Macrophyte assisted phytoremediation and toxicological profiling of metal(loid)s polluted water is influenced by hydraulic retention time.

The present study reports findings related to the treatment of polluted groundwater using macrophyte-assisted phytoremediation. The potential of three macrophyte species (Phragmites australis, Scirpus holoschoenus, and Typha angustifolia) to tolerate exposure to multi-metal(loid) polluted groundwater was first evaluated in mesocosms for 7- and 14-day batch testing. In the 7-day batch test, the polluted water was completely replaced and renewed after 7 days, while for 14 days exposure, the same polluted water, added in the first week, was maintained. The initial biochemical screening results of macrophytes indicated that the selected plants were more tolerant to the provided conditions with 14 days of exposure. Based on these findings, the plants were exposed to HRT regimes of 15 and 30 days. The results showed that P. australis and S. holoschoenus performed better than T. angustifolia, in terms of metal(loid) accumulation and removal, biomass production, and toxicity reduction. In addition, the translocation and compartmentalization of metal(loid)s were dose-dependent. At the 30-day loading rate (higher HRT), below-ground phytostabilization was greater than phytoaccumulation, whereas at the 15-day loading rate (lower HRT), below- and above-ground phytoaccumulation was the dominant metal(loid) removal mechanism. However, higher levels of toxicity were noted in the water at the 15-day loading rate. Overall, this study provides valuable insights for macrophyte-assisted phytoremediation of polluted (ground)water streams that can help to improve the design and implementation of phytoremediation systems.

Unveiling the capacity of bioaugmentation application, in comparison with biochar and rhamnolipid for TPHs degradation in aged hydrocarbons polluted soil.

Persistent, aged hydrocarbons in soil hinder remediation, posing a significant environmental threat. While bioremediation offers an environmentally friendly and cost-effective approach, its efficacy for complex contaminants relies on enhancing pollutant bioavailability. This study explores the potential of immobilized bacterial consortia combined with biochar and rhamnolipids to accelerate bioremediation of aged total petroleum hydrocarbon (TPH)-contaminated soil. Previous research indicates that biochar and biosurfactants can increase bioremediation rates, while mixed consortia offer sequential degradation and higher hydrocarbon mineralization. The present investigation aimed to assess whether combining these strategies could further enhance degradation in aged, complex soil matrices. The bioaugmentation (BA) with bacterial consortium increased the TPHs degradation in aged soil (over 20% compared to natural attenuation - NA). However, co-application of BA with biochar and rhamnolipid higher did not show a statistically prominent synergistic effect. While biochar application facilitated the maintenance of hydrocarbon degrading bacterial consortium in soil, the present study did not identify a direct influence in TPHs degradation. The biochar application in contaminated soil contributed to TPHs adsorption. Rhamnolipid alone slightly increased the TPHs biodegradation with NA, while the combined bioaugmentation treatment with rhamnolipid and biochar increased the degradation between 27.5 and 29.8%. These findings encourage further exploration of combining bioaugmentation with amendment, like biochar and rhamnolipid, for remediating diverse environmental matrices contaminated with complex and aged hydrocarbons.

Bioaugmentation and vermicompost facilitated the hydrocarbon bioremediation: scaling up from lab to field for petroleum-contaminated soils.

The biodegradation of total petroleum hydrocarbon (TPH) in soil is very challenging due to the complex recalcitrant nature of hydrocarbon, hydrophobicity, indigenous microbial adaptation and competition, and harsh environmental conditions. This work further confirmed that limited natural attenuation of petroleum hydrocarbons (TPHs) (15% removal) necessitates efficient bioremediation strategies. Hence, a scaling-up experiment for testing and optimizing the use of biopiles for bioremediation of TPH polluted soils was conducted with three 500-kg pilots of polluted soil, and respective treatments were implemented: including control soil (CT), bioaugmentation and vermicompost treatment (BAVC), and a combined application of BAVC along with bioelectrochemical snorkels (BESBAVC), all maintained at 40% field capacity. This study identified that at pilot scale level, a successful application of BAVC treatment can achieve 90.3% TPH removal after 90 days. BAVC's effectiveness stemmed from synergistic mechanisms. Introduced microbial consortia were capable of TPH degradation, while vermicompost provided essential nutrients, enhanced aeration, and, potentially, acted as a biosorbent. Hence, it can be concluded that the combined application of BAVC significantly enhances TPH removal compared to natural attenuation. While the combined application of a bioelectrochemical snorkel (BES) with BAVC also showed a significant TPH removal, it did not differ statistically from the individual application of BAVC, under applied conditions. Further research is needed to optimize BES integration with BAVC for broader applicability. This study demonstrates BAVC as a scalable and mechanistically sound approach for TPH bioremediation in soil.

