MULTIPLE SCENARIOS TO IMPROVE METAL TOLERANCE
From the previous sections it is clear that metal(loid) tolerance and detoxification is a multigenic phenomenon that is based on a plethora of mechanisms. Thus, the scope for improving tolerance and detoxification is enormous; on the one hand it includes generic routes such as adaptation of root architecture, improved antioxidant response and greater deposition of metal(loid)s in root vacuoles, and on the other hand, more specific processes such as the use of metal(loid)-specific microbes or alterations in transport for a particular element. Which approach is the most likely to be successful will depend on many factors, for example whether contamination comes from a specific metal(loid) or from multiple elements, whether the aim is to improve tolerance to increase yield or, for instance, to allow greater metal accumulation for remediation purposes. In the subsequent sections we will show examples of how metal tolerance has been successfully enhanced and provide a more detailed discussion of root based processes that look particularly promising.
Metal(loid) liganding and vacuolar sequestration Detoxification of heavy metal(loid)s in some cases requires reductant (e.g. in the case of Hg and As) but, since vacuolar deposition is typically in the form of metal(loid) complexes, relies on liganding to organic thiol groups that are found on compounds such as glutathione and phytochelatins. Many studies tested the effect of upregulating one or more of the components of this process; e.g. overexpression of reductases (Shi et al., 2016) or ABC transporters for vacuolar sequestration of metal(loid)s (Zhang et al., 2018). The outcomes of these studies are sometimes counterintuitive: Increased expression of phytochelatin synthases increased tolerance for As in several species but also causes Cd hypersensitivity in many cases (Li et al., 2004). This is possibly to do with different metal(loid)s requiring specific phytochelatins. Alternatively, there may be an imbalance between Cd liganding and subsequent transport into the vacuole. Either way, biosynthesis of phytochelatin (and other thiol ligands) is also likely to greatly increase the energetic cost in terms of generating reduced carbon.
Vacuolar sequestration provides metal(loid) tolerance by protecting sensitive biochemical machinery in the cytosol but also by preventing translocation of metal(loids) from root to shoot. This is exemplified by studies where the tonoplast ABCC1 and ABCC2 were knocked out; mutants showed hypersensitivity to Cd and Hg (Park et al., 2012) and, in contrast to WT plants, stored the majority of Cd in the cytoplasm. However, long distance metal translocation was also greater in the mutant (Park et al., 2012).
Alteration of transporters and transport pathways Reducing metal(loid) influx and/or increasing its efflux can both aid in providing tolerance. A significant reduction in AsV uptake can be achieved by manipulating Pi transporters from the Pht family (Shin et al., 2004, Remy et al., 2012) while that of AsIII is greatly lowered by loss of function in NIPs such as Lsi1 (Ma et al., 2008). For Zn, overexpression of the plasma membrane localised OsZIP5 enhanced Zn uptake from the soil which suggests that a loss of function approach could limit Zn toxicity when ambient levels are high (Lee et al., 2010).
Alternatively, one could focus on increasing metal(loid) efflux from the plant. Some members of the ZIP family function as efflux system; Liu et al. (2019) showed that excess Zn, Cu and Cd enhanced the expression level of the plasma membrane localised OsZIP1. Its overexpressing led to better rice growth during metal stress, most likely by functioning as a metal exporter. The finding that metal impact on ZIP1 transcription occurred via the methylating action of a specific histone (H3K9me2) (Liu et al., 2019) provides a potential vehicle for fine-tuning ZIP1 activity. To properly evaluate the efficacy of this system direct evidence for metal efflux, for example by using radio-isotopes, is urgently needed. The use of heterologous systems to enhance efflux is exemplified by expression of the yeast AsIII/H+antiporter ScACR3p in both Arabidopsis (Ali et al., 2012) and rice (Duan et al., 2012); it successfully increased AsIII efflux from the roots and, in the case of Arabidopsis , improved growth.
Decreasing HMA function could provide avenues to prevent toxic build-up of metal(loid)s in aboveground organs. For example, the presence of metals such as Cd and Zn leads to downregulation of HMA4 in the pericycle and was shown to be accompanied by lower accumulation of Cd and Zn in leaves of Thlaspi arvense (Hammond et al., 2006),Populus trihocarpa (Hammond et al., 2006, De Oliveira and Tibbett, 2018) and of P. nigra (Adams et al., 2011). Similarly, overexpression in rice of the tonoplast located OsHMA3 (Ueno et al., 2010) greatly enhances Cd sequestration in the root and hence drastically reduced grain Cd levels under field conditions (Lu et al., 2019).
