Sulfur-Oxidizing Bacteria Alleviate Salt and Cadmium Stress in Halophyte Tripolium pannonicum (Jacq.) Dobrocz.

The aim of this study was to investigate how introducing halophilic sulfur-oxidizing bacteria (SOB) Halothiobacillus halophilus to the growth substrate affects the physiological and biochemical responses of the halophyte Tripolium pannonicum (also known as sea aster or seashore aster) under salt and cadmium stress conditions. This study assessed the plant's response to these stressors and bacterial inoculation by analyzing various factors including the accumulation of elements such as sodium (Na), chloride (Cl), cadmium (Cd) and sulfur (S); growth parameters; levels of photosynthetic pigments, proline and phenolic compounds; the formation of malondialdehyde (MDA); and the plant's potential to scavenge 2,2-Diphenyl-1-picrylhydrazyl (DPPH). The results revealed that bacterial inoculation was effective in mitigating the deleterious effect of cadmium stress on some growth criteria. For instance, stem length was 2-hold higher, the growth tolerance index was 3-fold higher and there was a 20% increase in the content of photosynthetic pigments compared to non-inoculated plants. Furthermore, the SOB contributed to enhancing cadmium tolerance in Tripolium pannonicum by increasing the availability of sulfur in the plant's leaves, which led to the maintenance of an appropriate, about 2-fold-higher level of phenolic compounds (phenylpropanoids and flavonols), as well as chloride ions. The level of MDA decreased after bacterial application in all experimental variants except when both salt and cadmium stress were present. These findings provide novel insights into how halophytes respond to abiotic stress following inoculation of the growth medium with sulfur-oxidizing bacteria. The data suggest that inoculating the substrate with SOB has a beneficial effect on T. pannonicum's tolerance to cadmium stress.

Plant-Based Solutions for Non-Productive Sites Useful in the Management of Dry Land.

The article presents an overview of research conducted in recent years, i.e., from 2004 until now. The study has been prompted by the threat of drought over large land areas which, as a result of current climate change, may lead to desertification in dry and hot regions of the world. For the same reason, large areas of farmland are affected by drought stress. At the same time, rising air temperatures result in a significant intensification of evaporation and a gradual increase in soil salinity. This applies in particular to acres of farmland, forested areas, and green areas of cities, as well as degraded land or brownfields. As the crop stability is threatened, the food base of the world's population is at risk and, additionally, in areas of industrial districts, people's health is in decline. Due to these multistress conditions for plant growth, we propose a review of the current literature which addresses the possibility of counteracting these unfavorable phenomena through the appropriate selection of plant species and, when only applicable, also through specific agroecological treatments. A selection of herbaceous and woody plants useful for cultivation on saline marginal lands was proposed.

Beet Molasses Enhance Salinity Tolerance in Thymus serpyllum-A Study under Greenhouse Condition.

The growing demand for Thymus serpyllum biomass to produce drugs, cosmetics and spices necessitates the search for innovative methods mitigating the negative effects of environmental stressors in order to improve its yield under unfavorable conditions. Due to the exposure of plants to salinity stress (SS), we investigated the effect of sugar beet molasses (SBM) on the growth and biochemical parameters related to plants' response to SS. Wild thyme plants were treated for 5 weeks to sodium chloride and 3% molasses solution using two modes of application (soil irrigation or foliar sprays). Plants irrigated by SBM showed slighter stem growth inhibition than control plants, high stress tolerance index and maintained a constant root water content under salt stress. Moreover plants treated with 100 mM NaCl and soil-applied SBM had lower lipid peroxidation level, showed lower POD activity, higher total soluble protein content and maintained a more even free amino acids level, compared to the control treatments. The concentration of potassium ions was higher in the case of plant roots irrigation with sugar beet molasses compared to control plants. In this experiment, most of the growth and biochemical parameters from foliar molasses-sprayed plants did not differ significantly from the control. We provided evidence that soil-applied SBM beneficially changed the plant's biochemical response to salt stress. On the basis of the obtained results, we conclude that this soil amendment contributes to the strengthening of plant protection against this harmful environmental factor.

