt stress.

Salicylic acid (SA) may improve plant tolerance to abiotic stresses; however, little is known about the underlying mechanisms by which this is achieved. Here, we investigated the effects of exogenous SA application on seed germination in the halophyte Limonium bicolor (Kuntze) under salt stress. Specifically, we examined the effect of salt stress on seed germination, sugar and protein contents, amylase activity, and the contents of various hormones, both in the presence and absence of exogenous SA treatments. Germination was significantly suppressed by a 200 mM NaCl treatment but was significantly improved when 0.08 mM SA was concurrently applied. During germination, the seeds treated with SA had high levels of gibberellic acid (GA) and high levels of amylase and α-amylase activity, but low abscisic acid (ABA) contents. The SA treatment upregulated the expression of key genes involved in GA biosynthesis while downregulating those involved in ABA biosynthesis, thereby triggering a favorable hormonal balance between GA and ABA that enhanced seed germination under salt stress.

[Cloning and expression of a novel metallothionein gene LbMT2 from Limonium bicolor].

The full length cDNA of a novel metallothionein (LbMT2) gene was cloned from a cDNA library of Limonium bicolor. The LbMT2 gene cloned is 518 bp in length, which includes a 64 bp of 5' untranslated region (UTR) and a 205 bp of 3' untranslated region. This gene has an open reading frame (ORF) of 249 bp in length, encoding a protein of 82 amino acid residues with the molecular mass of 8.1 kDa and theoretical pI of 4.71. The expression of LbMT2 gene in L. bicolor in response to CuSO4, CdCl2, NaCl, cold, and PEG was further investigated using real time quantitative PCR. In both leaf and root of L. bicolor, the expression of LbMT2 was induced by CuSO4, CdCl2, NaCl, and cold, but inhibited by PEG stress. LbMT2 gene was inserted into a prokaryotic expression vector (pGEX-4T-2) to produce the recombinant expression vector pGEX-LbMT2. The expression of LbMT2 in E. coli BL21 was induced with IPTG, which produced a protein band with expected size of 35 kDa on SDS-PAGE.

Methyl jasmonate improves tolerance to high salt stress in the recretohalophyte Limonium bicolor.

Limonium bicolor is a typical recretohalophyte with salt glands in the epidermis, which shows maximal growth at moderate salt concentrations (100mM NaCl) but reduced growth in the presence of excess salt (more than 200mM). Jasmonic acid (JA) alleviates the reduced growth of L. bicolor under salt stress; however, the underlying mechanism is unknown. In this study we investigated the effects of exogenous methyl jasmonate (MeJA) application on L. bicolor growth at high NaCl concentrations. We found that treatment with 300mM NaCl led to dramatic inhibition of seedling growth that was significantly alleviated by the application of 0.03mM MeJA, resulting in a biomass close to that of plants not subjected to salt stress. To determine the parameters that correlate with MeJA-induced salt tolerance (assessed as the biomass production in saline and control conditions), we measured 14 physiological parameters relating to ion contents, plasma membrane permeability, photosynthetic parameters, salt gland density, and salt secretion. We identified a correlation between individual indicators and salt tolerance: the most positively correlated indicator was net photosynthetic rate, and the most negatively correlated one was relative electrical conductivity. These findings provide insights into a possible mechanism underlying MeJA-mediated salt stress alleviation.

High-performance liquid chromatographic quantification of plumbagin from transformed rhizoclones of Plumbago zeylanica L.: inter-clonal variation in biomass growth and plumbagin production.

