Discussion
In this study, we characterized CsGGCT2;1 gene in Camelina for
its role in providing arsenite tolerance by maintaining GSH homeostasis.
Under AsIII stress, CsGGCT2;1 OE lines had significantly higher
shoot and root biomass, improved chlorophyll content, lower lipid
peroxidation, and accumulated less arsenic than the WT. Further analysis
showed that relative to non-stress MS control conditions, resources in
the plants treated with AsIII shifted towards the γ-glutamyl cycle as
Glu, Cys, Gly, GSH, and 5-OP levels increased; some slightly to some
multiple fold differences.
Paulose et al . (2013) investigated the role of AtGGCT2;1and its involvement in GSH degradation and the recycling of glutamate in
Arabidopsis plants under AsIII stress. The authors observed thatAtGGCT2;1 gene was strongly upregulated in roots exposed to AsIII
treatment but not in the shoots. Overexpression of AtGGCT2;1 in
Arabidopsis led to increased tolerance to AsIII as evidenced by
significantly higher shoot biomass in the OE lines growing on media
containing AsIII, and a significant decrease in arsenic accumulation in
shoots with an increased accumulation in roots of OE plants relative to
WT. Overall levels of GSH were similar in WT and AtGGCT2;1 OE
lines. However, AtGGCT2;1 OE lines also had a significantly
higher accumulation of 5-OP than WT plants in both root and shoots, but
the level was up to 30-fold higher in shoots. Based on an
N15 Glu assay, Paulose et al . (2013) concluded
that the AtGGCT2;1 OE lines had better tolerance and less
accumulation of arsenic in shoot tissue due to an enhanced γ-glutamyl
cycle and more efficient glutamate recycling.
Building on the previous work from Paulose et al . (2013), we
translated the research from the model plant Arabidopsis thalianato the oilseed crop Camelina sativa . Both Camelina and
Arabidopsis belong to the Brassicaceae family, and each have three
homologs of GGCT s – GGCT1 (or GGCT2;3 ),GGCT2;1 , and GGCT2;2 (Figure S1). CsGGCT2;1 and AtGGCT2;1
have high amino acids sequence similarity (~92%)
(Figure S1a). This high protein sequence similarity with Arabidopsis may
be one reasons why we observed comparable results in Camelina. We used
20 µM AsIII concentration to analyze Camelina plants, as the
concentration used for Arabidopsis (35 µM), by Paulose et al .
(2013), was too toxic for the WT Camelina plants to grow. We found that
AsIII treatment upregulated CsGGCT2;1 gene in Camelina WT plants.
However, contrary to AtGGCT2;1 differential regulation in
Arabidopsis, we observed the upregulation of CsGGCT2;1transcripts in both roots and shoots (Figure 2). Camelina, being a
hexaploid, have three homeologs of GGCT2;1 and could have a
different expression profile compared to Arabidopsis. Relative to WT,
the overexpression of CsGGCT2;1 also provided stronger tolerance
to AsIII in Camelina (Figure 3). We observed a 40-60% less arsenic in
both roots and shoots in the Camelina GGCT2;1 OE lines compared
to the WT (Figure 5). We also found no difference in GSH levels between
the WT and CsGGCT2;1 OE lines (Figure 7a). Contrary to the high
levels of 5-OP in shoots reported for AtGGCT2;1 OE lines of
Arabidopsis (30-fold relative to WT) (Paulose et al ., 2013), we
detected only a 20% increase in 5-OP in combined root and shoot tissues
from 21 old-day seedlings of CsGGCT2;1 OE lines relative to CsWT
(Figure 6a). This combination of results indicates that the observed
tolerance to As was more robust in the Camelina lines overexpressingCsGGCT2;1 than that reported in Arabidopsis by Paulose et
al. (2013).
Arsenic is not a required element in plants, and once taken up by plants
leads to various adverse effects on plants’ growth and development (Bali
and Sidhu 2021; Farooq et al . 2016). Arsenic impacts the plants’
morphological, physiological, and metabolic attributes, e.g., lowering
shoot and root biomass, reducing plant height, impacting root growth,
decreasing chlorophyll content and photosynthetic rate, etc. (Ahmadet al . 2020a; Majumder et al . 2020; Singh et al .