A Dual Role of Vanadium in Environmental Systems-Beneficial and Detrimental Effects on Terrestrial Plants and Humans.

The importance of vanadium (V) in the functioning of land systems is extremely diverse, as this element may exert both positive and harmful effects on terrestrial organisms. It recently become considered an element of beneficial character with a range of applications for human welfare. The health-ameliorative properties of this transition element depend on its degree of oxidation and on optimal concentration in the target cells. It was found that a similar relationship applies to vascular plants. However, excessive amounts of vanadium in the environment contaminate the soil and negatively affect the majority of living organisms. A significantly elevated level of V results in the destabilization of plant physiological balance, slowing down the growth of biomass which significantly reduces yield. In turn, low doses of the appropriate vanadium ions can stimulate plant growth and development, exert cytoprotective effects, and effectively enhance the synthesis of some biologically active compounds. We present the scientific achievements of research teams dealing with such topics. The issues discussed concern the role of vanadium in the environment, particular organisms, and highlight its dualistic influence on plants. Achievements in the field of V bioremediation, with the use of appropriately selected microorganisms and plant species, are emphasized.

Distinct co-tolerance responses to combined salinity and cadmium exposure in metallicolous and non-metallicolous ecotypes of Silene vulgaris.

This study compared co-tolerance to salinity and cadmium and investigated its mechanisms in a facultative metallophyte Silene vulgaris originating from distinct habitats. Shoots of calamine (Cal) and non-metallicolous (N-Cal) ecotypes grown in vitro were exposed to 10 and 100 mM NaCl, 5 µM CdCl2 and their combinations. Stress effects were evaluated based on growth, oxidative stress parameters, and DNA content and damage. Tolerance mechanisms were assessed by analyzing non-enzymatic antioxidants, osmolytes and ion accumulation. Irrespective of the ecotype, Cd stimulated shoot proliferation (micropropagation coefficients MC = 15.2 and 12.1 for Cal and N-Cal, respectively, growth tolerance index GTI = 148.1 and 156.7%). In Cal ecotype this was attributed to an increase in glutathione content and reorganization of cell membrane structures under Cd exposure, whereas in N-Cal to enhanced synthesis of other non-enzymatic antioxidants, mainly carotenoids and ascorbate. Low salinity stimulated growth of Cal ecotype due to optimizing Cl- content. High salinity inhibited growth, especially in Cal ecotype, where it enhanced DNA damage and disturbed ionic homeostasis. Species-specific reaction to combined salinity and Cd involved a mutual inhibition of Na+, Cl- and Cd2+ uptake. N-Cal ecotype responded to combined stresses by enhancing its antioxidant defense, presumably induced by Cd, whereas the metallicolous ecotype triggered osmotic adjustment. The study revealed that in S. vulgaris Cd application ameliorated metabolic responses to simultaneous salinity exposure. It also shed a light on distinct strategies of coping with combined abiotic stresses in two ecotypes of the species showing high plasticity in environmental conditions.

Insight into mechanisms of multiple stresses tolerance in a halophyte Aster tripolium subjected to salinity and heavy metal stress.

The study aimed at comparing metabolic reactions of a halophyte Aster tripolium to abiotic stresses. Profiling of endogenous phytohormones, soluble carbohydrates and stress-related amino acids was conducted in plants exposed to moderate and high salinity (150 and 300 mM NaCl), and heavy metal salts CdCl2 or PbCl2 (100 and 200 µM). High NaCl and Pb doses inhibited growth of A. tripolium (Stress Tolerance Index STI) of 37% and 32-35%, respectively. The plants tolerated moderate salinity and Cd (STI = 91% and STI = 83-96%, respectively). Toxic metals accumulated mainly in the roots but Cd translocation to the shoots was also observed. The stressors did not affect total concentrations of the main growth promoting phytohormones but we observed enhanced deactivation of auxins and gibberellins, and reduced accumulation of jasmonate precursor. ABA content increased under stress except for moderate salinity. A common reaction was also activation of osmotic adjustment, however it was disparately manifested under salinity and metallic stress. The distinct responses to salinity and metallic stresses involved changes in carbohydrate profile and altered interplay between salicylic acid content and the pool of active gibberellins. The content of active jasmonates diversified A. tripolium reactions to salt excess and each of the heavy metals. This parameter was linked to the accumulation of ethylene precursor. The results of the study can be used to decipher potential co-tolerance mechanism of this halophyte species to multiple environmental stresses.

