Metabolomics Analyses Reveal Metabolites Affected by Plant Growth-Promoting Endophytic Bacteria in Roots of the Halophyte Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum L. (common ice plant) is an edible halophyte. However, if ice plants are used to phytoremediate salinity soil, there are problems of slow initial growth, and a long period before active NaCl uptake occurs under higher salinity conditions. Application of endophytic bacteria may improve the problem, but there remain gaps in our understanding of how endophytic bacteria affect the growth and the biochemical and physiological characteristics of ice plants. The aims of this study were to identify growth-promoting endophytic bacteria from the roots of ice plants and to document the metabolomic response of ice plants after application of selected endophytic bacteria. Two plant growth-promoting endophytic bacteria were selected on the basis of their ability to promote ice plant growth. The two strains putatively identified as Microbacterium spp. and Streptomyces spp. significantly promoted ice plant growth, at 2-times and 2.5-times, respectively, compared with the control and also affected the metabolome of ice plants. The strain of Microbacterium spp. resulted in increased contents of metabolites related to the tricarboxylic acid cycle and photosynthesis. The effects of salt stress were alleviated in ice plants inoculated with the endobacterial strains, compared with uninoculated plants. A deeper understanding of the complex interplay among plant metabolites will be useful for developing microbe-assisted soil phytoremediation strategies, using Mesembryanthemum species.

Nutritional and Functional Evaluation of Inula crithmoides and Mesembryanthemum nodiflorum Grown in Different Salinities for Human Consumption.

The nutritional composition and productivity of halophytes is strongly related to the biotic/abiotic stress to which these extremophile salt tolerant plants are subjected during their cultivation cycle. In this study, two commercial halophyte species (Inula crithmoides and Mesembryanthemum nodiflorum) were cultivated at six levels of salinity using a soilless cultivation system. In this way, it was possible to understand the response mechanisms of these halophytes to salt stress. The relative productivity decreased from the salinities of 110 and 200 mmol L-1 upwards for I. crithmoides and M. nodiflorum, respectively. Nonetheless, the nutritional profile for human consumption remained balanced. In general, I. crithmoides vitamin (B1 and B6) contents were significantly higher than those of M. nodiflorum. For both species, ß-carotene and lutein were induced by salinity, possibly as a response to oxidative stress. Phenolic compounds were more abundant in plants cultivated at lower salinities, while the antioxidant activity increased as a response to salt stress. Sensory characteristics were evaluated by a panel of culinary chefs showing a preference for plants grown at the salt concentration of 350 mmol L-1. In summary, salinity stress was effective in boosting important nutritional components in these species, and the soilless system promotes the sustainable and safe production of halophyte plants for human consumption.

Cutin:cutin-acid endo-transacylase (CCT), a cuticle-remodelling enzyme activity in the plant epidermis.

Cutin is a polyester matrix mainly composed of hydroxy-fatty acids that occurs in the cuticles of shoots and root-caps. The cuticle, of which cutin is a major component, protects the plant from biotic and abiotic stresses, and cutin has been postulated to constrain organ expansion. We propose that, to allow cutin restructuring, ester bonds in this net-like polymer can be transiently cleaved and then re-formed (transacylation). Here, using pea epicotyl epidermis as the main model, we first detected a cutin:cutin-fatty acid endo-transacylase (CCT) activity. In-situ assays used endogenous cutin as the donor substrate for endogenous enzymes; the exogenous acceptor substrate was a radiolabelled monomeric cutin-acid, 16-hydroxy-[3H]hexadecanoic acid (HHA). High-molecular-weight cutin became ester-bonded to intact [3H]HHA molecules, which thereby became unextractable except by ester-hydrolysing alkalis. In-situ CCT activity correlated with growth rate in Hylotelephium leaves and tomato fruits, suggesting a role in loosening the outer epidermal wall during organ growth. The only well-defined cutin transacylase in the apoplast, CUS1 (a tomato cutin synthase), when produced in transgenic tobacco, lacked CCT activity. This finding provides a reference for future CCT protein identification, which can adopt our sensitive enzyme assay to screen other CUS1-related enzymes.

The response of a model C3/CAM intermediate semi-halophyte Mesembryanthemum crystallinum L. to elevated cadmium concentrations.