2019). Arsenic also disrupts plants’ cellular oxidative state by making
reactive oxygen species (ROS) and causing lipid peroxidation (Ahmadet al . 2020a; Kofroňová, Mašková, and Lipavská 2018; Majumderet al . 2020; Singh et al . 2019). We observed a significant
reduction in the growth of WT Camelina plants exposed to AsIII, whereas
there was minimal impact on the growth of the CsGGCT2;1 OE lines;
empirically, the OE lines had growth almost similar to unchallenged
plants. Arsenic is known to induce oxidative stress in plants,
therefore, to understand the mechanism of CsGGCT2;1 role in protecting
plants from its toxicity, we explored the effects of AsIII treatment by
analyzing chlorophyll content, MDA, and ROS levels. Chlorophyll content
is a predictor of plants’ photosynthetic efficacy. Researchers have
found chlorophyll decreased due to arsenic exposure, thereby limiting
the growth and development of the plant (Ahmad et al . 2020b;
Anjum et al . 2017; Meselhy et al . 2021). We observed that
AsIII treatment decreased the chlorophyll content in the WT plants,
while there was no impact on chlorophyll in the CsGGCT2;1 OE
lines (Figure 4a).
Malondialdehyde (MDA) indicates lipid peroxidation and membrane damage
in living cells and many researchers have reported increased MDA levels
in plants exposed to AsIII (Anjum et al . 2017; Khare et
al . 2017; Pandey et al . 2016; Yadu et al . 2019). We
observed an increase in MDA levels in WT Camelina seedlings exposed to
AsIII treatment. However, MDA in CsGGCT2;1 OE lines was similar
under both MS control and AsIII treatment (Figure 4b). One reason for
this increase in the level of MDA is an increase in ROS due to oxidative
stress induced by the AsIII treatment. Arsenic exposure increased ROS
levels in Indian mustard (Pandey et al . 2016), Arabidopsis (Khareet al . 2017), and pigeon peas (Yadu et al . 2019). We also
visualized ROS levels using a fluorescent probe
(H2DCF-DA staining) in the plant’s root tips
(~1cm) and quantified them using ImageJ software. As
expected, we found increased ROS in the root tips of AsIII treated WT
Camelina relative to WT under control (MS) conditions. Also somewhat
expected, root tips of the CsGGCT2;1 OE lines had lower levels of
ROS as compared to WT, under both MS control and AsIII treatments,
however the differences were only significant for the AsIII treatment
(Figure 4c&d). Higher chlorophyll, lower lipid peroxidation, and lower
ROS levels under AsIII treatment could be why there was better growth ofCsGGCT2;1 OE lines relative to WT.
After arsenic uptake, plants limit its damaging effects on cellular
machinery by sequestering it in vacuoles. One crucial redox molecule
involved in AsIII detoxification via conjugation is GSH. Glutathione
levels are maintained in the plants through the γ-glutamyl cycle, a
continuous cycle of synthesis of GSH from glutamate, cysteine, and
glycine and its subsequent breakdown into component amino acids
(Hasanuzzaman et al . 2017; Noctor et al . 2012; Pauloseet al . 2013). Glutathione and its oligomer, PCs, complex with
AsIII in the cytosol and the complex is then transported to the vacuole
via protein transporters (ABCC transporters) (Bali and Sidhu 2021;
Noctor et al. 2012; Tang et al . 2019). Overexpression of
genes related to the γ-glutamyl and PCs synthesis pathway has resulted
in increased tolerance in Arabidopsis (Dhankher et al . 2002; Guoet al . 2008; Li et al . 2005; Paulose et al . 2013;
Song et al . 2010), Brassica (Gasic and Korban 2007; Reisingeret al . 2008) and Cottonwood (LeBlanc et al . 2011).
