Bioaccumulation, antioxidative response, and metallothionein expression in Lupinus luteus L. exposed to heavy metals and silver nanoparticles

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Soil contamination with heavy metals & phytoremediation

Pollution of soil with heavy metals becomes a fast-rising issue in many places due to a vast scope of anthropogenic activities (Pant et al., 2014). The major contribution to this problem is often divided into two main sources. The first one includes industrial activities such as mining and manufacturing. The second one is connected to modern agriculture (Tóth et al., 2016).
Most heavy metals (HMs) degrade slowly and cannot be decomposed naturally, resulting in their long-lasting presence in the environment. As a result, they can seep into the deeper layers of the soil and pose threat to groundwater reservoirs (Motuzova et al., 2014; Pant et al., 2014). In addition, such soil pollution can increase oxidative stress in plants which can slowly and gradually decrease plant growth causing a significant decrease in photosynthesis efficiency (Chabukdhara et al., 2013). Overall, metal contamination of soil and water creates a direct obstruct in normal growth of plants reducing crop yields (Bolan et al., 2014).
Continually increasing soil contamination with heavy metals affects all links in the food chain – from soil microorganisms through plants, animals, and humans. Heavy metals are one of the most prevalent abiotic stress factors affecting the growth, development, and productivity of plants, and are a direct threat not only to the soil environment but also groundwater and human health, causing damage by oxidative stress and inducing cancer in cells (Mahar et al., 2016). High availability and toxicity of heavy metals in the soil, sometimes lasting over a few hundred years, makes it impossible to use contaminated areas for agricultural purposes (Liu et al., 2014). Accumulation of metals such as Lead (Pb), Chromium (Cr), Nickel (Ni) can seep through food chain and cause DNA damage via their mutagenic properties which can lead to carcinogenic changes in bodies of animals and humans. Since long-term persistence of heavy metals in the environment causes a threat to human health and crop production, technologies involved in effective remediation of contaminated areas are increasingly gaining importance (Pant et al., 2014; Tóth et al., 2016). Moreover, due to the growing health concerns associated with heavy metals and their broad distribution in soil and water, more attention has been directed to identification of the sources of such pollution and the remediation of contaminated sites (Duan et al., 2017). The use of physical and chemical methods to deal with heavy metal contaminated areas includes the use of ion exchange, reverse osmosis, chemical reduction, precipitation and evaporation (Gong et al., 2018). Modern techniques that allow for a reduction in the concentrations of hazardous substances include processes that promote the desorption and subsequent removal in the liquid phase. Such actions include soil washing techniques, as well as electrokinetic methods and heat treatments (Habibul et al., 2019). All of them can be successfully applied to the process of decontamination, but they require a lot of external resources and in most cases are too expensive for large-scale use (Sharma et al., 2014). Due to those reasons, in recent decades, more and more attention has been given to phytoremediation, in which plants absorb and transform contaminants in order to detoxify the site and clean-up polluted environments (Yadava et al., 2018). It is worth mentioning that phytoremediation can also be applied to other contaminants – not solely to heavy metals but also to pesticides, explosives and crude oil. Overall, phytoremediation can takes advantage of the natural processes and requires less equipment and labor than other technologies (Burges et al., 2017, Jaskulak et al., 2019). In general, phytoremediation in accepted as a sufficient and cost-effective approach to clean up metal-contaminated soils.
Assisted phytoremediation is a wildly accepted technology used in remediation of heavy metals by use of hyperaccumulating plants and soil fertilization. Hyperaccumulators are able to store extremely high levels of heavy metals in their above-ground tissues without suffering its toxic effects. Such plants are characterized by their remarkable biochemical mechanisms which allow them to accumulate and translocate metals in their cells. Therefore, they show promise for the use in large-scale phytoremediation (Cortés-Eslava et al., 2018). Understanding the molecular mechanisms of hyperaccumulation may, consequently, help in enhancing the performance of hyperaccumulators for phytoremediation. During the process of phytoremediation, hyperaccumulators extract and accumulate heavy metals from soils, which is followed by harvesting biomass until the concentration of specific contaminant decreases to acceptable level (Chowdhary et al., 2018).
The identification of mechanisms by which plant respond to metal exposure is thus, a prime objective in plant research (Hattab et al., 2015). However, genes involved in the protection against metal toxicity and the molecular mechanisms underlying this protection are still largely undefined in plants. Especially in species other than model plants. Thus, understanding the gene expression and its regulation involved in metal toxicity is essential for understanding the genetic and molecular mechanisms involved in effective phytoremediation (Cortés-Eslava et al., 2018). To this day over 450 heavy metal accumulators have been identified, but overwhelmingly (80%) of them are tolerant to nickel. Hyperaccumulation of cadmium was noticed in two plant families: Brassicaceae and Crassulaceae. Hence, a number of physiological studies had been carried out to understand the hyper-tolerance and its mechanisms within these families. The development of high-throughput deep sequencing technology has enabled the large-scale RNA-seq of dynamic transcriptomes, even without fully sequenced reference genomes (Chowdhary et al., 2018).

