Cadmium-responsive genes in rice and their corresponding homologous arsenic-responsive genes in
Arsenic, a class-1 carcinogenic, is a ubiquitous metalloid found in the atmosphere, soils, natural waters, and organisms. The World Health Organization (WHO) estimates that hundred million people worldwide might be chronically exposed to arsenic in drinking water at concentrations above the safety standard. Conventionally applied techniques to remove arsenic species show low removal efficiency, high operational costs, and high-energy requirements. The biological methods, especially phytoremediation, could be cost-effective for protecting human health and the environment from toxic metal contamination. Plants, as sessile organisms, have developed an extraordinary capacity to tolerate arsenic through three main strategies: uptake repression, sequestration into the vacuole, or extrusion. Therefore, arsenic perception and tolerance require a coordinated response that involves arsenic transporters, extrusion pumps, vacuole transporters, and the activation of the phytochelatin biosynthetic pathway. For phytoremediation to become a feasible strategy for arsenic removal from contaminated sites, it is essential to completely understand the molecular mechanisms of arsenic uptake, extrusion, and sequestration, as well as how this response is coordinated. The new genome-wide technologies provide a unique opportunity to understand the molecular mechanisms underlying arsenic perception and accumulation in plants that will open up new possibilities for phytoremediation of arsenic-contaminated waters and soils.
- biotechnological approaches
- Arabidopsis thaliana
- Pteris vittata
Arsenic is a poison naturally present in the Earth’s crust, where it constitutes the 20th most abundant element. Contamination with this element is derived from natural activities, such as volcanism, erosion, or leaching into aquifers, as well as from anthropogenic activities, like mining, smelting ores, or industry. The most abundant forms of arsenic in the environment are the inorganic oxyanions of arsenite (As(III)) and arsenate (As(V)), both of them highly soluble in water [1, 2, 3]. As(III) is more toxic and relatively mobile in contaminated soils, whereas As(V) is considered relatively less toxic.
Arsenic from contaminated soil and water resources poses an environmental threat for all living beings, since it is bioavailable for crops and animals, eventually entering the food chain. Once inside the cell, As(V) interferes with phosphate metabolism, due to its structural analogy to this compound, while As(III) binds to sulfhydryl groups of proteins affecting their functions. Arsenic toxicity is, thus, mostly derived from its interference with enzymes involved in DNA synthesis and repair and cellular energy homeostasis, among others . This metalloid is a well-documented genotoxic agent and class I carcinogen affecting the skin and internal organs. Arsenic effects on human health have been a matter of comprehensive reviews [5, 6, 7, 8, 9, 10]. For these reasons, chronic arsenic exposure, reported in many regions of the world, constitutes an environmental and public health problem of devastating proportions, particularly in Bangladesh [11, 12]. Besides the exposure to contaminated water, another major source of chronic arsenic exposure is derived from dietary consumption of contaminated rice. This crop accumulates relatively high amounts of arsenic and constitutes the main staple food for over 3 billion people across the world, mostly in Asia, being also extensively used for infant feeding [13, 14, 15, 16, 17].
For all these reasons, it is imperative to develop strategies to efficiently limit the effects of arsenic contamination and its entry in the food chain. Physicochemical treatments have been assayed for arsenic-contaminated waters, and recently nanomaterials and metal-organic frameworks (MOFs) are emerging as new promising adsorbent materials. In general, physicochemical treatments are effective at high arsenic concentrations but fail to remove it when contamination levels are low. Another drawback of these techniques is their economic cost, hardly affordable in economically poor rural areas [18, 19].
An alternative approach is the use of living organisms to mitigate arsenic contamination, a strategy named bioremediation. This strategy takes advantage of the ability of microorganisms and plants to tolerate and accumulate arsenic present in nature . The use of plants to clean up contaminated environments is an environmentally friendly green technology, well-accepted sociologically, relatively easy to implement, and potentially cost-effective that can be used to treat both contaminated soils and waters [21, 22, 23]. The main drawbacks of this strategy include the requirement of prolonged periods of time to be effective, lack of reproducibility due to environmental constraints, and the handling of the plant-fixed arsenic waste disposal. Another limitation is that high concentrations of arsenic may inhibit plant growth and, therefore, phytoremediation performance. A recent report has shown that plants accumulating arsenic could efficiently extract and convert it into valuable compounds, such as arsenic nanoparticles, with potential medical applications . In addition, recent advances in the understanding of arsenic toxicity and the pathways of arsenic uptake, accumulation, and tolerance in different plant species, mostly in
In this review, we focus on the phytoremediation strategy to combat arsenic poisoning: we provide a brief overview on the mechanisms of arsenic perception and signaling in different organisms, as well as a survey of plants with proven or potential use for arsenic phytoremediation. Finally, we address several biotechnological approaches that could potentially improve efficiency of arsenic phytoextraction and enable the development of safer edible crops.
