Nitric oxide mediated transcriptional modulation enhances plant adaptive responses to arsenic stress Pradyumna Kumar Singh1,2, Yuvraj Indoliya1,2, Abhisekh Singh Chauhan1,2, Surendra Pratap Singh1, Amit Pal Singh1, Sanjay Dwivedi1, Rudra Deo Tripathi1,2*, Debasis Chakrabarty1,2* 1
Council of Scientific and Industrial Research - National Botanical Research Institute (CSIR-
NBRI), Rana Pratap Marg, Lucknow-226001, India 2
Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi
Marg, New Delhi-110 001, India * Corresponding author;
[email protected],
[email protected]
Supplementary Fig. 1: CLSM observation for NO on 4th and 12th day. DIC and overlay images of roots of NO observation in different treatment at both time intervals showed changes in NO level.
Supplementary Fig. 2: CLSM observation for superoxide on 4th and 12th day. DIC and overlay images of roots of superoxide in different treatment at both time intervals showed reduction in superoxide level in the AsIII+SNP treatment in comparison to AsIII treatment. 9
A
B 4 day
8
4.5
12 day
4 day
12 day
7
Relative fluorescence level
Relative fluorescence level
4 6
5 4 3 2
1
3.5 3 2.5 2 1.5 1 0.5 0
0
Control
SNP
AsIII
AsIII+SNP
Control
SNP
AsIII
AsIII+SNP
Supplementary Fig. 3: Relative fluorescence of NO (A) and superoxide (B) in different treatments at both time intervals. There is increase in NO level in the AsIII+SNP treatment in comparison to AsIII treatment while decrease in superoxide content in the AsIII+SNP treated root in comparison to AsIII treated root.
B
C
D
AsIII+SNP
AsIII
SNP
Control
A
Supplementary Fig. 4: Images of cell viability assay on 4th day. Image of viable cell of root (A), DIC image of root (B), Image of dead cell of root (C) and overlay of all images (D), showed less toxicity in the AsIII+SNP treated root in comparison to AsIII treated root.
B
C
D
AsIII+SNP
AsIII
SNP
Control
A
Supplementary Fig. 5: Images of cell viability assay on 12th day. Image of viable cell of root (A), DIC image of root (B), Image of dead cell of root (C) and overlay of all images (D), showed higher in the AsIII treatment in comparison to AsIII+SNP treatment.
A
B Quality Check
Base Trimming
Contamination Removal
Read Alignment
Gene and Transcript Differential Expression
Sample (Root)
Total Reads (paired-end)
GC (%)
% of data>= Q30
Raw read length (bp)
Aligned Read Count
Aligned (%)
4 day Control
63,013,448
51.3
84.6
100 x 2
46,146,574
83.78%
4 day SNP
33,517,798
52.81
91.4
100 x 2
27,825,128
88.41%
4 day AsIII
125,737,228
50.48
86.3
100 x 2
99,229,580
83.84%
4 day AsIII+SNP
143,136,926
51.78
87.9
100 x 2
117,978,838
86.03%
12 day Control
85,587,594
53.01
83.6
100 x 2
56,991,320
84.71%
12 day SNP
38,545,688
53
90.2
100 x 2
25,971,729
68.49%
12 day AsIII
106,858,902
49.21
87.2
100 x 2
88,491,141
84.32%
12 day AsIII+SNP
69,586,772
49.27
89.2
100 x 2
49,301,534
76.08%
Supplementary Fig. 6: Bioinformatics pipeline for processed samples (A) and Summary of the fastq file (B).
Relative abundance (Total FPKM)
120000
A
120000
Control SNP
B
100000
100000
AsIII 80000
80000
AsIII +SNP
60000
60000
40000
40000
20000
20000
0
0 1
2
3
4
5
6
7
8
9
10
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12
1
2
3
4
Chromosome number 5000
C
5000
4500
Total number of gene expressed
5
6
7
8
9
10
11
12
Chromosome number
D
4500
4000
4000
3500
3500
3000
3000
2500
2500
2000
2000
1500
1500
1000
1000
500
500
0
0 1
2
3
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6
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8
Chromosome number
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
Chromosome number
Supplementary Fig. 7: Relative abundance and total number of genes among different samples. A) Relative abundance (total FPKM) on 4th day, B) Relative abundance on 12th day, C) Total number of gene expressed per chromosome on 4th day, D) Total number of gene expressed per chromosome on 12th day.
