Massimo Zacchini

Growth, physiological and molecular traits in Salicaceae trees investigated for phytoremediation of heavy metals and organics

Worldwide, there are many large areas moderately contaminated with heavy metals and/or organics that have not been remediated due to the high cost and technical drawbacks of currently available technologies. Methods with a good potential for coping with these limitations are emerging from phytoremediation techniques, using, for example, specific amendments and/or plants selected from various candidates proven in several investigations to be reasonably efficient in extracting heavy metals from soil or water, or in co-metabolizing organics with bacteria flourishing or inoculated in their rhizospheres. Populus and Salix spp., two genera belonging to the Salicaceae family, include genotypes that can be considered among the candidates for this phytoremediation approach. This review shows the recent improvements in analytical tools based on the identification of useful genetic diversity associated with classical growth, physiological and biochemical traits, and the importance of plant genotype selection for enhancing phytoremediation efficiency. Particularly interesting are studies on the application of the phytoremediation of heavy metals and of chlorinated organics, in which microorganisms selected for their degradation capabilities were bioaugmented in the rhizosphere of Salicaceae planted at a high density for biomass and bioenergy production.

Spatial distribution of cadmium in leaves and its impact on photosynthesis: examples of different strategies in willow and poplar clones.

The interaction of cadmium (Cd) with photosynthesis was investigated in poplar (Populus x canadensis Mönch., clone A4A, Populus nigra L., clone Poli) and willow (Salix alba L., clone SS5) clones that had different leaf metal concentrations in preliminary experiments. Plants grown in the presence of 50 microm CdSO(4) for 3 weeks under hydroponic conditions were used to examine leaf gas exchange, chlorophyll fluorescence parameters and images, and for Cd detection using energy dispersive X-ray fluorescence (ED-XRF). Leaves were finally analysed for Cd and phytochelatin concentrations. Results showed that SS5 had the highest leaf Cd concentration and high gas exchange activity similar to that of Poli, which had the lowest Cd concentration. Leaf fluorescence images evidenced in large undamaged areas of SS5 corresponded to high values of F(v)/F(m), F(o), PhiPSII, qP and NPQ, while patches of dark colour (visible necrosis) close to the main vein corresponded to low values of these parameters. In A4A, these necrotic patches were more diffuse on the leaf blade and associated with a range of fluorescence parameter values. ED-XRF analysis indicated that Cd was only detectable in necroses of SS5 leaves, while in A4A it was relatively more diffuse. Phytochelatins (PCs) were not detected in SS5, while their concentration was high in both Poli and A4A. The absence of these molecules in SS5 is thought to favour confinement of high accumulations of Cd to necrotic areas and gives SS5 the ability to maintain high photosynthesis and transpiration in remaining parts of the leaf.

Screening of poplar clones for cadmium phytoremediation using photosynthesis, biomass and cadmium content analyses.

Variability of cadmium tolerance and distribution in plant organs was investigated in ten selected poplar clones from different species, hybrids and genotypes. To this end, plants were exposed for three weeks to 50 μM CdSO4 in hydroponics. Cadmium accumulation in roots, stem and leaves as well as biomass production and photosynthesis maintenance varied greatly among clones. The metal mostly accumulated in roots, up to 80% in some clones, and interestingly, only 50% in some others. In general, metal tolerance reflected the degree of photosynthesis inhibition, which was higher in clones with high cadmium accumulation in leaves. Biomass production, net photosynthesis, transpiration, and pigment content were differently reduced in cadmium-treated clones with respect to control. The variability of cadmium distribution among leaves, stem, and roots provides candidate poplars for specific phytoremediation processes of contaminated waters and soils.