Many areas exhibiting increased concentrations of soluble salts are simultaneously polluted with heavy metals (HM), and halophytes with extended tolerance to heavy metal toxicity seem to represent a promising tool for their phytoremediation. In this study, the response of the soil-grown C3-CAM (Crassulacean acid metabolism) intermediate halophyte Mesembryanthemum crystallinum (common ice plant) to increased concentrations of Cd (0.01-1 mM) was investigated. None of the tested Cd treatments affected growth parameters or tissue water content of either C3 or CAM-performing plants. Chlorophyll a fluorescence confirmed high tolerance of the photosynthetic apparatus of both metabolic states towards Cd. Plants performing both photosynthesis types accumulated significant Cd amounts only under the highest (1 mM) treatment, and the metal was primarily deposited in the roots, which are features typical of an excluding strategy. Upon the application of 1 mM Cd solution CAM-performing plants, due to the NaCl pre-treatment applied for CAM induction, were exposed to significantly higher amounts of bioavailable Cd in comparison with those of C3-performing plants. As a result, roots of CAM plants accumulated over 4-fold higher Cd amounts when compared with C3 plants. In our opinion, enhanced Cd-accumulating potential observed in CAM-performing plants was the effect of osmotic stress episode and resulting modifications e.g. in the detoxifying capacity of the antioxidative system. Increased antioxidative potential of NaCl pre-treated plants was pronounced with significantly higher activity of CuZnSOD (copper-zinc superoxide dismutase), not achievable in C3 plants subjected to high Cd concentrations. Moreover, the applied Cd doses induced SOD activity in a compartment-dependent manner only in C3 plants. We confirmed that none of the applied Cd concentrations initiated the metabolic shift from C3 to CAM.

Making Epidermal Bladder Cells Bigger: Developmental- and Salinity-Induced Endopolyploidy in a Model Halophyte.

Endopolyploidy occurs when DNA replication takes place without subsequent mitotic nuclear division, resulting in cell-specific ploidy levels within tissues. In plants, endopolyploidy plays an important role in sustaining growth and development, but only a few studies have demonstrated a role in abiotic stress response. In this study, we investigated the function of ploidy level and nuclear and cell size in leaf expansion throughout development and tracked cell type-specific ploidy in the halophyte Mesembryanthemum crystallinum In addition to developmental endopolyploidy, we examined the effects of salinity stress on ploidy level. We focused specifically on epidermal bladder cells (EBC), which are modified balloon-like trichomes, due to their large size and role in salt accumulation. Our results demonstrate that ploidy increases as the leaves expand in a similar manner for each leaf type, and ploidy levels up to 512C were recorded for nuclei in EBC of leaves of adult plants. Salt treatment led to a significant increase in ploidy levels in the EBC, and these cells showed spatially related differences in their ploidy and nuclear and cell size depending on the positions on the leaf and stem surface. Transcriptome analysis highlighted salinity-induced changes in genes involved in DNA replication, cell cycle, endoreduplication, and trichome development in EBC. The increase in cell size and ploidy observed in M. crystallinum under salinity stress may contribute to salt tolerance by increasing the storage capacity for sodium sequestration brought about by higher metabolic activity driving rapid cell enlargement in the leaf tissue and EBC.

The localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L. hypocotyls discloses their differing roles in rhizogenesis.

This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H2O2) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H2O2 production and blocked rhizogenesis. Ultrastructural studies revealed differences in H2O2 localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H2O2 level regulation in vascular cylinder cells, while peroxidase (POD) participates in H2O2 level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O2 (•-)) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3'-diaminobenzidine (DAB) staining of H2O2 was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H2O2 was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O2 (•-) was then limited to epidermis cells, while H2O2 was accumulated only in vascular tissue. These results indicate that O2 (•-) is engaged in processes of rhizogenesis induction involving division of competent cells, while H2O2 is engaged in developmental processes mainly involving cell growth.

Pattern of antioxidant enzyme activities and hydrogen peroxide content during developmental stages of rhizogenesis from hypocotyl explants of Mesembryanthemum crystallinum L.