Soil fertilization with sewage sludge and other waste products

In order to increase the efficiency of the assisted phytoremediation and at the same time deal with a constant rise in the quantity of produced wastewater worldwide, the application of sewage sludge to land is often seen as a viable option (Alvarenga et al., 2016). Since large volumes of sewage sludge have to be disposed or treated in some manner, the use of it as a fertilizer has become a more common practice in recent years. It was shown before that this kind of action improves soil properties and increases crop productivity (Bourioug et al., 2015). Beneficial application of organic waste like sewage sludge or animal manure in agricultural soils can allow to maintain and restore the quality of previously degraded soils, as well as reduce the need for application of synthetic fertilizers (Chowdhary et al., 2018). In recent decades, more and more lands have been chronically suffering from a severe decrease in organic matter caused mostly by overexploitation, and droughts (Peng et al., 2016). Therefore, the reuse of biosolids like manures and sewage sludge is proposed as an alternative solution for improvement in organic matter content and soil quality at low costs (Saveyn et al., 2014). In the past 20 years, the idea of reusing nutrients by applying sewage sludge to agricultural soil has become a more common practice and it is currently considered as a leading alternative to other disposal methods such as incineration or storing and processing at landfills. The application of organic waste such as sewage sludge as soil amendments is also a crucial strategy to the “end of waste” policy in Europe, that will contribute to an increase in the levels of soil organic matter content that tends to lower across almost all Europe mostly as a result of overproduction, industrialization, contamination with heavy metals (HMs) and droughts (Chowdhary et al., 2018). On the contrary, the idea of using sewage sludge as a fertilizer has also a couple of important drawbacks that must be taken into consideration. Inadequately processed sewage sludge can have a wide variety of undesired traits and in consequence, adverse effects on given environment (Saveyn et al., 2014). Due to this risk, it is crucial to gather knowledge about reducing the hazards associated with using organic wastes such as sewage sludge as a soil amendments (Alvarenga et al., 2016). Thus, further knowledge on biochemical and physiological responses of plants to stress helps develop new strategies for purification of contaminated areas and overall improvement of the environment.

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Aims of the work

The main aim of the thesis concerns the identification of mechanisms by which plants respond to metal exposure and to soil supplementation with waste products (manures, sewage sludge). The designed studies aimed to evaluate the impact of complex metal contamination, on the level of abiotic stress, activity of antioxidative enzymes, efficiency of photosynthesis, genotoxicity and expression of selected genes in higher plants and mutual correlation of these variables. The practical purpose of the project was focused mostly on proposing new physiological biomarkers as short-term toxicity tests which will allow for a more precise application of waste products into soil during remediation processes. The overall goal of the project is also to identify the ways in which sewage sludge application is influencing plants on gene expression level (including metal chelators and transporters), to broaden our understanding on the environmental impact and safety of such supplementation of both agricultural and degraded soil (Chapters 2 & 3).
Moreover, since phytoremediation, despite still being a novel technology, has already entered the stage of modeling, the presented work highlights the main advantages, and limitations of existing models, to explore their applicability in given circumstances and proposes a simple model for the prediction of cadmium removal during assisted phytoremediation with sewage sludge supplementation (Chapter 4).
Current research gaps:
1. The need to identify and validate the usability of new biomarkers with high sensitivity and selectivity in order to increase the efficiency of the assisted phytoremediation process and precisely determine the degree of risk resulting from the presence of contaminants;
2. Lack of known sequences coding key genes associated with heavy metal detoxification processes in plants exposed in natural conditions with complex contamination;
3. Lack of information on potential reference genes for plants other than model species, especially in the case of complex contamination of natural soil;
4. Scarce information on the impact of soil additives, including sewage sludge, on changes in the level of gene expression in plants;
Specific goals:
1. Identification of key metal responsive genes in species other than model plants and important for the remediation purpose;
2. Assessment of the sensitivity of selected stress markers, along with the identification of the genes encoding them in higher plants;
3. Creation of an information gateway about metal detoxification and defense pathways, which will help to develop strategies for environmental clean-up and rehabilitation of contaminated soils;
4. Assessment of the impact of soil supplementation with sewage sludge and manures on genotoxicity and expression of selected genes in higher plants and the mutual correlation of these variables;