2. Arsenic perception and signaling
In this section, we present an overview of the known molecular mechanisms involved in the arsenic response particularly in yeast and plants that will be the basis to design new applied strategies in the near future.
2.1 Arsenic response studies in prokaryotes: the pioneer studies of ars operon in
The identification of arsenic resistance genes in prokaryotes was first reported over 50 years ago . This laboratory identified one strain of
In this review, we will describe the main genes composing the
One of the most important components of the
Another component of the operon,
The last component is
In conclusion, in non-arsenic conditions, ArsR stays attached to the operator of the operon and halts the transcription and expression
Saccharomyces cerevisiae: a suitable model to study arsenic response in eukaryotes
Similarly to ArsR in prokaryotes, Yap8 is considered the arsenic sensor in yeast. There is only one difference between the mechanisms of action of both transcription factors: while ArsR is a repressor, Yap8 acts as an activator. The identification of Yap8 implied an important contribution, since this protein was the first arsenic sensor described in eukaryotes, giving rise to a promising model on how other arsenic sensors may act in higher organisms including plants.
2.3 Arsenic response in eukaryotes:
We will focus this section on the mechanisms of arsenic uptake and detoxification in the plant model
The mechanisms of incorporation and extrusion of As(V) and As(III) have been extensively described in
Once inside the cell, As(V) is rapidly transformed into As(III) through an As(V) reductase enzyme. Initially, AtACR2 was thought to be the main arsenate reductase in plants , which was identified by sequence homology with the yeast ACR2. A few years later, whether AtACR2 was the main As(V) reductase raised many questions among the scientific community , and a major role of AtACR2 remains to be confirmed. Later on, our group identified the gene (At2g21045) which encodes the major As(V) reductase in
Once As(V) is reduced to As(III), it can be extruded by the previously mentioned NIP transporters. Even though As(V) tolerance in
In conclusion, arsenic in plants is incorporated into the cells either through the high-affinity phosphate transporters PHT (As(V)) or through NIPs (As(III)). Once inside the cell, it is reduced by ARQ1/HAC1 and subsequently gets sequestered by PCs. The resulting PCs-As(III) complexes interact with ABCC transporters mediating PCs-As(III) transport into the vacuoles. Alternatively, a fraction of As(III) can be extruded into the media through the NIPs transporters (Figure 1).
Overall, the detoxification mechanisms have been designed with the aim of protecting living organisms from the most dangerous chemical form of arsenic, As(III). Nevertheless, a novel phosphate vacuolar transporter has been recently identified . Those mutants show an As(V) resistant phenotype since they cannot accumulate phosphate inside the vacuoles, and as a consequence it is likely that phosphate present in the cytoplasm provoked the repression of the As(V)/phosphate transporter
3. Plant species with potential use for arsenic phytoremediation
The mechanisms of arsenic tolerance in plants—discussed above—confer adaptive responses that enable plant growth in the presence of this metalloid. There is an immense natural diversity in the arsenic response among different plant species. Some of them have developed an extraordinary capacity to grow in the presence of arsenic and hyperaccumulate this metalloid, holding a great potential to be used for phytoremediation strategies. The most critical parameter in plant species required for phytoremediation is a high extraction capacity. This can be achieved in different ways: via hyperaccumulation of the contaminant inside the cells or via high growth potential or high biomass production in the presence of the toxic compound. They should also be robust and adapted to a variety of biogeochemical environments, seasonal fluctuations, and climatic conditions, in order to obtain good extraction rates in different locations over the years. In addition, the ability to detoxify a variety of xenobiotics in complex degraded environments would be desirable.
A plant species is considered arsenic hyperaccumulator if it is able to accumulate more than 1000 μg g−1 of dry weight [61, 62]. The brake fern
Other species have been evaluated for their arsenic phytoremediation potential. Many of them are endemic species that have been selected because of their ability to grow in arsenic-contaminated areas, and thus they are good candidates for their use as arsenic phytoremediators [61, 63, 84, 85, 86, 87]. As mentioned above, the remediation of arsenic-contaminated waters and paddy soils deserves special attention due to safety concerns. Several wetland plants and aquatic macrophytes have been shown to hyperaccumulate arsenic when growing in contaminated environments and could contribute to solving this devastating trouble [66, 88, 89, 90, 91, 92, 93, 94].
