Cadmium-tolerant rhizospheric bacteria of the C3C_{3}/CAM intermediate semi-halophytic common ice plant (Mesembryanthemum crystallinum L.) grown in contaminated soils

The common ice plant, Mesembryanthemum crystallinum L., has recently been found as a good candidate for phytoremediation of heavy-metal polluted soils. This semi-halophyte is a C(3)/CAM (Crassulacean acid metabolism) intermediate plant capable of tolerating extreme levels of cadmium in the soil. The aim of the work was to obtain and characterize novel, Cd-tolerant microbial strains that populate the root zone of M. crystallinum performing different types of photosynthetic metabolism and growing in Cd-contaminated substrates. The plants exhibiting either C(3) or CAM photosynthesis were treated for 8 days with different CdCl(2) doses to obtain final Cd concentrations ranging from 0.82 to 818 mg⋅kg(–1) of soil d.w. The CAM phase was induced by highly saline conditions. After treatment, eighteen bacterial and three yeast strains were isolated from the rhizosphere and, after preliminary Cd-resistance in vitro test, five bacterial strains were selected and identified with a molecular proteomics technique. Two strains of the species Providencia rettgeri (W6 and W7) were obtained from the C(3) phase and three (one Paenibacillus glucanolyticus S7 and two Rhodococcus erythropolis strains: S4 and S10) from the CAM performing plants. The isolates were further tested for Cd-resistance (treatment with either 1 mM or 10 mM CdCl(2)) and salinity tolerance (0.5 M NaCl) in model liquid cultures (incubation for 14 days). Providencia rettgeri W7 culture remained fully viable at 1 mM Cd, whereas Rh. erythropolis S4 and S10 together with P. glucanolyticus S7 were found to be resistant to 10 mM Cd in the presence of 0.5 M NaCl. It is suggested that the high tolerance of the common ice plant toward cadmium may result from the synergic action of the plant together with the Cd/salt-resistant strains occurring within rhizospheral microbiota. Moreover, the isolated bacteria appear as promising robust microorganisms for biotechnological applications in bio- and phytoremediation projects

Ozone fumigation increases the abundance of nutrients in Brassica vegetables : broccoli (Brassica oleracea var. italica) and Chinese cabbage (Brassica pekinensis)

Brassicaceae vegetables, among them broccoli and Chinese cabbage, are well recognized due to the nutritional properties. Four-week-old Chinese cabbage and broccoli seedlings were fumigated with O3 for 3 days before being transplanted into the field. The effect of O3 treatment was determined after reaching marketable quality (ca. 10 weeks). The inflorescences of O3-treated broccoli were enriched in vitamin E (α-tocopherol and γ-tocopherol), whereas Chinese cabbage heads had an increased content of anthocyanins and β-carotene. Ozone treatment did not significantly affect the productivity of both examined vegetables

Expression of genes involved in heavy metal trafficking in plants exposed to salinity stress and elevated cd concentrations

Many areas intended for crop production suffer from the concomitant occurrence of heavy metal pollution and elevated salinity; therefore, halophytes seem to represent a promising perspective for the bioremediation of contaminated soils. In this study, the influence of Cd treatment (0.01–10.0 mM) and salinity stress (0.4 M NaCl) on the expression of genes involved in heavy metal uptake (irt2–iron-regulated protein 2, zip4–zinc-induced protein 4), vacuolar sequestration (abcc2–ATP-binding cassette 2, cax4–cation exchanger 2 pcs1–phytochelatin synthase 1) and translocation into aerial organs (hma4–heavy metal ATPase 4) were analyzed in a soil-grown semi-halophyte Mesembryanthemum crystallinum. The upregulation of irt2 expression induced by salinity was additionally enhanced by Cd treatment. Such changes were not observed for zip4. Stressor-induced alterations in abcc2, cax4, hma4 and pcs1 expression were most pronounced in the root tissue, and the expression of cax4, hma4 and pcs1 was upregulated in response to salinity and Cd. However, the cumulative effect of both stressors, similar to the one described for irt2, was observed only in the case of pcs1. The importance of salt stress in the irt2 expression regulation mechanism is proposed. To the best of our knowledge, this study is the first to report the combined effect of salinity and heavy metal stress on genes involved in heavy metal trafficking

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

KEY MESSAGE: H(2)O(2)is necessary to elicit rhizogenic action of auxin. Activities of specific catalase and manganese superoxide dismutase forms mark roots development. ABSTRACT: 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 H(2)O(2) level, inhibited rhizogenesis and induced non-regenerative callus, suggesting that certain level of H(2)O(2) 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 H(2)O(2) 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

Pathways of ROS homeostasis regulation in "Mesembryanthemum crystallinum" L. calli exhibiting differences in rhizogenesis

