RESULTS
Biothiol concentrations in roots and shoots varied greatly depending onArabidopsis genotypes according to the HPLC-DAD analysis (Fig. 1;
Supplementary Table 2). Glutathione was present in all genotypes, but it
was at remarkably low concentrations in the γECS mutants cad2-1 ,pad2-1 and rax1-1 , when compared with the levels of the
wild type Col-0 (all mutants were unequivocally identified by PCR
amplification and sequence alignment; Supplementary Fig. 3). In the
phytochelatin-defective mutant cad1-3 , the GSH concentration in
shoots was almost 2-fold compared with the wild-type. In the presence of
3 µM Hg, PC2 ((γGlu-Cys)2-Gly),
PC3 ((γGlu-Cys)3-Gly) and
PC4 ((γGlu-Cys)4-Gly) were found in
Col-0 roots, whereas only PC2 and PC3appeared in rax1-1 roots. However, none of those PCs were
observed in cad2-1 and pad2-1 mutants. As expected, we
could not detect PCs in cad1-3 under Hg stress, in spite of the
fact that GSH concentrations were the highest observed both in shoots
and roots, doubling the concentration found in Arabidopsis Col-0
(Fig. 1; Supplementary Table 2) .
The decrease of GSH levels in pad2-1 , cad 2-1 andrax1-1 and the inability to synthesize PCs in cad1-3 were
accompanied by significant changes in ascorbic acid (ASA), reduced (GSH)
and oxidized glutathione (GSSG) concentrations, as measured by
HPLC-ESI-MS(TOF) (Table 1). Arabidopsis mutants treated with 3 µM
Hg had ASA concentrations in roots well above of values found in Col-0,
which almost doubled in shoots. On the other hand, GSH concentrations
followed the same pattern found using HPLC-DAD, with pad2-1 ,cad2-1 and rax1-1 having the lowest values both in roots
and shoots, whereas the GSH concentration in cad1-3 was 2-fold
higher than Col-0. Exposure to 3 µM Hg led to general increases in GSH
concentrations in shoots and roots of all mutant genotypes, effect
particularly intense in cad1-3 . With respect to GSSG,
concentrations were one order of magnitude lower than those of GSH, but
they changed with a similar pattern. As a result, there were minimal
changes in the relative content of GSSG irrespective of genotype and
occurrence of Hg stress.
The marked changes in ASA and GSH/GSSG contents observed in response to
Hg suggested possible alterations in the redox balance of mutant shoots
and roots. We firstly analysed the concentrations of Hg in shoots and
roots of Arabidopsis , which accumulated largely in roots (shoots
Hg concentrations was less than 1% of that found in roots; Fig. 2a).
All γECS mutants had similar Hg levels in roots, which were
approximately 50% of the concentration found in Col-0 and cad1-3plants (Fig. 2a). However, Hg concentrations in shoots were not
statistically different between genotypes. In parallel, we determined
chlorophyll fluorescence parameters, and observed that non-photochemical
quenching was severely impaired in γECS and PCS mutant genotypes both in
control and 3 µM Hg-treated plants (Fig. 2b), confirming that limiting
biothiols metabolism led to stress in leaves. GR activity, an enzyme
specifically sensitive to this Hg (Sobrino-Plata et al. 2009), was not
affected in roots (Fig. 3a), but was impaired by Hg in Col-0 andcad1-3 roots with an almost complete inhibition in cad2-1 ,pad2-1 , and rax1- 1 mutants (Fig. 3a). Despite such
inhibition, the amount of GR protein did not change appreciably in
shoots and roots even under Hg-stress independently of the genotype
(Fig. 3b). With regard to γECS in shoots, protein accumulation under
control conditions was remarkably lower (40-50%) in γECS andcad1-3 mutants than in Col-0. Mercury stress also led to a marked
decrease in Col-0 leaves, reaching similar values in all genotypes (Fig.
3b). However, there were no differences among genotypes and Hg stress
levels in root γECS, probably due to the low signal obtained by α-γECS
immunodetection (high background; Fig. 3b).
Our previous study established that part of the ability of plants to
withstand Hg toxicity depends on the formation of Hg-PCs complexes, such
as HgPC2 (Hg(γGluCys)2Gly) and
HgPC3 (Hg(γGluCys)3Gly) (Carrasco-Gil et
al., 2011). Full HPLC-ESI-MS(TOF) analysis of biothiol ligands and
Hg-biothiol complexes (Hg-PCs) in shoots and roots of showed clear
differences between all studied Arabidopsis genotypes (Fig. 4).