Phytostabilization of metal(loid)s by ten emergent macrophytes following a 90-day exposure to industrially contaminated groundwater.

Better understanding of macrophyte tolerance under long exposure times in real environmental matrices is crucial for phytoremediation and phytoattenuation strategies for aquatic systems. The metal(loid) attenuation ability of 10 emergent macrophyte species (Carex riparia, Cyperus longus, Cyperus rotundus, Iris pseudacorus, Juncus effusus, Lythrum salicaria, Menta aquatica, Phragmites australis, Scirpus holoschoenus, and Typha angustifolia) was investigated using real groundwater from an industrial site, over a 90-day exposure period. A "phytobial" treatment was included, with 3 plant growth-promoting rhizobacterial strains. Plants exposed to the polluted water generally showed similar or reduced aerial biomass compared to the controls, except for C. riparia. This species, along with M. aquatica, exhibited improved biomass after bioaugmentation. Phytoremediation mechanisms accounted for more than 60% of As, Cd, Cu, Ni, and Pb removal, whilst abiotic mechanisms contributed to ∼80% removal of Fe and Zn. Concentrations of metal(loid)s in the roots were generally between 10-100 times higher than in the aerial parts. The macrophytes in this work can be considered "underground attenuators", more appropriate for rhizostabilization strategies, especially L. salicaria, M. aquatica, S. holoschoenus, and T. angustifolia. For I. pseudacorus, C. longus, and C. riparia; harvesting the aerial parts could be a complementary phytoextraction approach to further remove Pb and Zn. Of all the plants, S. holoschoenus showed the best balance between biomass production and uptake of multiple metal(loid)s. Results also suggest that multiple phytostrategies may be possible for the same plant depending on the final remedial aim. Phytobial approaches need to be further assessed for each macrophyte species.

Comparative toxicological assessment of three soils polluted with different levels of hydrocarbons and heavy metals using in vitro and in vivo approaches.

The biological effects induced by the pollutants present in soils, together with the chemical and physical characterizations, are good indicators to provide a general overview of their quality. However, the existence of studies where the toxicity associated to soils contaminated with mixtures of pollutants applying both in vitro and in vivo models are scarce. In this work, three soils (namely, Soil 001, Soil 002 and Soil 013) polluted with different concentrations of hydrocarbons and heavy metals were evaluated using different organisms representative of human (HepG2 human cell line) and environmental exposure (the yeast Saccharomyces cerevisiae, the Gram-negative bacterium Pseudomonas putida and, for the in vivo evaluation, the annelid Enchytraeus crypticus). In vitro assays showed that the soluble fraction of the Soil 001, which presented the highest levels of heavy metals, represented a great impact in the viability of the HepG2 cells and S. cerevisiae, while organic extracts from Soils 002 and 013 caused a slight decrease in the viability of HepG2 cells. In addition, in vivo experiments showed that Soils 001 and 013 affected the survival and the reproduction of E. crypticus. Altogether, these results provide a general overview of the potential hazards associated to three specific contaminated sites in a variety of organisms, showing how different concentrations of similar pollutants affect them, and highlights the relevance of testing both organic and soluble extracts when in vitro safety assays of soils are performed.

Evaluation of biostimulation, bioaugmentation, and organic amendments application on the bioremediation of recalcitrant hydrocarbons of soil.