KEY MESSAGE: H2O2 is necessary to elicit rhizogenic action of auxin. Activities of specific catalase and manganese superoxide dismutase forms mark roots development. Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots on medium containing 2,4-dichlorophenoxyacetic acid. Explants became competent to respond to the rhizogenic action of auxin on day 3 of culture, when hydrogen peroxide content in cultured tissue was the highest. L-Ascorbic acid added to the medium at 5 µM lowered the H2O2 level, inhibited rhizogenesis and induced non-regenerative callus, suggesting that certain level of H2O2 is required to promote root initiation. Coincident with the onset of rhizogenic determination, meristemoids formed at the periphery of the hypocotyl stele and the activity of the manganese form of superoxide dismutase, MnSOD-2 was induced. Once induced, MnSOD-2 activity was maintained through the post-determination phase of rooting, involving root growth. MnSOD-2 activity was not found in non-rhizogenic explants maintained in the presence of AA. Analyses of the maximum photochemical efficiency of photosystem II and the oxygen uptake rate revealed that the explants were metabolically arrested during the predetermination stage of rhizogenesis. Respiratory and photosynthetic rates were high during root elongation and maturation. Changes in catalase and peroxidase activities correlated with fluctuations of endogenous H2O2 content throughout rhizogenic culture. Expression of a specific CAT-2 form accompanied the post-determination stage of rooting and a high rate of carbohydrate metabolism during root growth. On the other hand, the occurrence of MnSOD-2 activity did not depend on the metabolic status of explants. The expression of MnSOD-2 activity throughout root development seems to relate it specifically to root metabolism and indicates it as a molecular marker of rhizogenesis in M. crystallinum.

Effect of salt treatment on phenolic compounds and antioxidant activity of two Mesembryanthemum edule provenances.

Mesembryanthemum edule L. is an edible and medicinal halophyte widespread in Tunisia seashore. In this study, parameters of oxidative stress, phenolic compounds and antioxidant activities were comparatively investigated in two M. edule provenances (Jerba and Bizerte, respectively sampled from arid and humid bioclimatic stages). Plants were subjected to 0, 300 and 600mM NaCl treatment under glasshouse conditions. Results showed that M. edule response to salinity depends on provenance (P), salt treatment (T) and their interaction (P×T). (T) affected more significantly the oxidative stress parameters and antioxidant activities than (P) and (P×T). Conversely, (P) was much affluent for tannin polymerization degree and interaction between the two factors (P×T) was more determinants for analyzed antioxidant parameters. The higher salt tolerance of Jerba plants was associated with low levels of malondialdehyde and of electrolyte leakage mainly at 600mM NaCl. Besides, antioxidant activities of Jerba provenance, were more efficient than Bizerte. In addition, avicularin was the major phenolic in both provenances. This compound concentration increased with salinity in Jerba shoots, while it was reduced in Bizerte especially at 600mM NaCl. Overall, the higher salt tolerance of plants from Jerba provenance, and to a lower extent of those from Bizerte, may be partly related to their better capacity to limit oxidative damage when salt-challenged, and this is likely the result of redistribution in phenolic composition. Besides, abiotic factors such as salinity could be determinant in antioxidant potentiality of this medicinal plant.

A CAM- and starch-deficient mutant of the facultative CAM species Mesembryanthemum crystallinum reconciles sink demands by repartitioning carbon during acclimation to salinity.

In the halophytic species Mesembryanthemum crystallinum, the induction of crassulacean acid metabolism (CAM) by salinity requires a substantial investment of resources in storage carbohydrates to provide substrate for nocturnal CO(2) uptake. Acclimation to salinity also requires the synthesis and accumulation of cyclitols as compatible solutes, maintenance of root respiration, and nitrate assimilation. This study assessed the hierarchy and coordination of sinks for carbohydrate in leaves and roots during acclimation to salinity in M. crystallinum. By comparing wild type and a CAM-/starch-deficient mutant of this species, it was sought to determine if other metabolic sinks could compensate for a curtailment in CAM and enable acclimation to salinity. Under salinity, CAM deficiency reduced 24 h photosynthetic carbon gain by >50%. Cyclitols were accumulated to comparable levels in leaves and roots of both the wild type and mutant, but represented only 5% of 24 h carbon balance. Dark respiration of leaves and roots was a stronger sink for carbohydrate in the mutant compared with the wild type and implied higher maintenance costs for the metabolic processes underpinning acclimation to salinity when CAM was curtailed. CAM required the nocturnal mobilization of >70% of primary carbohydrate in the wild type and >85% of carbohydrate in the mutant. The substantial allocation of carbohydrate to CAM limited the export of sugars to roots, and the root:shoot ratio declined under salinity. The data suggest a key role for the vacuole in regulating the supply and demand for carbohydrate over the day/night cycle in the starch-/CAM-deficient mutant.