4. Potential transgenic/biotechnological approaches for phytoremediation and arsenic-free crop development
As mentioned above, huge efforts have been made in recent years in order to understand the underlying molecular mechanisms of accumulation and tolerance of arsenic in plants. These initiatives provide a basic knowledge that is crucial to develop novel phytoremediation strategies for environmental cleanup and for producing safe staple crops grown in contaminated lands. The key elements of the arsenic response in the model plant
Phytoremediation of arsenic-contaminated areas requires the development of new plant varieties with enhanced uptake and accumulation of this metalloid in order to remove as much arsenic as possible from the environment. On the contrary, inhibiting the arsenic uptake and translocation to the edible parts and promoting its extrusion outside the cells will be imperative for the development of safe and productive crops grown on arsenic-contaminated lands [25, 96].
4.1 Generation of arsenic transgenic plants for phytoremediation purposes
One of the drawbacks of the phytoremediation approach is that it may have low efficiency as a consequence of phytotoxicity when plants are exposed to high arsenic levels. Moreover, some natural hyperaccumulators do not produce enough biomass, which is crucial for successful phytoremediation, and are also restricted to very specific climatic conditions . Next-generation “omic” approaches are paving the way to increase plant tolerance and extraction of arsenic, holding promising results for phytoremediation . Arsenic uptake, accumulation, and tolerance can be augmented through the modulation of influx/efflux plasma membrane transporters, the regulation of the arsenate reductase activity, and the increase of the amount of PCs and glutaredoxins . The coordination of this element in the arsenic perception and tolerance is a key aspect to sequester arsenic from the cytosol into the vacuoles or its translocation from root to shoot via xylem loading [96, 99].
4.1.1 Arsenic uptake enhancement: phytoextraction
The knowledge acquired in studying the mechanisms of arsenic uptake and translocation by hyperaccumulators such as
Another interesting example of a biotechnological application has been provided by the overexpression of the
Importantly, the next-generation gene-editing CRISPR/Cas9 technology is nowadays an emerging tool to obtain improved crops . This technology is target-specific and allows targeting multiple genes in the genome with high efficiency and specificity. Thereby, this system opens up the possibility to obtain precisely edited crops with enhanced arsenic extraction and accumulation. For example, engineering the aquatic plant
4.1.2 Arsenic plant tolerance improvement: increasing thiol-rich compound production
As(III) chelated with sulfhydryl-rich proteins forms complexes that get sequestered into the vacuoles through vacuolar transporters. Therefore, arsenic tolerance in plants can be enhanced by modifying GSH and PCs . The constitutive expression of the PC biosynthetic gene
In plants, arsenic exposure increases GSH content, which has been correlated with the feedback induction and increased expression of glutathione S-transferases (GSTs) . GSTs quench reactive molecules with the addition of GSH and protect the cell from oxidative damage. Indeed, overexpression of a rice
4.1.3 Rhizoremediation: a beneficial plant-microbe interaction for arsenic phytoextraction
Rhizoremediation takes advantage of the interaction between plants and bacteria living in the rhizosphere for phytoremediation purposes [96, 106]. Arsenic-resistant bacteria associated to the rhizosphere have been demonstrated to play an important role in promoting plant growth and arsenic phytoextraction capacities from contaminated soils [107, 108]. Some bacteria and fungi increase the capability of plants to cope with arsenic by the release of phytohormones such as indole-3-acetic acid and/or essential vitamins and iron. These nutrients promote plant growth and reduce arsenic cytotoxicity. In addition, some microorganisms play an important role in arsenic bioavailability by catalyzing redox reactions that enhance the efficiency of arsenic uptake by the plant roots .
Bacterial strains isolated from the rhizosphere of autochthonous plants grown on arsenic-contaminated industrial lands have been used to enhance phytoremediation in
4.2 Development of arsenic-free food crops
Overall, the generation of arsenic-free crops in the aboveground organs involves increasing root sequestration or extrusion in order to reduce root-to-shoot translocation of the metalloid. Basic breakthroughs have been made in
4.2.1 Arsenate reductases and inositol transporters: from
AtARQ1/HAC1 constitutes the main As(V) reductase in the roots of
4.2.2 Arsenic extrusion strategies
In yeast, ACR3 from
4.2.3 Decreasing arsenic accumulation in rice kernel: Lsi1 and Lsi2 knockout approach
In rice roots, the aquaporin OsNIP2;1/Lsi1 is a major entry route for Si(IV) . This carrier also mediates As(III) uptake and methylated arsenic compounds, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) . Knockouts of this gene show significant lower levels of these arsenic species than wild-type rice plants at short times of exposure . However at longer expositions, these knockout rice plants start to accumulate arsenic since there are bidirectional solute transporters driven by concentration gradient. Lsi2 is an additional aquaporin found in rice. Lsi2 mediates Si(IV) efflux and As(III) transport into the symplast avoiding the Casparian strip and then being easily loaded into the xylem . A knockout mutation of this gene in rice leads to a dramatic reduction on As(III) transport to the xylem and its accumulation in the shoots and grains .