In shoots, we could only detect free PC2([PC2-H]−; m/z 538.1) and
PC3 ([PC3-H]−;m/z 770.2) ligands, whereas in roots there were oxidized variants
of free PCs, such as ([PC3oxd-H]−;m/z 768.2), Hg-PC complexes like HgPC2([HgPC2-H]−; m/z 738.1) and
HgPC3 ([HgPC3-H]−;m/z 970.1). The graphical table included in Fig. 4b shows the
groups of free ligands, oxidised PCs and Hg-biothiol complexes, found in
shoots and roots of all Arabidopsis genotypes. The results inrax1-1 and cad2-1 roots closely resembled those found for
Col-0, where we detected
[HgPC2-H]− and
[HgPC3-H]−, albeit with a rather
weak signal (data not shown). On the other hand, in pad2-1 we
only found GSH ([GSH+H]+; m/z 308.1) and
GSSG ([GSSG+H]+; m/z 613.3) in roots and
shoots, which were better detected in positive mode, in addition to
PC2, that was just over the background signal. As
expected, cad1-3 did not accumulate free PCs or Hg-PCs complexes.
Recent studies indicated that toxic elements (Cd and As) are chelated
with PCs in roots impeding translocation to shoots and potentially
helping plants to attenuate stress, in a manner that metal(loid)-PCs
complexes would mass in root vacuoles (Liu et al., 2010; Mendoza-Cózatl
et al., 2008) However, to some extent metal(loid)s may travel to shoots
bound to organic ligands such as PCs (Shi et al., 2019). To determine
whether Hg had a similar behaviour, we studied the possible occurrence
of biothiols and Hg-PCs complexes in xylem sap by HPLC-ESI-MS(TOF) using
both positive and negative modes. Since Hg blocks water movement through
plant vascular tissues, we used a Schölander pressure chamber to
generate sufficient root pressure to impulse xylem water movement. All
xylem sap samples were checked for phloem or broken cells fluids
contamination by measuring MDH activity, which indicated that
cross-contamination was negligible in all Arabidopsis genotypes
under Hg stress (Supplementary Fig.3). The compounds GSH
([GSH+H]+; m/z 308.1) and GSSG
([GSSG+H]+; m/z 613.3) appeared in the
xylem sap of all genotypes, albeit signals were lower in γECS mutants
(data not shown). The characteristic PC2 peak
([PC2+H]+; m/z 540.1)
appeared in xylem sap of Col-0 and, at very low intensity, also inrax1-1 (Fig. 5). This compound coeluted with another ofm/z 538.1, which was tentatively identified as oxidized
PC2 (PC2oxd). However,
PC2 or PC2oxd were not detected incad2-1 , pad2-1 and, as expected, PCS mutant cad1-3 .
To confirm the nature of PC2oxd we run in parallel a
hydroponic experiment with Col-0 Arabidopsis treated with 10 µM
Cd for 72 h. In this case, we got a better signal in MS(TOF) in negative
mode with a m/z 536.1
([PC2oxd-H]−) (Supplementary Fig.
4a); molecular ion that was subjected to tandem MS
(-MS2), and was compared with those obtained using
PC2 (m/z 538.13) and PC2oxd
(m/z 536.1) standards, which had characteristic daughter ions atm/z 254.1 and 128.0 (Supplementary Fig. 4c). Incidentally, we
were unable to observe any Cd-PC complex, in spite of using ESI-MS(TOF)
settings appropriate for detection of CdPC2, as we
obtained the characteristic peaks associated with the natural Cd
isotopic distribution (major
[CdPC2-H]− peak at m/z650.0) by direct injection of a Cd:PC2 standard
(Supplementary Fig. 4b).