Structural, physiological and genetic diversification of Silene vulgaris ecotypes from heavy metal-contaminated areas and their synchronous in vitro cultivation.

MAIN CONCLUSION: Results provide significant comparison of leaf anatomy, pigment content, antioxidant response and phenolic profile between individuals from miscellaneous populations and describe unified cultivation protocols for further research on stress biology. The plant communities growing on heavy metal-polluted areas have attracted considerable attention due to their unique ability to tolerate enormous amounts of toxic ions. Three ecotypes of Silene vulgaris representing calamine (CAL), serpentine (SER) and non-metallicolous (NM) populations were evaluated to reveal specific adaptation traits to harsh environment. CAL leaves presented a distinct anatomical pattern compared to leaves of SER and NM plants, pointing to their xeromorphic adaptation. These differences were accompanied by divergent accumulation and composition of photosynthetic pigments as well as antioxidant enzyme activity. In CAL ecotype, the mechanism of reactive oxygen species scavenging is based on the joint action of superoxide dismutase and catalase, but in SER ecotype on superoxide dismutase and guaiacol-type peroxidase. On the contrary, the concentration of phenylpropanoids and flavonols in the ecotypes was unchanged, implying the existence of similar pathways of their synthesis/degradation functioning in CAL and SER populations. The tested specimens showed genetic variation (atpA/MspI marker). Based on diversification of S. vulgaris populations, we focused on the elaboration of similar in vitro conditions for synchronous cultivation of various ecotypes. The most balanced shoot culture growth was obtained on MS medium containing 0.1 mg l-1 NAA and 0.25 mg l-1 BA, while the most abundant callogenesis was observed on MS medium enriched with 0.5 mg l-1 NAA and 5.0 mg l-1 BA. For the first time, unified in vitro protocols were described for metallophytes providing the opportunity to conduct basic and applied research on stress biology and tolerance mechanisms under freely controlled conditions.

Changes in proteolytic activity and protein carbonylation in shoots of Alyssum montanum ecotypes under multi-metal stress.

The aim of present study was to evaluate the proteolytic response of metallicolous (M) and nonmetallicolous (NM) ecotypes of Alyssum montanum under heavy metals (HMs) stress. Therefore, shoot cultures of tested species grown on medium enriched simultaneously with 0.7 mM ZnSO4, 3.0 µM Pb(NO3)2 and 16.4 µM CdCl2 and these concentration corresponded to the content of their soluble forms marked in calamine substrate. After 8 weeks of cultivation, the overall protease activity (azocaseinolytic) and determination of the proteolytic (gelatinolytic) enzymes profile were estimated in HMs-treated and untreated specimens. The proteins of NM specimens were more susceptible to proteolysis induced by HMs than proteins of M ones. It was found that applied HMs ions caused an increase of protease activity in HMs-treated shoots of NM ecotype that was accompanied by diminished total soluble proteins content and their higher carbonylation. In contrast, the activities of the neutral proteases and metal-dependent serine proteases decreased in HMs-treated shoots of M ecotype. Our results have revealed significant differences at the protein metabolism level in contrasting A. montanum ecotypes cultured in vitro in the presence of HMs.

Evaluation of the protective role of exogenous growth regulators against Ni toxicity in woody shrub Daphne jasminea.