4.2.4 Arsenic sequestration in root vacuoles to avoid translocation
4.2.5 Conversion of arsenic to less toxic volatile forms
Microorganisms are able to volatilize metals and metalloids into the atmosphere through methylation . Several phytoremediation approaches for mercury and arsenic volatilization have been proposed as a suitable strategy to produce safe crops grown in contaminated crops. However, plants do not have in their genome genes encoding As(III) methyltransferases . Therefore, the methylated species of arsenic (MMAs and DMAs) found in plants are the result of microbial activity in the rhizosphere. The volatile arsenic species produced by these microorganisms are then taken up, translocated, and released to the atmosphere by the plant . The reduction of arsenic accumulation through volatilization in rice has been achieved by overexpression of As(III) methyltransferases from bacteria and fungi [121, 122]. Although this approach is very efficient, the release of contaminants into the atmosphere is controversial.
4.2.6 Controlling the AsV/Pi transporter specificity
It has been recently shown that the phosphate transporter PvPHT1;2 from
4.2.7 Phytochelatin and GSH pathway to decrease As accumulation
As mentioned above, one of the most important peptides involved in arsenic accumulation and detoxification of arsenic is the PC, forming As(III)-PC complexes that can be sequestered in the vacuoles. Endosperm-specific intron-containing hairpin RNA-mediated gene silencing of
4.2.8 Iron plaques on rice roots restrict arsenic availability
The oxygenation of roots as a consequence of oxygen diffusion from aerenchyma to the rhizosphere and microbial growth leads to the oxidation of iron and the subsequent formation of iron plaques on the surface of roots particularly in rice . The formation of these plaques is highly variable among different rice genotypes . These iron plaques promote arsenic adsorption and sequestration on the root surface through the formation of Fe-As complexes, thus restricting arsenic bioavailability for plants. Hence, there is a direct relationship between iron plaque formation and reduced arsenic accumulation in the aboveground biomass in rice . Elucidating the genetic mechanisms that affect iron plaque formation would encourage the generation of genetically modified rice with low arsenic accumulation.
4.2.9 Transcription factors: the key coordinators of the arsenic response
Transcriptional regulation is a major factor in the regulation of the capacity of plants to tolerate and accumulate arsenic . Therefore a suitable approach to enhance plant phytoremediation performance will be the identification of key transcriptional regulators of the arsenic response. In
A coordinated network of arsenic, transport, chelation, trafficking, and sequestration mechanisms is crucial to uptake, translocate, and detoxify arsenic. To achieve this, there must be a strong transcriptional regulation following arsenic exposure . However, sensing of arsenic and the signal transduction pathway remains completely unknown. As a matter of fact, a master regulator controlling the expression of other key transcription factor molecules in response to arsenic has not been identified yet. Discovering such transcription factor would be fundamental for developing genetically modified crops that trigger the expression of arsenic detoxification genes to adapt plants to the stress in a synchronized manner.
4.2.10 Relevance of transcriptomic approaches for phytoremediation strategies
The basic discoveries of the arsenic signaling pathway drawn from model plants such as
|Arsenic- and cadmium-responsive gene in rice||Arsenic-responsive orthologue in |
|Glutathione metabolism-related genes||Os09g0367700||AT2G29490, AT2G29450|
|Cell wall biogenesis-related genes||Os11g0592000|
|Expression regulation-related genes||Os02g0168200||AT1G14600|
|Transmembrane transport-related genes||Os01g0695800||AT2G36910|
Arsenic contamination poses a global threat for all living organisms; for this reason, different strategies have been developed to cope with this serious challenge. Among them, phytoremediation is a promising approach. In this chapter, we have provided a brief overview on the status of arsenic phytoremediation from polluted soils and waters, with special focus on the mechanisms of arsenic perception and tolerance in several organisms, plant species used for arsenic phytoremediation, and biotechnological approaches that are driven to increase phytoremediation efficiency as well as crop protection. Although much knowledge and experience have been gained over the last years, there are still many aspects to be discovered and improved. We have exciting times ahead: the exploitation of natural variation, the use of “omic” technologies and biotechnological approaches, a holistic perspective of plant-soil-microbiota interactions, and valorization of plant-fixed arsenic will provide unique opportunities to boost this green strategy.
We apologize to all colleagues whose studies were not cited in this review because of space constraints. We gratefully acknowledge funding by the Spanish Ministry of Economy and Competitiveness (project BIO2014-55741-R) and by the LIFE Programme of the European Commission (Grant number LIFE15 ENV/ES/000382) to AL. CN was supported by project BIO2014-55741-R, CM was supported by project LIFE15 ENV/ES/000382, and MN was supported by a FPU grant (FPU17/05202) from the Spanish Ministry of Science and Innovation.
Conflict of interest
The authors declare no conflict of interest.