In Col-0 xylem sap, along with to PC2 and
PC2oxd we found only a compound with the characteristic
Hg-isotopic fingerprint that could correspond to Hg-PCs complexes, which
was tentatively assigned to HgPC2, eluting separately
from free biothiol ligands (Fig. 5a). The MS(TOF) spectrum (in positive
mode) of the detected compound
([HgPC2+H]+; m/z 740.1)
fitted well with theoretical data and also with a Hg:PC2standard mixture (1:1) (Fig. 5b). The identity of the m/z 740.1
ion peak of Col-0 xylem samples was confirmed using tandem MS/MS
analysis. The same Hg:PC2 standard mixture was used to
set up analytical conditions, and the m/z 740.1 mother ion was
selected and sent to the collision cell for fragmentation
(MS2). Several major daughter ions appeared withm/z 609.1, 536.1 and 508.1 both in the HgPC2standard and the Col-0 xylem sap (Fig. 5c). Some of these ions were
tentatively identified by comparing with those detected in Hg-biothiol
complexes analysis as follows: m/z 609.1 was assigned to
[HgPC2-Glu]+; m/z 536.1
matched [PC2oxd-2H]+, andm/z 508.1 was assigned to [HgGSH+H]+.
Further identification of the m/z 536.1 ion, with the highest
intensity peak, was obtained after a second fragmentation
(MS3) resulting in various ions. The
MS3 spectra of both the xylem sap and the standard
mixture were also very similar, with a major m/z 507.1 daughter
ion (possibly [GSH-H]+), with a second m/z489.1 ion also present in both samples (Fig. 5d). Therefore, we can
assert that HgPC2 complexes could be transferred from
roots to shoots via xylem flux, process that did not occur inrax1-1 , cad2-1 and cad1-3 mutants. Nevertheless, we
could not determine to what extent Hg flows to shoots via xylem, since
our ICP-MS analysis failed to detect Hg above background levels,
probably due to the small volume of sample collected (10-50 µL).
In view of the relevant role that biothiol metabolism has in tolerance
to Hg and Hg speciation in plants, we analysed the expression pattern of
20 genes involved in sulphur uptake, assimilation and incorporation to
biothiols under Hg-stress (Gigolashvili & Kopriva, 2014). The
expression pattern was organ-dependent, with some genes being
over-expressed in the shoots of certain mutants treated with Hg (Fig.
6), whereas in the roots we only detected gene down-regulation under Hg
stress (Fig. 7). Regarding transcription factors in shoots, MYB28was induced only in the γECS-mutants cad2-1 and pad2-1under Hg exposure, whereas MYB51 was suppressed in rax1-1 .
On the other hand, both MYB28 and MYB51 were
down-regulated in roots under Hg-stress, especially in rax1-1 andcad1-3 mutants (Fig. 7). We also found significant
down-regulation of SLIM1 in roots of all mutant Arabidopsisgenotypes (Fig. 7; Suppl. Tables 3 and 4).
Among the genes involved in sulphur incorporation and assimilation in
shoots, the sulphur transporter SULTR1;2 had the highest
over-expression in Hg-treated cad2-1 , rax1-1 andcad1-3 plants, whereas a strong repression was observed in Col-0
(Fig. 6; Suppl. Table 3). A similar repression appeared in Col-0 for ATP
sulphurylase (ATPS3 ) and APS reductase (APR1 andAPR3 ) (Fig. 6). On the other hand, ATPS4 (only inpad2-1 ), APR1 , APR2 and APR3 (only inrax1-1 ), were over-expressed in pad2-1 , rax1-1 andcad1-3 plants treated with 3 µM Hg. With regard to GSH and PCs
metabolism, we only observed a minor down-regulation under Hg stress,
particularly significant for cad2-1 and pad2-1O-acetylserine (thiol) lyase (OASTLA and OASTLB ) genes.
Interestingly, expression of the phytochelatin synthase genesPCS1 and PC2 decreased in leaves in Hg-treated plants,
being particularly significant in pad2-1 , rax1-1 andcad 1-3 (Fig. 6, Supplementary Table 3). Finally, in roots under
Hg stress we only observed significant gene down-regulation, mostly in
the mutant genotypes. Especially relevant was the down-regulation of
sulphate transporters, including a remarkable decrease forSULTR1;2 in cad2-1 , rax1-1 and cad1-3 (Fig.
7). The expression of other sulphur transporters decreased, including
that of SULTR2;1 in rax1-1 and cad1-3 , andSULTR3;5 , which was very intense in all Hg-exposedArabidopsis genotypes. With regard to sulphur assimilation genes,
the most consistent changes occurred in cad1-3 , whereATPS1 , ATPS3 , SiR , OASTLB , OASTLC ,γECS , GSH-S , PCS1 and PCS2 expression
decreased in plants treated with 3 µM Hg (Fig. 7, Supplementary Table
4).