Introduction
The often indiscriminate use of mercury (Hg) in several human
activities, mostly related with chemical industries and gold-mining, and
the use of ineffective waste removal practices has caused a progressive
contamination of soils and groundwater worldwide (Selin, 2010).
Contamination by this hazardous metal needs to be tackled by using
costly cleaning approaches that result in numerous environmental side
effects (Chaney et al., 1997), whereas plant innate ability to take up
metals can be exploited for soil phytoremediation (Krämer, 2005), in a
sustainable low cost manner particularly appealing in Hg polluted areas
(He et al., 2015). However, this requires tolerant plants able to
withstand cellular damages caused by toxic metal(loid)s (Rascio &
Navari-Izzo, 2011). Among other mechanisms, Hg and other toxic
metal(loid)s activate the rapid synthesis of thiol-rich peptides
(biothiols) such as glutathione (GSH; γGlu-Cys-Gly) and phytochelatins
(PC; (γGlu-Cys)n-Gly, n ranging from 2 to 11)
(Cobbett, 2000). Biothiols play a critical role in toxic metal tolerance
by maintaining the intracellular redox balance and binding toxic metals
to form less harmful chemical species (Hernández et al., 2015), which
are translocated to vacuoles limiting the cytosolic concentration of
free metal (Sharma, Dietz, & Mimura, 2016). Sulphur assimilation and
biothiol metabolism are thought to contribute to Hg tolerance and
homeostasis (Carrasco-Gil et al., 2011), but there is still limited
knowledge on regulatory mechanisms and how those metabolites mitigate
Hg-induced stress.
The overall sulphur acquisition and assimilation pathway is highly
conserved in the course of evolution, and starts with sulphate up-take
by plant roots, but due to its large reduction energetic costs, it is
mostly assimilated in leaves after xylematic transportation viadifferent classes of sulphate transporters (SULT) (Gigolashvili &
Kopriva, 2014). Arabidopsis has up to 14 sulphate transporter
genes distributed in five groups (AtSULTR1-5 ), with Groups 1 and
2 being more related with S-assimilation (Kopriva, 2006). Once sulphate
accumulates at the cytosol, assimilation starts with the synthesis of
adenosine phosphosulphate (APS) by adenosine triphosphate sulphurylase
(ATPS) from ATP (see pathway shown in Figs. 6 and 7). APS is reduced
subsequently by APS reductase (APR), and then sulphite is reduced by
sulphite reductase (SiR). The generated sulfhydryl ion binds to
O-acetylserine in a step catalysed by OAS-thiol lyase (OAS-TL),
synthesizing cysteine (Cys). This thiol-containing amino acid is then
used by the enzyme γ-glutamylcysteine synthetase (γECS), which ligates
Cys to glutamate (Glu), to generate γ-glutamylcysteine (γEC).
Subsequently, glutathione synthetase (GSH-S) forms GSH from Gly and γEC
(Kopriva, Malagoli, & Takahashi, 2019).
In the present study we evaluate the response of differentArabidopsis thaliana genotypes with altered GSH levels under Hg
stress, using three γECS mutant alleles cad2-1 , pad2-1 andrax1-1 , which contain limited amounts of GSH relative to the wild
type (Col-0) (Parisy et al., 2007), and a cad1-3 PCS mutant
unable to produce PCs (Cobbett, 2000). It is already known that Hg leads
to specific stress alterations in biothiol metabolism in comparison with
other toxic metals (Sobrino-Plata et al., 2009), but little information
is available about its influence on sulphur metabolism. Hence, we
analysed the changes in the transcriptional regulation of sulphate
uptake and sulphur assimilation pathway under Hg stress. In addition, we
determined Cys, Glu-Cys, GSH, and PCs accumulation in roots and shoots
to assess plant biothiol distribution. Mercury is taken up by roots
where is strongly retained (Carrasco-Gil et al., 2011, 2013), and only a
small portion is thought to be translocated to shoots via xylem,
as occurs with other metal(loid)s (Khodamoradi, Khoshgoftarmanesh, &
Maibody, 2017); but it may be loaded to the xylem as chelated ions
(Álvarez-Fernández, Díaz-Benito, Abadía, López-Millán, & Abadía, 2014).
Here, we analysed xylem sap samples from different Arabidopsisgenotypes for detection of Hg-biothiol complexes, and we show the
presence of HgPC2 complexes in Arabidopsis wild
type, suggesting that Hg could be transported from the roots to the
shoots not only as free ions but also as a biothiol chelated form.