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.