Similarly, we observed that overexpressing CsGGCT2;1 , a gene
involved in the γ-glutamyl cycle, enhanced AsIII tolerance in Camelina.
On analyzing levels of thiols i.e., GSH, γ-EC, and cysteine, we observed
no significant differences between WT and CsGGCT2;1 OE lines,
except for γ-EC under MS control conditions, which was significantly
lower than WT plants (Figure 7b). The decrease in γ-EC may have been due
to the direct breakdown of γ-EC by GGCTs. Kumar et al . (2015)
also reported the breakdown of γ-EC by Arabidopsis GGCTs inin-vivo functional assays in yeast and the authors found very low
activity of GGCT2;1 directed towards γ-EC breakdown, theGGCT2;1 homeolog in Camelina could have more effectively
increased activity towards γ-EC breakdown than that in Arabidopsis
(AtGGCT2;1 ) (Kumar et al . 2015). It could also be that the
heightened activity of CsGGCT2;1 leads to the breakdown of GSH,
triggering the upregulation of the glutathione synthase (GS) gene. As a
result, the synthesis of additional GSH from γ-EC and Gly is induced.
Further, no difference in γ-EC levels was observed under AsIII-treated
conditions because the overall γ-glutamyl pathway was induced to
counteract AsIII toxicity. The lack of difference in the levels of GSH
and Cys between the WT and OE lines could be due to the efficient
recycling of these by γ-glutamyl cycle, as reported by Paulose et
al. (2013). In response to AsIII treatment, we observed an overall 1.5
to 3-fold increase in the level of these thiols for both CsWT andCsGGCT2;1 OE lines, indicating a shift of resources towards the
γ-glutamyl cycle (Figure 7). The same trend was observed for Glu and Gly
as well, the remaining amino acid components of GSH, also indicative of
a resource shift towards the γ-glutamyl cycle for enhanced survival
under AsIII stress.
Further, we found a 19-33% increase in 5-OP levels in OE lines relative
to WT under MS control and AsIII treatments. GGCT2;1 is shown to
directly acts on GSH and γ-Glu-AA and converts both into 5-OP
(Ohkama-Ohtsu et al . 2008; Paulose et al . 2013). Though
Paulose et al. (2013) reported a substantial build-up of 5-OP
levels in the AtGGCT2;1 OE lines, there was not a considerable
accumulation in Camelina (Figure 6a). Based on our qRT-PCR analysis,CsOXP1 expression was upregulated in the OE lines (Figure 6b),
which indicates that the overexpression of CsGGCT2;1 has
upregulated the downstream gene OXP1 , which in turn increased the
recycling of 5-OP to maintain steady state levels of GSH in the
seedlings.
Better survival of OE lines (relative to WT) under AsIII treatment also
seems to be attributable to lower arsenic uptake and accumulation within
the transformed plants. CsGGCT2;1 OE lines had ~
40 to 60% lower arsenic accumulation relative to WT while maintaining a
similar level of GSH (Figures 5 & 7). This could be because of
efficient γ-glutamyl cycle via faster degradation of γ-EC and GSH in theCsGGCT2;1 OE lines, thus faster recycling of Cys, Glu, and Gly
for subsequent GSH synthesis. This efficient degradation and synthesis
of γ-EC and GSH might prevent the opportunity of binding of AsIII to
these thiols for sequestration and accumulation of As in vacuole. When
calculated as GSH per unit of arsenic, the CsGGCT2;1 OE lines had
more GSH available Therefore, the damaging effects of As at the cellular
level were more efficiently limited in the OE lines. Low accumulation of
arsenic could also be due to the role of CsGGCT2;1 in GSH
distribution in the root and root architecture. Joshi et al.(2019) noted differences in the growth and GSH distribution in the roots
of Arabidopsis WT and ggct2;1 knockdown lines (Joshi et
al . 2019). Less accumulation could also due to induction of some
aquaporin channels involved in both the uptake and efflux of As back
into the environment (Kumar et al . 2018; Lindsay and Maathuis
2016). Further investigation is needed to investigate the relationship
between GGCTs and metal transporters/channel genes.