MAIN CONCLUSION: The results provide a significant verification of the activity of exogenously applied phytohormones: gibberellic acid, jasmonic acid, and brassinolide in the modulation of the plant's response to nickel treatment. The study investigated nickel accumulation and its toxicity to Daphne jasminea shoots cultured in vitro with or without exogenous supplementation with phytohormones: gibberellic acid (GA3), jasmonic acid (JA), and brassinolide (BL). The aim was to verify the modulatory effect of exogenous plant growth regulators (PGRs) on plant reaction to Ni excess. The combined action of Ni and PGRs was evaluated at the anatomical, ultrastructural, and biochemical levels. Nickel toxicity was manifested in decreased biomass accretion and growth tolerance index (83-53.6%), attributed to enhanced synthesis of growth inhibitors, mainly abscisic acid. As a defence reaction, endogenous gibberellins accumulated. Exogenous GA3 ameliorated the plant reaction to Ni stress, inducing proliferation and growth rate. Ni tolerance in the presence of GA3 was attributed to peroxisomal reactions that stimulated the synthesis of endogenous JA. In contrast, the application of BL caused enhanced Ni accumulation. Plants suffered from pronounced stress due to massive oxidation. The defence strategy of plants subjected to Ni and BL involved cell wall rearrangements. Exogenous JA stimulated the synthesis of active auxins and salicylic acid, contributing to enhanced mitotic activity within explants. However, JA disturbed the integrity of chloroplasts and lamellar compartments. Our study revealed that an action of exogenous PGRs may either enhance tolerance to Ni or increase metal toxicity in D. jasminea. Particularly in in vitro culture, where explants are subjected to external phytohormonal stimuli, the combined effects of supplemental PGRs may enhance stress and substantially affect plant development. Our results provide a significant verification of exogenous PGRs activity in the modulation of plant response to nickel.

Heavy metal tolerance in contrasting ecotypes of Alyssum montanum.

The response of metallicolous (M) and nonmetallicolous (NM) Alyssum montanum ecotypes to multi-metal stress was investigated under in vitro condition and compared in this study. Shoot cultures were simultaneously treated with 0.7 mM ZnSO4, 3.0 µM Pb(NO3)2 and 16.4 µM CdCl2 for 8 weeks and evaluated for their morphogenetic and ultrastructural reaction, growth tolerance as well as ability to Zn, Pb, and Cd uptake. Moreover, tissue localization and concentrations of antioxidant compounds were determined in order to elucidate the potential role of ROS-scavenging machinery in plant tolerance to metal toxicity. The results clearly demonstrated that M specimens treated with heavy metals showed less phytotoxic symptoms and low level of lipid peroxidation than reference NM one. The enhanced tolerance of M ecotype resulted from heavy metals detoxification in trichomes and intracellular leaf compartments as well as balanced ROS accumulation. The inactivation of ROS in M plants was based on peroxidase-flavonoid system, while in NM plants such relationship was not detected and amounts of antioxidant enzymes or phenolic compounds was comparable to untreated specimens or decreased significantly. Considering the procumbent growth of such hemicryptophyte which reproduce effectively in the presence of heavy metals but is characterized by low biomass production, it is proposed to exploit M ecotype of A. montanum in revegetation schemes of polluted calamine wastes to provide the prompt stabilization of areas prone to erosion.

The acclimatization strategies of kidney vetch (Anthyllis vulneraria L.) to Pb toxicity.

Kidney vetch (Anthyllis vulneraria L.) is a well-known Zn hyperaccumulator. Zn often occurs with Pb in one ore; thus, plants inhabiting waste dumps are exposed not only to Zn but also to Pb toxicity. While the response of kidney vetch to Zn toxicity is relatively well known, the Pb survival strategy of Anthyllis vulneraria has not been the subject of investigations. The aim of presented research was to determine the survival strategy of kidney vetch exposed to high lead concentrations. Shoot explants of a calamine kidney vetch ecotype were placed on agar media containing 0.0, 0.5, 1.0, and 1.5 mM Pb. Morphological, physiological, and biochemical responses, in particular photosynthetic apparatus of plantlets, were examined. The most pronounced changes were observed in plants grown on media supplemented with 1.5 mM Pb after 8 weeks of culture. Increased dry weight and high lead accumulation were observed in roots. Similarly, in shoots, increased dry weight and a decreased number of newly formed shoots were recorded. The accumulation of lead was many times lower in shoots than in roots. In leaf cells' ultra-structure, looser arrangement of chloroplast thylakoid grana was observed. Despite the decrease in chlorophyll a and carotenoid content, the photosynthetic apparatus remained efficient due to the lack of photoinhibition and increased electron transport rate beyond photosystem II (PSII). For the first time, an acclimatization mechanism based on maintaining the high efficiency of photosynthetic apparatus resulting from increasing of electron transport rate was described.