A comparison of the hydrogen peroxide (H2O2) content, proline and betacyanin concentration and activities of some antioxidant enzymes (catalase, superoxide dismutase, guaiacol and ascorbate peroxidases) was made in Mesembryanthemum crystallinum L. calli differing in rhizogenic potential. Callus was induced from hypocotyls of 10-day-old seedlings on a medium containing 1 mg l−1 2,4-dichlorophenoxyacetic acid and 0.2 mg l−1 kinetin, which was either supplemented with 40 mM NaCl (CIM-NaCl medium) or did not contain any salt (CIM medium). The callus obtained on CIM-NaCl was rhizogenic, whereas the callus induced on the medium without salt was non-rhizogenic throughout the culture. The rhizogenic callus differed from the non-rhizogenic callus in lower betacyanin and H2O2 content, but the rhizogenic callus displayed a higher proline level. The activity of H2O2 scavenging enzymes, such as catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (POD), was markedly higher in the rhizogenic callus than in the non-rhizogenic callus, but the total activity of superoxide dismutase (SOD) was higher in the non-rhizogenic callus than in the rhizogenic callus. Aminotriazole (CAT inhibitor) and diethyldithiocarbamate (SOD inhibitor) were added solely to the CIM and CIM-NaCl media to manipulate the concentration of reactive oxygen species (ROS) in the cultured tissues. Both CAT and SOD inhibitors brought about an increase in H2O2 content in calli cultured on CIM-NaCl and the loss of rhizogenic potential. Conversely, the addition of inhibitors to the medium without salt led to a decrease in H2O2 content. This corresponded with a significant decrease in the endogenous concentration of betacyanins, but did not change the lack of rhizogenic ability

The fungal endophyte Epichloë typhina improves photosynthesis efficiency of its host orchard grass (Dactylis glomerata)

According to the results presented in this paper the fungal endophyteEpichloë typhinasignificantly improves the growth, PSII photochemistry and C assimilation efficiency of its hostDactylis glomerata. In this paper, we present a comprehensive study of the impact of the endophytic fungi Epichloë typhina on its plant hosts’ photosynthesis apparatus. Chlorophyll a fluorescence, gas exchange, immuno-blotting and spectrophotometric measurements were employed to assess photosynthetic performance, changes in pigment content and mechanisms associated with light harvesting, carbon assimilation and energy distribution in Dactylis glomerata colonized with Epichloë typhina. According to the results presented in this study, colonization of D. glomerata results in improved photosynthesis efficiency. Additionally, we propose a new mechanism allowing plants to cope with the withdrawal of a significant fraction of its energy resources by the endophytic fungi. The abundance of LHCI, LHCII proteins as well as chlorophyll b was significantly higher in E+ plants. Malate export out of the chloroplast was shown to be increased in colonized plants. To our knowledge, we are the first to report this phenomenon. Epichloë colonization improved PSII photochemistry and C assimilation efficiency. Elevated energy demands of E+ D. glomerata plants are met by increasing the rate of carbon assimilation and PSII photochemistry. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00425-015-2337-x) contains supplementary material, which is available to authorized users

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

Alterations in Chlorophyll a Fluorescence and Pigments Concentration in the Leaves of Cauliflower and Broccoli Transplants Subjected to Chilling

Chlorophyll a fluorescence parameters and photosynthetic pigments content in leaves of broccoli (Brassica oleracea L. var. italica) cv. ‘Monaco’ F1 and cauliflower (Brassica oleracea L. var. botrytis) cv. ‘Bruce’ F1 transplants were analyzed to investigate the effects of chilling temperatures (6 and 10 °C) and the exposure length (1 or 2 weeks) on the photosynthetic apparatus condition in the 3-year experiment. Data were compared to control plants exposed to 14 °C for 1 and 2 weeks. The lowest values of maximum quantum efficiency of photosystem II (Fv/Fm) and the performance index (PI) were observed in cauliflower chilled at 6 °C for 2 weeks. This was not accompanied by any substantial reduction in chlorophylls concentration, however, significant correlations between Fv/Fm or PI and chlorophylls content in cauliflower leaves were found. There were no negative effects of chilling on photosynthetic activity and chlorophyll content in broccoli leaves, the amount of carotenoids significantly increased in the broccoli chilled for 2 weeks at 6 °C. The chlorophyll a fluorescence indices and accelerated carotenoids synthesis showed that broccoli had less vulnerable photosynthetic apparatus to stress-induced temperatures and more efficient protection mechanisms in comparison to cauliflower transplants

Is the redox state of the PQ pool involved in regulating the ET biosynthesis pathway of CAM facultative semi-halophytes?

The mechanisms and factors regulating ethylene (ET) biosynthesis and its action remain largely unexplored, particularly in CAM-facultative and (semi)halophytic plants. The use of Mesembryanthemum crystallinum L. (ice plant) provides a unique opportunity to examine plastoquinone (PQ) - ET interactions in semi-halophytes and CAM-facultative plants simultaneously. Here, we present the results of an analysis of the common ice plant’s response to prolonged (14-day) salinity stress and DCMU, which maintains the PQ pool in a more oxidised state, thereby mimicking darkness conditions. Differentially expressed gene (DEG) analysis showed that the expression of genes involved in ET regulation was not significantly altered after DCMU application. However, in $C_{3}$ plants not affected by salinity stress, the expression of genes related to both photosystems, photosynthesis, and the photosynthetic electron transport chain was significantly affected by DCMU. We propose that sustained salinity stress and the occurrence of CAM photosynthesis render physiological processes insensitive to disruptions caused by a modified PQ pool redox state. The UPLC-MS analysis of the ET biosynthesis pathway central intermediate - 1-aminocyclopropane-1-carboxylic acid (ACC) - confirmed the molecular analysis results, as ACC content was similarly affected in salinity untreated and treated plants. Moreover, the analysis of key antioxidative system components, namely catalase and superoxide dismutases, suggests that PQ pool redox state does not directly regulate them. Instead, an alternative regulation mechanism involving reactive oxygen species (ROS) accumulation and a ROS-induced signalling cascade has been proposed