In the present work, the operational conditions for improving the degradation rates of Total Petroleum Hydrocarbons (TPHs) in contaminated soil from a machinery park were optimized at a microcosms scale along a 90-days incubation period. In this study, bioremediation strategies and an organic amendment have been tested to verify the remediation of soil contaminated with different hydrocarbons, mineral oils, and heavy metals. Specifically, designed biostimulation and bioaugmentation strategies were compared with and without adding vermicompost. The polluted soil harboring multiple contaminants, partially attenuated for years, was used. The initial profile showed enrichment in heavy linear alkanes, suggesting a previous moderate weathering. The application of vermicompost increased five and two times the amounts of available phosphorus (P) and exchangeable potassium (K), respectively, as a direct consequence of the organic amendment addition. The microbial activity increased due to soil acidification, which influenced the solubility of P and other micronutrients. It also impacted the predominance and variability of the different microbial groups and the incubation, as reflected by phospholipid fatty acid (PLFA) results. An increase in the alkaline phosphatases and proteases linked to bacterial growth was displayed. This stimulation of microbial metabolism correlated with the degradation rates since TPHs degradation' efficiency after vermicompost addition reached 32.5% and 34.4% of the initial hydrocarbon levels for biostimulation and bioaugmentation, respectively. Although Polycyclic Aromatic Hydrocarbons (PAHs) were less abundant in this soil, results also decreased, especially for the most abundant, the phenanthrene. Despite improving the degradation rates, results revealed that recalcitrant and hydrophobic petroleum compounds remained unchanged, indicating that mobility, linked to bioavailability, probably represents the limiting step for further soil recovery.

Differential response of silencing HvIcy2 barley plants against Magnaporthe oryzae infection and light deprivation.

BACKGROUND: Phytocystatins (PhyCys) act as endogenous regulators of cysteine proteases (CysProt) involved in various physiological processes. Besides, PhyCys are involved in plant reactions to abiotic stresses like drought or darkness and have been used as effective molecules against different pests and pathogens. The barley PhyCys-CysProt system is considered a model of protease-inhibitor regulation of protein turnover. Thirteen barley cystatins (HvCPI-1 to HvCPI-13) have been previously identified and characterized. Among them HvCPI-2 has been shown to have a relevant role in plant responses to pathogens and pests, as well as in the plant response to drought. RESULTS: The present work explores the multiple role of this barley PhyCys in response to both, biotic and abiotic stresses, focusing on the impact of silencing this gene. HvIcy-2 silencing lines behave differentially against the phytopathogenic fungus Magnaporthe oryzae and a light deprivation treatment. The induced expression of HvIcy-2 by the fungal stress correlated to a higher susceptibility of silencing HvIcy-2 plants. In contrast, a reduction in the expression of HvIcy-2 and in the cathepsin-L and -B like activities in the silencing HvIcy-2 plants was not accompanied by apparent phenotypical differences with control plants in response to light deprivation. CONCLUSION: These results highlight the specificity of PhyCys in the responses to diverse external prompts as well as the complexity of the regulatory events leading to the response to a particular stress. The mechanism of regulation of these stress responses seems to be focused in maintaining the balance of CysProt and PhyCys levels, which is crucial for the modulation of physiological processes induced by biotic or abiotic stresses.

Arabidopsis response to the spider mite Tetranychus urticae depends on the regulation of reactive oxygen species homeostasis.

Reactive oxygen species (ROS) are molecules that play a prominent role in plant response to numerous stresses, including plant interactions with herbivores. Previous findings indicate that Arabidopsis plants showed an increase in H2O2 accumulation after Tetranychus urticae infestation. Despite its importance, no information has been reported on the relationships between ROS-metabolizing systems and the spider mite-triggered plant-induced responses. In this work, four ROS-related genes that were differentially expressed between the resistant Bla-2 and the susceptible Kon Arabidopsis accessions were selected for the analysis. These genes encode proteins putatively involved in the generation (BBE22) and degradation (GPX7 and GSTU4) of H2O2, and in the degradation of ascorbate (AO). Overexpressing BBE22 and silencing GPX7, GSTU4 and AO resulted in higher leaf damage and better mite performance relative to the wild-type plants. Minor effects on H2O2 accumulation obscure major effects on the expression of genes related to ROS-metabolism and JA and SA signaling pathways, and on ROS-related enzymatic activities. In conclusion, the integration of ROS and ROS-related compounds and enzymes in the response of Arabidopsis to the spider mite T. urticae was confirmed. However, the complex network involved in ROS signaling makes difficult to predict the impact of a specific genetic manipulation.