A
Control Vs SNP
Control Vs AsIII
Control Vs SNP
B
Control Vs AsIII+SNP
Control Vs AsIII+SNP C Control Vs SNP
Control Vs AsIII
Control Vs AsIII+SNP
Control Vs AsIII
D
Control Vs SNP
Control Vs AsIII
Control Vs AsIII+SNP
Supplementary Fig. 8: Analysis of up-regulated and down regulated differentially expressed (p≤ 0.05, fold change ≥±log 2) genes by comparison between control and treated samples. A) Upregulated genes and B) down-regulated genes on 4th day. C) Up-regulated genes and D) downregulated genes on 12th day.
Supplementary Fig. 9: Validation of genes by real time PCR using actin as internal control on 4th day.
Supplementary Fig. 10: Validation of genes by real time PCR using actin as internal control on 12th day.
Supplementary Fig. 11: Validation of genes by real time PCR using Os01g0610100 as internal control on 4th day. The additional internal control Os01g0610100 was identified by using Normfinder (moma.dk/normfinder-software). The results showed similar expression patterns of genes with RNASeq data.
Supplementary Fig. 12: Validation of genes by real time PCR using Os01g0610100 as internal control on 12th day. The additional internal control Os01g0610100 was identified by using Normfinder (moma.dk/normfinder-software). The expression patterns of genes were matched with trancriptome data.
AsIII+SNP
AsIII
AsIII+SNP SNP
AsIII
SNP
AsIII+SNP
AsIII
AsIII+SNP SNP
AsIII
AsIII+SNP
SNP
AsIII
SNP
Secondary metabolisms
Hormone metabolisms
TFs
B
Secondary Transporters metabolisms
Hormone metabolisms
TFs
Transporters
A
CLUSTER I
CLUSTER II
CLUSTER III
CLUSTER IV
CLUSTER V
Supplementary Fig. 13: K-means clustering of different sets of genes on 4th day (A) and 12th day (B) showed expression behaviour of genes in categorized clusters.
9
A c
9
8
8
B a
a
7 JA (µg/g fresh weight)
7 6
6
5
b
5
a 4
4
3
3
b 2
d
1
c
2 1
0
0 Cont
SNP
AsIII
AsIII+SNP
Cont
SNP
AsIII
AsIII+SNP
Supplementary Fig. 14: Quantification of JA content by HPTLC. The JA content on 4th day (A) and 12th day (B) showed reduction in JA level in the AsIII+SNP treatment in comparison to AsIII treatment. One Way ANOVA (SPSS 16.0 software) was used to analyze the significance level in samples.
AsIII Vs AsIII+SNP
C Vs AsIII+SNP
C Vs AsIII
C
C Vs SNP
AsIII Vs AsIII+SNP
C Vs AsIII+SNP
C Vs AsIII
B
C Vs SNP
AsIII Vs AsIII+SNP
C Vs AsIII+SNP
C Vs AsIII
C Vs SNP
AsIII Vs AsIII+SNP
C Vs AsIII+SNP
C Vs AsIII
C Vs SNP
A
D
Figure 8.
Supplementary Fig. 15: Differential expression analysis of genes of transcription factors (TFs), secondary and lipid metabolism. A) Represented genes expression of TFs on 4th day and B) represented genes expression of TFs on 12th day. C) and D) showed secondary and lipid metabolism genes on 4th and 12th day, respectively.
14
Root length (cm)
12
cde
def
f
ef bc
bcd
ab
10
a
8 6 4 2
0 Control
SNP 10 µM
SNP 20 µM
SNP 30 µM
SNP 40 µM
SNP 50 µM
SNP 75 µM SNP 100 µM
Supplementary Fig. 16: Selection of SNP concentration by using different concentration of SNP on 12th day showed maximum increase in 30 µM SNP treatment.
