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.