Silencing barley cystatins HvCPI-2 and HvCPI-4 specifically modifies leaf responses to drought stress.

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.

HvPap-1 C1A Protease Participates Differentially in the Barley Response to a Pathogen and an Herbivore.

Co-evolutionary processes in plant-pathogen/herbivore systems indicate that protease inhibitors have a particular value in biotic interactions. However, little is known about the defensive role of their targets, the plant proteases. C1A cysteine proteases are the most abundant enzymes responsible for the proteolytic activity during different processes like germination, development and senescence in plants. To identify and characterize C1A cysteine proteases of barley with a potential role in defense, mRNA and protein expression patterns were analyzed in response to biotics stresses. A barley cysteine protease, HvPap-1, previously related to abiotic stresses and grain germination, was particularly induced by flagellin or chitosan elicitation, and biotic stresses such as the phytopathogenic fungus Magnaporthe oryzae or the phytophagous mite Tetranychus urticae. To elucidate the in vivo participation of this enzyme in defense, transformed barley plants overexpressing or silencing HvPap-1 encoding gene were subjected to M. oryzae infection or T. urticae infestation. Whereas overexpressing plants were less susceptible to the fungus than silencing plants, the opposite behavior occurred to the mite. This unexpected result highlights the complexity of the regulatory events leading to the response to a particular biotic stress.

Plant senescence and proteolysis: two processes with one destiny.

Senescence-associated proteolysis in plants is a complex and controlled process, essential for mobilization of nutrients from old or stressed tissues, mainly leaves, to growing or sink organs. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and dynamic protein traffic to ensure the complete transformation of proteins of high molecular weight into transportable and useful hydrolysed products. Protease activities are strictly regulated by specific inhibitors and through the activation of zymogens to develop their proteolytic activity at the right place and at the proper time. All these events associated with senescence have deep effects on the relocation of nutrients and as a consequence, on grain quality and crop yield. Thus, it can be considered that nutrient recycling is the common destiny of two processes, plant senescence and, proteolysis. This review article covers the most recent findings about leaf senescence features mediated by abiotic and biotic stresses as well as the participants and steps required in this physiological process, paying special attention to C1A cysteine proteases, their specific inhibitors, known as cystatins, and their potential targets, particularly the chloroplastic proteins as source for nitrogen recycling.

HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses.

Protein breakdown and mobilization from old or stressed tissues to growing and sink organs are some of the metabolic features associated with abiotic/biotic stresses, essential for nutrient recycling. The massive degradation of proteins implies numerous proteolytic events in which cysteine-proteases are the most abundant key players. Analysing the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. In this study, dark and nitrogen starvation treatments were selected to induce stress in barley. Results show that C1A proteases participate in the proteolytic processes triggered in leaves by both abiotic treatments, which strongly induce the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Differences in biochemical parameters and C1A gene expression were found when comparing transgenic barley plants overexpressing or silencing the HvPap-1 gene and wild-type dark-treated leaves. These findings associated with morphological changes evidence a lifespan-delayed phenotype of HvPap-1 silenced lines. All these data elucidate on the role of this protease family in response to abiotic stresses and the potential of their biotechnological manipulation to control the timing of plant growth.

HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination.

Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors.

C1A cysteine protease-cystatin interactions in leaf senescence.

Senescence-associated proteolysis in plants is a crucial process to relocalize nutrients from leaves to growing or storage tissues. The massive net degradation of proteins involves broad metabolic networks, different subcellular compartments, and several types of proteases and regulators. C1A cysteine proteases, grouped as cathepsin L-, B-, H-, and F-like according to their gene structures and phylogenetic relationships, are the most abundant enzymes responsible for the proteolytic activity during leaf senescence. Besides, cystatins as specific modulators of C1A peptidase activities exert a complex regulatory role in this physiological process. This overview article covers the most recent information on C1A proteases in leaf senescence in different plant species. Particularly, it is focussed on barley, as the unique species where the whole gene family members of C1A cysteine proteases and cystatins have been analysed.