Municipal solid waste compost application improves productivity, polyphenol content, and antioxidant capacity of Mesembryanthemum edule.

Organic wastes were successfully used as soil amendment to improve agrosystems productivity. Yet, the effectiveness of this practice to enhance plant antioxidant capacities has received little attention. Here, we assess the effect of municipal solid waste (MSW) compost (at 40 t ha(-1)) on growth, polyphenol contents and antioxidant activities of Mesembryanthemum edule. MSW compost application significantly increased the soil contents of carbon, nitrogen, calcium, phosphorus and potassium. This was associated with higher nutrient (N, P, and K) uptake, which likely led to the significant improvement of the plant biomass and relative growth rate (RGR) (+93% on average) as compared to the control. In the same way, the fertilizing effect of the added organic matter significantly enhanced the antioxidant potential M. edule, assessed by radical scavenging activity, iron reducing power and ß-carotene bleaching capacity. This was associated with significantly higher antioxidant contents, mainly total phenols and flavonoids. Heavy metal (Pb, Cd, Cu, and Zn) concentrations were slightly increased upon compost application, but remained lower than phytotoxic values. Overall, our results point out that short-term MSW compost application at 40 t ha(-1) is efficient in enhancing the productivity together with the antioxidant potentiality of M. edule without any adverse environmental impact.

Dosage of 2,6-bis (1.1-dimethylethyl)-4-methylphenol (BHT) in the plant extract Mesembryanthemum crystallinum.

A naturally occurring BHT was identified in the leaves of the halophyte plant Mesembryanthemum crystallinum. This phenol was extracted in this study by two methods at the different plant growth stages. One of the methods was better for BHT extraction; the concentration of this phenol is plant growth stage dependent. In this study, the floraison stage has the highest BHT concentration. The antioxidant activity of the plant extract was not related to BHT concentration. The higher antioxidant activity is obtained at seedlings stage.

Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

Effects of Cd2+ on K+, Ca2+ and N uptake in two halophytes Sesuvium portulacastrum and Mesembryanthemum crystallinum: consequences on growth.

One of the limits of Cd2+-phytoextraction is the high toxicity of this metal to plants. Growth restriction, chlorosis and necrosis are usually accompanied with a large disturbance of the uptake of essential elements. This work aims to study the effects of cadmium (Cd2+) on potassium (K+), calcium (Ca2+) and nitrogen (N) acquisition, and their consequences on growth in two halophytes species: Sesuvium portulacastrum and Mesembryanthemum crystallinum. Seedlings were grown for 30 days in split-root conditions. One half of the root system was immersed in complete nutrient solution (Basal medium (B)) supplemented with 100 microM Cd2+, and the other half was immersed in a Cd2+-free medium, containing all nutrients (B/Cd plants) or deprived of potassium ((B-K)/Cd) or calcium ((B-Ca)/Cd) or nitrogen ((B-N)/Cd). Using this approach, we demonstrated that K+ and Ca2+ uptake was impaired in roots exposed to Cd2+. Concerning N, we noticed no indication of uptake inhibition by Cd2+. However, restriction of K+ uptake by roots was compensated by an increase in the K+-use efficiency, so that growth was not inhibited. Calcium uptake was strongly limited by Cd2. This inhibition was accompanied by a reduction in growth of ((B-Ca)/Cd) plants. Thus, we conclude that Cd2+ limits growth of both halophytes through restriction imposed on Ca2+ uptake. We suggest that the increase of Ca2+ availability in soils could improve the growth of both species in the presence of Cd2+. This would be essential for improving their utility for extraction of this metal by from salty contaminated soils.

Environment or development? Lifetime net CO2 exchange and control of the expression of Crassulacean acid metabolism in Mesembryanthemum crystallinum.