An optimized protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Plumbago zeylanica L. was developed through selection of suitable explant type and the bacterial strain. The infection of internodal explants from an in vivo plant and leaves of in vitro origin with the A4 strain resulted in the emergence of hairy roots at a transformation frequency of 86.33 and 42.33 %, respectively. Independent transformed root somaclones (rhizoclones) capable of sustained growth were maintained under a low illumination in auxin-free agar-solidified Murashige and Skoog (MS) medium through subcultures at periodic intervals. The presence of pRi T L-DNA rolB or rolC genes and pRi T R-DNA mas2 gene in the transformed rhizoclone genome was ascertained by PCR amplification. Concentrations and type of carbon source, auxin and media strength were optimized for root biomass growth. Five independent rhizoclones each from A4- and LBA9402-transformed root lines were studied for their plumbagin accumulation at different growth phases, using HPLC analysis. The potential for plumbagin biosynthesis was expressed in all the tested rhizoclones, although distinct inter-clonal variations were noted. It was evident that maturation of hairy roots was more important for plumbagin accumulation; slow-growing and early-maturing rhizoclones accumulated more plumbagin compared to fast-growing and late-maturing rhizoclones. A4-induced rhizoclone HRA2B5 was identified as the most superior clone with a higher plumbagin yield potential in comparison with other tested hairy root clones, in vitro-grown non-transformed roots and in vivo roots of naturally occurring P. zeylanica.

Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption.

Cell cultures of Plumbago rosea were immobilized in calcium alginate and cultured in Murashige and Skoog's basal medium containing 10 mM CaCl(2) for the production of plumbagin, an important medicinal compound. Studies were carried to find out the impact of immobilization on the increased accumulation of this secondary metabolite. Immobilization in calcium alginate enhanced the production of plumbagin by three, two and one folds compared to that of control, un-crosslinked alginate and CaCl(2) treated cells respectively. Cell loading at a level of 20% to the polymer volume (Na-alginate) was optimal and maximum plumbagin was obtained. At higher cell loading (40-50%), lower plumbagin accumulation was noticed. Addition of 200 mg l(-1) chitosan as an elicitor to the immobilized cells resulted in eight and two folds higher accumulation of plumbagin over control and immobilized cells. Also, more than 70% of the plumbagin was released into the medium, which is highly desirable for easy recovery of the product. Sucrose utilization rate of the cells was higher when cells were subjected to in situ product removal using Amberlite XAD-7. This may indicate that the toxicity of plumbagin was reduced on cells when it was removed from the medium. Cells subjected to combined treatments of chitosan, immobilization and in situ extraction showed a synergistic effect and yielded 92.13 mg g(-1) DCW of plumbagin which is 21, 5.7, 2.5 times higher than control, immobilized, immobilized and elicited cells respectively.

The role of herbicides in the erosion of salt marshes in eastern England.

Laboratory studies and field trials were conducted to investigate the role of herbicides on saltmarsh vegetation, and their possible significance to saltmarsh erosion. Herbicide concentrations within the ranges present in the aquatic environment were found to reduce the photosynthetic efficiency and growth of both epipelic diatoms and higher saltmarsh plants in the laboratory and in situ. The addition of sublethal concentrations of herbicides resulted in decreased growth rates and photosynthetic efficiency of diatoms and photosynthetic efficiency of higher plants. Sediment stability also decreased due to a reduction in diatom EPS production. There was qualitative evidence that diatoms migrated deeper into the sediment when the surface was exposed to simazine, reducing surface sediment stability by the absence of a cohesive biofilm. Sediment loads on leaves severely reduced photosynthesis in Limonium vulgare. This, coupled with reduced carbon assimilation from the effects of herbicides, could have large negative consequences for plant productivity and over winter survival of saltmarsh plants. The data support the hypothesis that sublethal herbicide concentrations could be playing a role in the increased erosion of salt marshes that has occurred over the past 40 years.

Contrasting physiological responses of dwarf sea-lavender and marguerite to simulated sea aerosol deposition.