Recent strategies of increasing metal tolerance and phytoremediation potential using genetic transformation of plants.

Avoidance and reduction of soil contamination with heavy metals is one of the most serious global challenges. Nowadays, science offers us new opportunities of utilizing plants to extract toxic elements from the soil by means of phytoremediation. Plant abilities to uptake, translocate, and transform heavy metals, as well as to limit their toxicity, may be significantly enhanced via genetic engineering. This paper provides a comprehensive review of recent strategies aimed at the improvement of plant phytoremediation potential using plant transformation and employing current achievements in nuclear and cytoplasmic genome transformation. Strategies for obtaining plants suitable for effective soil clean-up and tolerant to excessive concentrations of heavy metals are critically assessed. Promising directions in genetic manipulations, such as gene silencing and cis- and intragenesis, are also discussed. Moreover, the ways of overcoming disadvantages of phytoremediation using genetic transformation approachare proposed. The knowledge gathered here could be useful for designing new research aimed at biotechnological improvement of phytoremediation efficiency.

Differential Tolerance to Lead and Cadmium of Micropropagated Gypsophila fastigiata Ecotype.

In vitro techniques may provide a suitable tool for effective propagation and conservation of plant species representing various ecological niches. The elaboration of such protocols is also prerequisite for selection of heavy-metal-tolerant plant material that could be afterwards used for restoration or remediation of polluted sites. In this study, culture protocol for Gypsophila fastigiata propagation was developed. The highest multiplication coefficient, which reached 6.5, and the best growth parameters were obtained on modified MS medium supplemented with 1.0 mg L-1 2iP and 0.2 mg L-1 IAA. The obtained cultures were treated with different concentrations of lead nitrate (0.1, 0.5, and 1.0 mM Pb(NO3)2) or cadmium chloride (0.5, 2.5, and 5.0 µM CdCl2). The growth parameters, photosynthetic pigments, and phenolic compound content were examined in order to evaluate whether tested metal salts can have an adverse impact on studied culture. It was ascertained that Pb ions induced growth disturbances and contributed to shoot wither. On the contrary, the proliferative shoot cultures were established on media containing Cd ions and the multiplication coefficients and shoot length increased on all media enriched with CdCl2. Chlorophylls and carotenoid contents were negatively affected by application of 5.0 µM of cadmium; nevertheless, in shoots treated with 2.5 µM CdCl2, increased accumulation of photosynthetic pigments occurred and their amount was similar to untreated culture. Adaptation to Cd was associated with stimulation of phenolic compound synthesis. Hence, we have reported on unambiguous positive result of in vitro selection procedure to obtain vigorous shoot culture tolerant to cadmium.

From laboratory to field studies - The assessment of Biscutella laevigata suitability to biological reclamation of areas contaminated with lead and cadmium.

The aim of the work was to evaluate the usefulness of the in vitro multiplication of Biscutella laevigata calamine ecotype for in situ reclamation of post-flotation wastes polluted with Pb and Cd. The experiment was conducted on three steps: (i) plant shoots' production under in vitro condition, (ii) establishment of the material in greenhouse experiment, and finally (iii) field cultivation directly on the mining-waste heap of Olkusz Ore-Bearing Region, Poland. This region is known to be one of the most chemically-degraded area in central Europe. The laboratory-set in vitro analysis enabled to obtain the high-quality plant shoots, which multiply the most effectively (with growth tolerance index 130-150%) on medium containing 5.0µM CdCl2 and 0.5mM Pb(NO3)2. These plants were used for the next two ex vitro experiments. Several biometric and physiological analysis (i.e. of photosystem II activity Fv/Fm and PI, photosynthetic pigment contents) were done to indicate plant physiological status during these experiments. The main novelty of the work was to prove that in vitro-multiplied shoots of B. laevigata - the representative of native flora from Olkusz Ore-Bearing Region - can be successfully implemented in situ for the restoration of these degraded area. Moreover, the addition of sewage sludge as a source of organic compounds significantly improved plants' growth and development what is especially important due to the lack of other legal solutions for the management of the sewage sludge in some countries.