Immobilization and exclusion

Immobilization is the first barrier against toxicity of metal ions including cadmium. It occurs mostly on a root level (Dheri et al. 2007). Exclusion generally means all the mechanisms that can prevent Cd ions from entering the cytosol through the membranes (Kranner et al. 2011). Cd can be immobilized by means of the cell wall or for example extracellular carbohydrates (Fusconi et al 2006). The importance of this processes varies significantly among different concentrations of Cd, plant species and other environmental factors involved (Fusconi et al. 2006, Dheri et al. 2007). In early stages of seed germination in several species, the entrance of Cd to cells was noticed through the Ca channels in the cell membrane (Kranner et al. 2011).


Compartmentalization in vacuoles plays one of the crucial roles in Cd detoxification and tolerance by preventing further free circulation of metal ions in cell cytosol (Lang et al. 2011). Cd exposure causes a rapid synthesis of phytochelatins and metallothionein which in their way create complexes with Cd ions and are successfully transported to the vacuole (Bolan et al. 2013). After that, due to the acidic pH in cells vacuole, formed complex dissociates, and metal can be further complexed by organic acids and possibly amino acids (Dias et al. 2013). Hydrolases can degrade used phytochelatins in vacuoles, or they can return to the cytosol to continue with their role (Clemens et al. 2006).

Stress ethylene

Exposure to cadmium can stimulate the ethylene biosynthesis mostly via MSAE pathway (methionine, S-adenosylmethionine, 1-aminocyclopropane-1-carboxylic acid, ethylene) (Iqbal et al. 2015). Production of ethylene causes an increase in the activity of some antioxidant enzymes including guaiacol peroxidase and greater accumulation of insoluble and soluble phenolics (Masood et al 2012). In most species, the stimulation of ethylene production after Cd exposure peaks within only 5-10 hours after the initial exposure and declines after that (Iqbal et al. 2015). It is the hypothesis that in in vitro conditions the decrease in ethylene production is related to Cd-Sequestration and thus diminished stress. Due to the scarcity of available data about this response, it is impossible to assess at a molecular and cellular level the interactions between synthesis of ethylene and Cd stress (Masood et al 2012, Iqbal et al. 2015).

Table of contents :

CHAPTER I – General introduction
1.1 Soil contamination with heavy metals & phytoremediation
1.2 Soil fertilization with sewage sludge and other waste products
1.3 Aims of the work
1.4 Research hypotheses
CHAPTER II – Biomarkers of abiotic stress as tools for planning and monitoring of phytoremediation efficiency
2.1 Cadmium phytotoxicity – biomarkers
2.2 Enzymatic assays confirm the toxicity reduction after manure treatment of heavy metals contaminated soil
2.3 Antioxidative enzymes and expression of rbcL gene as tools to monitor heavy metal-related stress in plants
CHAPTER III – The influence of contaminants on the expression of genes encoding metal chelators & metal transporters in plants
3.1 Implementation of omics research to enhance phytoremediation efficiency – a review
3.2 Bioaccumulation, antioxidative response, and metallothionein expression in Lupinus luteus L. exposed to heavy metals and silver nanoparticles
3.3 Gene expression, DNA damage and other stress markers in Sinapis alba L. exposed to heavy metals with special reference to sewage sludge application on contaminated sites
3.4 Effects of sewage sludge supplementation on heavy metal accumulation and the expression of ABC transporters in Sinapis alba L. during assisted phytoremediation of contaminated sites
CHAPTER IV – Modeling of the assisted phytoremediation of metal contaminated soils
4.1 Modeling assisted phytoremediation of soils contaminated with heavy metals – main opportunities, limitations, decision making and future prospects
4.2 Modeling and optimizing the removal of cadmium by Sinapis alba L. from contaminated soil via Response Surface Methodology and Artificial Neural Networks during assisted phytoremediation with sewage sludge
CHAPTER V – Conclusions and perspectives
5.1 Main conclusions
5.2 Recent work and future perspectives


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