Plants of two wild native species from littoral areas, marguerite [Argyranthemum coronopifolium (Willd.) C.J. Humphries] and dwarf sea-lavender [Limonium pectinatum (Aiton) O. Kuntze], grown in an unheated plastic greenhouse, were sprayed 2 to 3 min per day over a 7-d period with different aqueous solutions containing (i) an anionic surfactant (S1); (ii) a solution simulating the composition of sea aerosol (S2); (iii) a solution simulating sea aerosol with anionic surfactant (S3), and (iv) deionized water alone (control). The plant resistance to sea aerosol and the ability to recover from treatments were studied. By the end of the spraying period, marguerite showed a significant reduction in growth compared with control. However, most of the growth parameters were significantly unaffected in dwarf sea-lavender when plants were treated with sea aerosol containing surfactant. Measurements of water relations variables in marguerite showed a slight decrease in leaf turgor potential after spraying with sea aerosol containing surfactant. The surfactant enhanced the foliar absorption of salt in marguerite plants, inducing reductions in leaf stomatal conductance and causing such damage in the photosynthetic apparatus that the level of net photosynthesis decreased and had not recovered by the end of the experiment. The treatments had no effect on leaf stomatal conductance and photosynthesis rate in dwarf sea-lavender plants. The response of the species studied to sea aerosol was related to the degree of salinity tolerance. Although both species are wild native plants from littoral areas, marguerite is not salt tolerant and was the most sensitive to the sea aerosol treatments, while dwarf sea-lavender, a halophyte species, was more efficient at decreasing the toxic salt content of the tissues as its growth and ornamental characteristics were not affected.

Evaluation of phytotoxicity of seaport sediments aged artificially by rotary leaching in the framework of a quarry deposit scenario.

In the framework of an ecological risk assessment of seaport sediments for terrestrial ecosystems when deposited in quarries, we simulated the "ageing" of sediments exposed to rain. This experiment highlighted an inflection point at the solid/liquid ratio 1/25, after which the extraction of pollutants increases moderately. The raw sediments studied inhibited the germination of Lolium perenne and Armeria maritima (a halophytic species) seeds. Furthermore, they affected the early development of L.perenne. The same sediments, leached at a ratio of 1/25, presented a reduction of acute (germination) and chronic (growth) phytotoxicity. The bioconcentration factors of the metals studied decreased with the leached sediment, except for Cu which was still clearly identified in root parts. Thus rotary leaching tests and phytotoxicity bioassays can be used to provide an initial assessment of the ability of plants, particularly halophytes, to colonize deposits of dredged seaport sediments.

Effect of salt stress on seed germination and seedling growth of three salinity plants.

Seeds of three halophyte species included Limonium sinense Kuntze, Glycine soja sieb. and Sorghum sudanense Stapf. were used to investigate the effects of different salinity concentrations on their germination percentage, germination energy, germination index, relative germination rate, relative salt-injury rate, radicle length and hypocotyl length. Results showed that seeds of G. soja can germinate well and rapidly at lower level of salt (< 200 mmol L(-1)), 50 mmol L(-1) salt concentrations is better to S. sudanense, suitable salt concentrations was beneficial to germinate of S. sudanense seeds. Seeds of L. sinense and S. sudanense can germinate at higher level of salt (400 mmol L(-1)). Three plant seeds had different physiological mechanism for germination under salt stress, radicle hypocotyl ratio of L. sinense and S. sudanense increased with increasing salt suggested that they had the higher tolerance in shoot growth under NaCl stress, which is perhaps the reason for their wide utilisation for saline soil rehabilitation.

Armeria maritima from a calamine heap--initial studies on physiologic-metabolic adaptations to metal-enriched soil.

Plants of Armeria maritima are found both on unpolluted sites and on soils strongly polluted with heavy metals. Seedlings of A. maritima from a zinc-lead calamine heap in ore-mining region (Boleslaw population) and from unpolluted area (Manasterz population) were tested to determine the zinc, cadmium and lead tolerance. In hydroponic experiments Boleslaw population was more tolerant to zinc, cadmium and lead. Localization of heavy metals in roots was determined using the histochemical method for detecting metal-complexes with dithizone. Their accumulation was found in root hairs, rhizoderma and at the surface of the central cylinder. Glutathione level in plants increased after metal treatment of both populations. However, its high level was not correlated with phytochelatin production. These metal-binding complexes were not detected in plants exposed to zinc, cadmium or lead. Changes of organic acids concentrations in Armeria treated with metals may suggest their role in metal translocation from roots to shoots. The content of organic acids, especially malate, decreased in the roots and increased in the leaves. These changes may be important in Pb-tolerance of Manasterz population and in Zn-, Cd-tolerance of calamine population from Boleslaw.

A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica.

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25+/-2 degrees C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.