Phytostabilization of Zn-Pb ore flotation tailings with Dianthus carthusianorum and Biscutella laevigata after amending with mineral fertilizers or sewage sludge.

Zinc-lead mining wastes remain largely unvegetated and prone to erosion for many years because of phytotoxic levels of residual heavy metals, low nutrient status and poor physical structure. The optimal solution for these areas is to restore plant cover using species which spontaneously appear on the spoils. These species are adapted to the conditions of tailings, and their establishment will promote further vegetation by increasing soil organic matter and development of a soil system capable of supporting the nutrient and water requirements of plants and microoorganisms. The potential of Dianthus carthusianorum and Biscutella laevigata to stabilize mine spoils was analysed in a three-year pot experiment. Post-flotation wastes accumulated after Zn and Pb recovery from ores, were collected from tailings and used as a substrate for plant growth. Seeds for seedling production were collected from plants growing spontaneously on mine tailings. Prior to the establishment of the three-year pot experiment, the substrate was amended with fertilizer NPK or municipal sewage sludge, supplemented with K2O (SS). Substrate samples were collected for chemical analyses, dehydrogenase and urease activities measurements each year at the end of the growing season. The plants were harvested three years after the amendments. Both tested plant species were equally suitable for revegetation of the tailings. The amendment including both SS and NPK resulted in an increase of Corg, Nt, available P, K, Mg contents, an increase of dehydrogenase (DHA) and urease activities and a decrease in the concentrations of the soluble forms of Zn, Pb and Cd. However, nutrient content, DHA activity and plant biomass were higher with SS than NPK addition. NPK application enhanced the substrate properties after the first growing season, while positive effects of SS use were still observed after three years. A longer-lasting positive effect of SS than NPK application was probably due to the high organic matter content in SS, which was gradually decomposing and releasing nutrients.

Organic amendments enhance Pb tolerance and accumulation during micropropagation of Daphne jasminea.

The study investigated the effects of organic amendments: pineapple pulp (PP) and agar hydrolyzate (AH), on micropropagation and Pb bioaccumulation and tolerance in a woody shrub Daphne jasminea cultured in vitro. The amendments were analyzed for their content of carbohydrates, phenolic acids, and phytohormones and added at a dose of 10 mL L-1 to the medium containing 1.0 mM lead nitrate. Micropropagation coefficient increased by 10.2-16.6 % in PP and AH variants, respectively. Growth tolerance index increased by 22.9-31.8 % for the shoots and by 60.1-82.4 % for the roots. In the absence of Pb, the additives inhibited multiplication and growth of microplantlets. PP and AH facilitated Pb accumulation in plant organs, especially in the roots. PP enhanced bioconcentration factor and AH improved Pb translocation to the shoots. Adaptation to Pb was associated with increased accumulation of phenolics and higher radical scavenging activity. Medium supplementation, particularly with AH, enhanced antiradical activity of Pb-adapted lines but reduced the content of phenolic compounds. The study results indicated that supplementation with organic amendments may be beneficial in in vitro selection against lead toxicity.

Chromium(VI) bioremediation by aquatic macrophyte Callitriche cophocarpa Sendtn.

Callitriche cophocarpa (water-starwort)--aquatic widespread macrophyte--was found to be an excellent chromium accumulator. The plants were exposed to various chromium(VI) concentration ranging from 50 to 700 microM in a hydroponic culture up to ca. 3 weeks. Physiological conditions of shoots were monitored via measuring potential photosynthesis quantum efficiency (F(v)/F(m)) and photosynthetic pigment contents. Additionally, the structure of leaves was analyzed using optical and atomic force microscopy (AFM). It has been shown that plants grown in 50 microM Cr(VI) solution exhibited photosynthetic activity and shoot and leaf morphology similar to control plants. Moreover, at the same time the average Cr concentration in their shoots reached about 470 mg kg(-1)d.w. after 10d and up to 1000 mg kg(-1)d.w. after 3 weeks of culture while in control plants did not exceed a few mgkg(-1)d.w. Our results point to Callitriche cophocarpa as a very promising species to be used in the investigation of chromium(VI) phytoremediation mechanisms as well as a good candidate for wastewaters remediation purpose.