Improved growth and water use efficiency of cherry saplings under reduced light intensity

Cherry (Prunus avium L.) saplings were grown under natural sunlight (controls) or moderate shading (up to 30%, depending on the incident light intensity and the hour of the day). Reduced light intensity increased the dry mass of each of the plant components studied. Consequently, the total dry mass of shaded plants was significantly greater than that of controls at the end of the growing season. However, the diurnal trend in the level of photosynthesis (per unit of leaf area) of shaded plants was similar to the controls in August, but lower in September. As the growing season proceeded, reduced photosynthetic rates, thinner mesophyll and larger specific leaf area in the shaded plants indicated that leaf development had adapted to shaded conditions throughout the growing season. It is suggested that increased growth of shaded plants was caused by a higher initial relative growth rate and a greater whole-plant photosynthesis. Shading consistently reduced transpiration over the season, therefore improving water use efficiency of shaded leaves. Our results suggest that a moderate reduction in light intensity can be a useful method for improving growth and saving water in hot and dry environments.

Cadmium accumulation and tolerance in Populus nigra and Salix alba

Rooted cuttings of Populus nigra L. clone Poli and Salix alba L. clone SS5 were treated for three weeks with 50 μM CdSO4 in nutrient solution. The willow showed a far higher Cd tolerance, expressed as tolerance index (Ti), than the poplar in both roots and leaves. The root Cd content was higher in poplar than in willow, whereas in leaves the opposite was found. As a consequence, the translocation factor (Tf) revealed a greater ability of Cd transport in willow than in poplar. Cd treatment enhanced cysteine, γ-glutamylcysteine and reduced glutathione contents in roots of both species, whereas in leaves they were only enhanced in poplar. Furthermore, only poplar leaves showed an enhanced content of phytochelatins, whereas malic and citric acids rose in response to Cd only in the willow leaves. Cd treatment increased putrescine, spermidine and spermine contents in both roots and leaves of the willow, whereas in poplar only the putrescine content was enhanced in roots.

Intraspecific variation of physiological and molecular response to cadmium stress in Populus nigra L.

Little is known about the variability of response to heavy metal stress within tree species, although it could be a key for a better understanding of tolerance mechanisms and for breeding. The aim of the present study was to characterize the natural variation of response to cadmium (Cd) in Populus nigra L. in order to understand the mechanisms of Cd tolerance. For that, two P. nigra genotypes, originating from contrasting environments in northern (genotype 58-861) and southern (genotype Poli) Italy, were exposed to Cd stress in hydroponics for 3 weeks. The effect of stress was estimated by measuring biomass production, photosynthetic performance and accumulation and translocation of Cd at the end of the experiment. To better understand the mechanisms of Cd tolerance, the expression of some candidate genes involved in the ascorbate-glutathione cycle (ascorbate peroxidase, glutathione reductase, glutathione S-transferase) and in metal sequestration (metallothioneins) was analyzed in leaves. Biomass production and photosynthesis were affected by the treatment in both clones but the southern clone was markedly more tolerant to Cd stress than the other. Nevertheless, the Cd content in leaves was not significantly different between the two clones and was quite low compared to other species. The content of thiols and phytochelatins (PCs), associated with the transcription profile of the glutathione S-transferase gene, indicated relevant differences in the use of the PCs pathway under Cd stress, which could explain the different tolerance to Cd. The northern clone accumulated thiols but down-regulated the GST gene, whereas the southern clone accumulated PCs and up-regulated the GST gene, which can be useful to complex and detoxify Cd. These results suggest that the glutathione pathway is involved in the differential Cd tolerance of the two genotypes. The natural germplasm of P. nigra represents a valuable resource for understanding tolerance to Cd and for selection of plant material for phytoremediation.

Ibuprofen exposure in Lemna gibba L.: Evaluation of growth and phytotoxic indicators, detection of ibuprofen and identification of its metabolites in plant and in the medium

Ibuprofen (IBU) is detected worldwide in water bodies due to the incomplete removal by wastewater treatments. Contrasting results have been reported on the toxicity of IBU on aquatic biomonitor plants such as duckweed, and no data about IBU detection and metabolism in plants has been reported. In this work, the effects of 1 mg L(-1) IBU on Lemna gibba L. were monitored in an 8-day laboratory test. In particular, an increase in frond number (+12%) and multiplication rate (+10%) while no variations in photosynthetic pigment content were observed. Moreover, UPLC-HRMS analysis of the presence of IBU and its metabolites in plants and in the growth medium was performed. The results showed that, besides IBU, 11 IBU metabolites were detected in plants. Among the IBU metabolites, hydroxyl- and dihydroxyl-IBU were found, whereas carboxyl-IBU was undetectable. Interestingly, some IBU metabolites were detected in the plant growth solution at the end of the IBU treatment, while no IBU products were found in the IBU solution without plants, suggesting a role for L. gibba in IBU metabolism. The findings of this work represent an important step for a better evaluation of the effects of IBU and its metabolites in duckweed, with notable implications for the eco-toxicological assessment of IBU in the aquatic ecosystem.