Proteins other than transporters per se , may also be relevant in the endeavour to maximise metal(loid) efflux and reduce translocation to the shoot: loss of function in enzymes involved in biosynthesis of glutathione and phytochelatins in Arabidopsis promoted As efflux from roots. Likewsie, knockout mutations in arsenate reductases such as HAC1 resulted in a reduced ability to efflux AsIII from the roots (Chao et al., 2014) indicating that HAC1 upregulation could enhance this function. Knockout mutations of HAC genes in rice had remarkably similar effects (Shi et al., 2016, Xu et al., 2017).
The problem of transport selectivity Unfortunately, the similarity between metal(loid)s and essential nutrients means many of the above strategies run a considerable risk of negatively affecting plant nutrition. Such a ‘cure is worse than the disease’ effect is exemplified by the reduced As toxicity in Pi transporter mutants (Shin et al., 2004, Remy et al., 2012). The latter can only thrive if Pi supply is drastically increased. Thus, keeping the “bad metals” out, while allowing the “good ones” in, critically relies on transport selectivity. Theoretically selectivity could be optimised in a number of ways, for instance via selection of appropriate alleles, the implementation of targeted mutations that increase substrate specificity or use of (more selective) heterologous systems. One of the few known examples that illustrate how this benefits plant tolerance is with IRT1, a primary Fe transporter localized to the plasma membrane of root rhizodermal cells (Dubeaux et al., 2018). IRT1 functions as a sensor for Fe but reports on excess Zn and Mn ions in the cytosol. Binding of these non-iron metals to the histidine-rich loop of IRT1 induces its phosphorylation by the CBL-interacting serine/threonine-protein kinase 23 (CIPK23) and subsequent binding to E3 ligase for polyubiquitination and degradation in the vacuole (Dubeaux et al., 2018). In this manner, IRT1 protects the plant from excess metals like Zn and Mn, whilst safeguarding efficient Fe uptake.
Mutational studies on OsHMA2 identified C-terminal cysteines that impact on metal selectivity (Satoh-Nagasawa et al., 2011). Altered sequences positively impacted on the proportion of Zn that is translocated to the shoot while limiting the amount of Cd transferred to above ground organs (Satoh-Nagasawa et al., 2011). Cysteines in the C-terminus of another HMA (HMA4) also are likely to impact on selectivity and are involved in Cd transport (Lekeux et al., 2018, Ceasar et al., 2020). Mutations in the histidine-rich loop or in the transmembrane domain of the barley Zn transporter HvMTP1 altered metal specificity by shifting it away from Zn toward Mn and Co (Podar et al., 2012).
The above examples clearly show that there is great scope to optimise transport selectivity in order to discriminate better between toxic and beneficial metal(loid)s
Root morphology/architecture Prolonged exposure to metal(loid)s can alter root cell wall composition. The synthesis and deposition of callose in response to As, Cd or Pb forms a physical barrier that inhibits cell-to-cell transport and prevents large scale metal(loid) incursion (Fahr et al., 2013). Similar to callose formation is the accelerated endodermal development in the form of increased suberin deposition and lignification (Lux et al., 2011). In both these cases, creation of a non-specific physical barrier can provide protection against a range of heave metal(loid), including many nutrients. However, such a mechanism may still be beneficial as long a nutrient uptake into the symplast has sufficient selectivity.
Most studies on metal(loid) stress, whether in vitro, hydroponics or compost, are conducted using homogenous distribution of nutrients and contaminants. Yet, natural soils are highly heterogeneous substrates with respect to both chemical and physical components (Hodge, 2009). The heterogeneous distribution of toxic metals under field conditions is exploited by plants by adjusting root architecture in order to avoid contact with contaminants. This ‘avoidance response’ typically inhibits the primary root growth and stimulates the formation of lateral roots in patches of low metal concentration. Avoidance responses have been observed in several species and for a range of elements, that includes Ni, Cd, Zn and Cu (Remans et al., 2012, Khare et al., 2017, Tognacchini et al., 2020, Palm et al., 2021). A great variability in response is evident across species, subspecies and accessions, and may depend on previous adaptation. For example, Tognacchini et al. (2020) found a much greater Ni-response in a Stellaria media accession that originated in a non-metalliferous soil compared to an accession adapted to ultramafic soil.
Although there may be parallels with more common root tropisms, the metal(loid) sensing and signal pathways that culminate in avoidance responses remain to be discovered. Elegant studies by Khare et al (Khare et al., 2017) using a collection of silenced transcription factor mutants in Arabidopsis , showed that the GPL4 (Glabra1 Enhancer Binding Protein‐like 4) transcription factor is involved in the suppression of root growth in areas that contain Cd. Root growth inhibition is likely mediated via generation of ROS that impact on the root apical meristem activity. These authors found that GPL4 not only orchestrated a Cd avoidance response but also suppressed root growth when roots were exposed to essential metals such as Cu and Zn.