The relative influence of plant age and environmental stress signals in triggering a shift from C(3) photosynthesis to Crassulacean acid metabolism (CAM) in the annual halophytic C(3)-CAM species Mesembryanthemum crystallinum was explored by continuously monitoring net CO(2) exchange of whole shoots from the seedling stage until seed set. Plants exposed to high salinity (400 mm NaCl) in hydroponic culture solution or grown in saline-droughted soil acquired between 11% and 24% of their carbon via net dark CO(2) uptake involving CAM. In contrast, plants grown under nonsaline, well-watered conditions were capable of completing their life cycle by operating in the C(3) mode without ever exhibiting net CO(2) uptake at night. These observations are not consistent with the widely expressed view that the induction of CAM by high salinity in M. crystallinum represents an acceleration of preprogrammed developmental processes. Rather, our study demonstrates that the induction of the CAM pathway for carbon acquisition in M. crystallinum is under environmental control.

Cadmium effects on growth and mineral nutrition of two halophytes: Sesuvium portulacastrum and Mesembryanthemum crystallinum.

Growth, cadmium accumulation and potassium and calcium status were studied in two halophytes from Aizoaceae family: Sesuvium portulacastrum and Mesembryanthemum crystallinum. After multiplication, the seedlings were cultivated on nutrient solution supplemented with NaCl (100mM) and CdCl2 (0, 50, 100, 200 and 300 microM). After 1 month of treatment, plants were harvested and the dry weight, as well as the Cd, K and Ca concentrations in tissues were determined. Results showed that S. portulacastrum, a perennial halophyte with slow growth, is significantly more tolerant to Cd than M. crystallinum, an annual plant. Cd severely inhibited Mesembryanthemum growth even at the lowest Cd concentration in culture medium (50 microM), and did not modify significantly that of Sesuvium. For both halophytes, Cd accumulation was significantly higher in the roots than in the shoots. However, Cd concentration reached 350-700 microg g(-1) DM in the shoots, values characteristic of Cd hyperaccumulator plants. The addition of Cd in the culture medium led to a disturbance of Ca and especially K nutrition, suggesting the possibility to improve plant growth and Cd phytoextraction of both halophytes by increasing nutrient availability in the culture medium.

Differences in the activities of some antioxidant enzymes and in H2O2 content during rhizogenesis and somatic embryogenesis in callus cultures of the ice plant.

Callus was obtained from hypocotyls of Mesembryanthemum crystallinum seedlings cultured on two types of medium-germination medium (GM) and callus induction medium (CIM). Following subculture on shoot induction medium SIM1, the callus formed on CIM medium regenerated roots or somatic embryos, while that obtained on GM medium was non-regenerative. The activities of CuZn-superoxidase dismutase (SOD) were comparable in all calli, but the activities of FeSOD and MnSOD varied according to the activity of photosystem II and the regenerative potential of the tissues. Catalase (CAT) activity was related to H2O2 concentration and affected by both the culture conditions and the morphogenic potential of the calli. The possible role of CAT, SODs and H2O2 in the regeneration of M. crystallinum from callus is discussed.

Early salt stress effects on the changes in chemical composition in leaves of ice plant and Arabidopsis. A Fourier transform infrared spectroscopy study.

A technique based on Fourier transform infrared (FT-IR) spectrometry was developed to detect the corresponding changes in chemical composition associated with the rapid changes in sodium and water content in 200 mM NaCl-stressed halophyte ice plants (Mesembryanthemum crystallinum). The changes in glycophyte Arabidopsis stressed with 50 mM NaCl were also examined for comparison. The obtained IR spectra were further processed by deconvolution and curve fitting to examine the chemical nature of the responding sources in the leaves. Using three stages of ice plant leaves, absorption bands corresponding to carbohydrates, cell wall pectin, and proteins were identified, with distinct IR spectra representing each developmental stage. Within 48 h of mild salt stress, the absorption band intensities in the fingerprint region increased continuously in both plants, suggesting that the carbon assimilation was not affected at the early stage of stress. The intensities of ester and amide I absorption bands decreased slightly in Arabidopsis but increased in ice plant, suggesting that the cell expansion and protein synthesis ceased in Arabidopsis but continued in ice plant. In both plants, the shift in amide I absorption band was observed hourly after salt stress, indicating a rapid conformational change of cellular proteins. Analyses of the ratio between major and minor amide I absorption band revealed that ice plant was able to maintain a higher-ordered form of proteins under stress. Furthermore, the changes in protein conformation showed a positive correlation to the leaf sodium contents in ice plant, but not in Arabidopsis.