Arsenic in As(V) response using natural variation have not

Arsenic is taken up by the plants and accumulates in
different tissues leading to toxicity and contamination of food chain 47, 48.
Studies suggest that As treated rice genotypes differ from each other at phenotypic
and genetic level with differential expression of number of genes 19, 49. However,
detailed studies to elucidate processes and molecular networks involved in As(V)
response using natural variation have not been carried out. In this study, the
effect of As uptake and accumulation on Arabidopsis
natural variation was investigated in contrasting accessions. Analysis showed
variation in the As translocation potential of Col-0 and Slavi-1, inspite of
the same level of As accumulation in the root tissue. (Fig. 2C). This suggests
that natural variation in As transport and accumulation mechanism might be
regulating the differential response in these accessions.

Genome-wide
transcriptional profiling identified set of genes, which might be involved in the
contrasting response of accessions towards As(V) stress. In Col-0, several
genes related to stress response, osmoprotectants and antioxidant system were
found to be up regulated (Supplementary Fig. S4). However, in Slavi-1,
stress-responsive genes such as HSPs, GSTs, and CYPs were modulated. Studies
have reported modulated expression of HSPs, CYPs and GSTs in response to heavy
metal stress in different plant species 18, 50, 51, 52. In this study, GSTs
from different classes were observed to be up regulated (Supplementary Fig. S3).
A number of differentially
expressed genes encoding transporter proteins were identified in Col-0 (Supplementary
Fig. S4) and Slavi-1 (Supplementary Table S8). Genes associated with transport
of sulfate 53, metals 54, 55, sugar 56 and lipid transfer proteins are
known to play key role in heavy metal stress in plants. The modulation in expression
of sugar transmembrane transporters, AtSWEET2
and AtSWEET16, in As tolerant
accession Col-0 suggest the restricted release of sugars especially glucose in
response to As stress, which may restricts sugar efflux from the cells in
response to As stress thereby conferring As tolerance. Further, the expression
of phosphate transporters such as PHT4;1 and PHT2;1 are down-regulated in
Col-0. Studies suggest the important role of PHT4;1 in chloroplast  compartmentation of phosphate and ATP
synthesis 57. Since it plays important role in shoot, its down-regulation in
Col-0 may limit root to shoot transportation of As(V), thereby minimizing the
sensitivity in As- tolerant accession as compared to Slavi-1.

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Expression of lipid
transfer proteins is known to be induced during stress owing to rapid
peroxidation due to the production of reactive oxygen species 47. These were
down-regulated in Col-0 whereas up-regulated in Slavi-1, suggesting stress
perception is higher in Slavi-1, leading to increased production of ROS. Contrary,
in Slavi-1, enhanced expression of genes encoding major facilitator superfamily
was observed under As(V) stress. Studies suggest their role in ATP dependent
efflux of xenobiotic compounds in the bacterial system 58 and provide resistance
towards stress.

Further, sulfate
transporters were highly up-regulated in Slavi-1 as compared to Col-0. It has
been reported that sulfate enters via sulfate transporter and contributes in
synthesis of metal chelating molecules such as GSTs and PCs for the detoxification
of toxic compounds 1, 37, 52. This enhancement is co-related with the abundance
of GST transcripts in Slavi-1, which suggests that increased GST expression
profiles might account for As-sensitive accession Slavi-1 being highly affected
by As(V) stress. Specific
induction of osmoprotectant-related gene, Galactinol synthase 1 (AtGolS1) in As(V)-tolerant accession
Col-0, is in accord with its role in protecting plant cells against abiotic
stress induced oxidative damage 59,60,61.

Expression of many
stress-related genes is regulated by transcription factors (TFs), via
interaction with cis-elements present
in the promoter regions. In this study, a set of regulatory genes with
differential expression were identified in Col-0 (Supplementary Table S10) and
Slavi-1 (Supplementary Fig. S3). Previous studies have identified role of some
key TFs including MYB, HSF, Zinc finger/CCCH, bHLH, Aux/IAA, WRKYs and AP2/ERF
involved in As stress responses 33, 50, 62. In this study, expression of
members of MYB transcription factor family was found to be modulated
exclusively in Slavi-1. MYB genes may play important role in As(V) stress
tolerance, as some of these are known to enhance anti-oxidant potential by regulating
phenylpropanoid/ flavonoids biosynthetic pathway 63,64. In addition, a number
of members of WRKY 65 and Zinc finger/C2H2 or CCCH, AP2/ERF and NAC 66, 67 were
differentially expressed in Slavi-1. Several transcriptomic studies on rice and
Arabidopsis identified the important
roles of members of these transcription factor gene families in regulating
plant responses to various stresses 51, 66.

The ERF subfamily has
been known to participate in the ethylene signaling pathway in plants in
response to abiotic stresses 68 including Cd and As stress in Arabidopsis and rice 33, 69. Previous
studies suggest DREB subfamily of AP2/ERF family under heavy metal stress has
inverse relation with osmotic potential in cell 70.
Intriguingly, up-regulated expression of members of this gene family in Slavi-1
suggests a possibility of enhanced heavy-metal inflow in the cells, which might
be a reason for the enhanced accumulation in shoot and increased sensitivity in
this accession.

Differential expression
of hormone-related genes, regulatory as well as functional components of
proteasome-related mechanisms might lead to contrasting As(V) responses of
Col-0 (Supplementary Fig. S4) and Slavi-1 (Supplementary Fig. S3). It is reported
that several phytohormones including ethylene play an important role during As
induced oxidative stress 50, 71, 72. Genes encoding ethylene biosynthesis and
signaling components were significantly modulated in As-tolerant accession
(Col-0) (Supplementary Fig. S4).  Studies
suggest brassinosteroids (BRs) inhibits ROS induced membrane peroxidation and
enhances the production of antioxidants 73, 74. In Slavi-1, genes encoding
BRs are down-regulated (Supplementary Fig. 3), which might be responsible for
increased sensitivity towards As stress. Additionally, enhanced expression of
the components and intermediates of the ubiquitin/proteasome pathway in Slavi-1
confirms extent of sensitivity in this accession. Protein modification and
degradation associated genes belonging to RING/U-box protein superfamily and
RING-H2 finger protein family (Supplementary Table S4) were also up-regulated
in Slavi-1. Taken together, our results suggest that genetic variations in accessions
leads to the induction of specific pathways along with the common networks in
response to As(V) stress. Therefore, the studies carried out using natural
variation 75, 76 for the genetic regulation in response to As stress and
nutrient deficiency 77 opens up a new avenue for better understanding of
mechanisms of As uptake, tolerance and detoxification in plants.