Of course, many non-metal stresses also generate ROS but fail to alter root architecture so the question of response specificity remains unanswered. It would also be very useful to know if and how GPL4 activity integrates into other models that have been advanced such as the localised production of ethylene to decrease lateral root formation (Remans et al., 2012) or the more general role of auxin to sustain root tropisms (Muthert et al., 2020, Lux et al., 2011). Further intriguing issues include the manner in which avoidance responses impact on the acquisition of nutrients and what the signalling components upstream of GPL4 are, particularly the primary sensor(s) that registers initial metal toxicity.
Research into the occurrence of avoidance responses and the opposite behaviour (i.e. active foraging for metals which is common in many hyperaccumulators; Tognacchini et al., 2020) has great promise but needs urgent answers to a host of questions: So far it remains unclear whether major crops show avoidance responses, and if so, how relevant they are in overall metal tolerance, particularly with respect to yield. We’re also largely ignorant of the genetic basis for this phenomenon and whether this behaviour can be optimised via selection and breeding.
Plant-growth-promoting rhizobacteria/fungi Microbes in the rhizosphere can greatly affect plant growth in a multitude of manners. Bacteria can alter growth through general mechanisms that include synthesising plant growth hormones (i.e. auxin) or compounds that affect plant growth hormones such as ACC (1-aminocyclopropane-1-carboxylate) deaminase that reduces the levels ethylene (Wu et al., 2018). Soil bacteria and fungi can usually withstand much higher concentrations of toxic metal(loid)s than plants and therefore are able to proliferate even in highly contaminated soil. This resilience is partly the result of robust export mechanisms to remove pollutants from the cytosol. In addition, microbes may reduce metal availability through specific detoxification pathways that are not found in plants but nevertheless assist plant tolerance. For example, the bacterial Mer operon is responsible for Hg methylation and subsequent volatilisation that reduces the soil Hg. The Cellulosimicrobium cellulans bacterium can reduce Cr6+ to the non-toxic Cr3+ (Chatterjee et al., 2009) whereas other bacteria can convert AsIII into the far less toxic AsV (e.g. (Majumder et al., 2013).
Microbes can greatly reduce metal(loid) bioavailability through release of chelating exudates and via sorption (Turnau et al., 2012) and thus improve plant growth. Several studies have shown positive effects of (ecto)mycorrhizas to protect trees and plants from heavy metal toxicity particularly by immobilising metals in the extraradical mycelium or hyphal mantle (Krupa and Kozdroj, 2004). Colonisation by arbuscular mycorrhizal fungi (AMF) can also increase plant tolerance by inducing plant genes that are involved in metal detoxification: Rhizophagus irregularis AMF promoted expression of the metallothionein related genePtMT2b in roots of Populus trihocarpa, irrespective of the Cd and Zn concentrations in soil (De Oliveira et al., 2020). Furthermore, AMF reduced the accumulation of Cd in the leaves by 40%, presumably via its ability to bind Cd either to the cell wall or intracellularly, thus limiting the Cd translocation to the shoot.
Soil bacteria with metal tolerance are common amongstPseudomonas , Bacillus , Enterobacter ,Ralstonia and other soil dwelling species. As for fungi, the mitigating effect of bacteria is primarily due to metal(loid) binding to exudates and sorption by cell wall fractions to lower metal(loid) bioavailability.
An in situ study on Lupin (Dary et al., 2010) showed that inoculation with a consortium of metal resistant bacteria generated an increase in plant biomass which was paralleled by a decrease in root and shoot levels of Cd, Cu, Pb and Zn. In contrast, a field study using rape cultivated in contaminated agricultural soil recorded improved growth of the plants in the presence of metal resistant bacteria, but in this case, bacterial activity increased metal bioavailability and hence drastically raised tissue Cu content (Ren et al., 2019). Some of this contradiction may stem from rape (Brassica napus ) being relatively metal tolerant but it stresses the importance of choosing appropriate rhizosphere organisms that are tailored to specific edaphic conditions and plant hosts.
These examples point to opportunities to exploit soil bacteria and mycorrhization of crops as strategies to aid in their cultivation during metal stress. Where mycorrhizas are concerned, part of the benefits may be via improved nutrition rather than metal detoxification per se, and further studies are needed to tease apart direct and indirect effects. Irrespective of the mechanistic details, we also need far more and better information about the efficacy and (economic) feasibility of manipulating the rhizosphere as an approach to raise plant heavy metal(loid) resistance. Inoculating soils with metal(loid) tolerant bacteria or fungi ‘in the field’ may be impractical and could be prohibitively expensive. Rhizosphere bacteria may need addition of nutrients to be effective, altering cost-benefit analyses. Alternatively, plants may be selected and/or engineered, to increase microbial activity in the rhizosphere but this is likely to attract a large penalty in reduced carbon and lacks specificity.