Assessment of ibuprofen tolerance and removal capability in Populus nigra L. by in vitro culture

Release of pharmaceuticals in the environment has been emerging as a great concern for ecosystem and human health. Ibuprofen (IBU) represents one of the most widespread pharmaceuticals in surface waters and sediments in spite of the high removal rates occurring in conventional wastewater technologies. To assess the potentiality of phytoremediation in assisting these technologies, the screening of plant species for tolerance and removal ability of pollutants is a very important issue. In this study, the effects of different IBU concentrations on callus cultures of Populus nigra L., a pioneer tree species in the riparian ecosystem, were investigated. Results evidenced a notable tolerance of poplar cells to IBU, especially at high concentrations (IBU 30 mg L(-1)), which even stimulated growth. At this concentration, the ability to withstand IBU was accompanied by inhibition of lipoxygenase (LOX) activity, reduction of lipid peroxide content and increase of membrane redox activity. Irrespective of initial IBU concentrations, a complete removal of this compound from the growth medium by poplar cells during a subculture occurred. Antioxidative enzyme activities and polyamine content were stimulated by IBU 0.03 mg L(-1), while no effect was found in cells exposed to IBU 30 mg L(-1), except for a decrease of guaiacol peroxidase (GPX) activity. These findings put in evidence a notable potential of this plant species for the phytoremediation of IBU-contaminated substrates.

Growth, physiological response and phytoremoval capability of two willow clones exposed to ibuprofen under hydroponic culture.

Ibuprofen (IBU) is one of the most widespread pharmaceuticals in the aquatic ecosystem, despite the high removal rate that occurs in wastewater treatment plants. Phytoremediation represents a technology to improve the performance of existing wastewater treatment. This study was conducted under hydroponics to evaluate the ability of Salicaceae plants to tolerate and reduce IBU concentration in contaminated water. To this end, we combined growth, physiological and biochemical data to study the effects of different IBU concentrations on two clones of Salix alba L. Data demonstrated that clone SS5 was more tolerant and showed a higher ability to reduce IBU concentration in the solution than clone SP3. The high tolerance to IBU shown by SS5 was likely due to several mechanisms including the capacity to maintain an elevated photosynthetic activity and an efficient antioxidative defence. These results illustrate the remarkable potential of willow to phytoremediate IBU-contaminated waters in natural and constructed wetlands.

Response of transglutaminase activity and bound putrescine to changes in light intensity under natural or controlled conditions in Quercus ilex leaves

In order to further study a previously observed relationship between polyamine (PA) content and changes in irradiation, we examined the level of free and bound PAs, the activity of transglutaminase (TGase, EC 2.3.2.13) and chlorophyll fluorescence in holm oak (Quercus ilex L.) leaves in response to different levels of light intensity and amount. A diurnal trend of free and bound putrescine (F-Put and B-Put, respectively) and TGase activity was observed in plants under natural conditions in the forest, with the highest value corresponding to the maximum light intensity and amount of light received by the leaves. In another set of experiments, potted Q. ilex plants in experimental fields were subjected to a range of periods of natural photosynthetic photon flux density (PPFD) by covering or not covering the whole trees. Under a natural photoperiod (uncovered leaves), B-Put content and TGase activity paralleled the diurnal PPFD pattern, reaching a maximum at the highest PPFD; prior to this maximum, free PAs showed a significant rise. Plants that were in darkness until midday and suddenly exposed to high light intensity showed enhanced TGase activity, resulting in the maximum accumulation of B-Put. The involvement of the accumulation of B-Put reflected in the changes of the B-Put/bound spermidine ratio during the photoprotective responses to high light stress in forest plants is discussed in relation to the chlorophyll fluorescence parameters observed.