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Expression of rice CYP450-like gene (Os08g01480) in Arabidopsis
modulates regulatory network leading to heavy metal and other
abiotic stress tolerance
Arti Rai, Ruchi Singh, Pramod Arvind Shirke, Rudra Deo Tripathi, Prabodh Kumar Trivedi,
Debasis Chakrabarty
Supporting Information S1 File
Figures A–M
Tables A-C
1
2
3
4
5
6
Figure A. An unrooted, maximum likelihood phylogenetic tree of CYPs. All CYP
sequences of Arabidopsis and rice CYP Os08g01480 was aligned using ClustalW and
phylogenetics tree was created using MEGA v5.2. Branch lengths are proportional to
evolutionary distance with each other. Os08g01480 is encircled with red border.
Tolerant
HARG
CN1646-2
NAYANMONI
CN1646-5
BRG-20
BRG-12
Sensitive
BRG-15
B
A
LARG
C
7
8
9
10
11
12
13
14
15
16
17
18
19
Figure B Expression profile of Os08g01480 (A) Expression in roots of two different japonica
rice cultivars [Azucena (As(V)-sensitive) and Bala (As(V)-tolerant)] exposed to 13.3 mM
concentration of As(V). The detailed description of the datasets utilized for the study is
provided in Supplementary Table S1. (B) Expression profile in roots of six contrasting rice
genotypes exposed to 50 µM concentration of As(V) for 24 h under standard physiological
conditions of 16 h light (115 μmol m-2 s-1) and 8 h dark photoperiod at 25 ± 2°C temperature.
HARG and LARG denotes for High arsenic accumulating rice genotypes and Low arsenic
accumulating rice genotypes. (C) Differential expression pattern of Os08g01480 during
abiotic Stresses. Expression profile analysis of Os08g01480 during control conditions (C),
cold (CS), drought (DS) and salinity (SS) stresses. The colour scale (representing log signal
values) is shown at the top.
20
A
LB
SacI
XbaI
Pnos
nptII
Tnos
B
CaMV35S
C
Os08g01480
Tnos
RB
20
WT
L1
L2
L3
Os08g01480
Actin
Relative transcript level
(Os08g01480
/Actin)
pBI: Os08g01480 expressing lines
16
12
8
4
0
D
21
22
23
24
25
26
27
28
L1
WT
L1
L2
L2
L3
L3
Figure C. Development of Os08g01480 expressing Arabidopsis lines (A) Schematic
representation of construct used for transformation of Arabidopsis. (B) Expression analysis of
Os08g01480 in transgenic lines using semi-quantitative PCR analysis and actin is taken as
endogenous control. (C) Relative expression analysis of Os08g01480 in transgenic lines
through qRT-PCR analysis. Data are shown as mean ± SD of three biological replicates in
each independent experiment. (D) Growth of WT and Os08g01480 expressing Arabidopsis
lines (L1, L2 and L3) in soilrite after two-week post germination.
A
C
WT
L1
L2
L1
L3
B
120
L3
D
100
d
c c
Root Length (cm)
9
b
a
a
6
3
30
31
32
33
34
35
36
37
38
80
bc
60
40
b
WT
d d d d
c
d
WT
bc
L1
b b
b
L2
L3
a
20
0
29
% Germination
12
b
L2
0
WT
L1
L2
L3
1 DAY
2 DAY
3 DAY
4 DAY
Figure D. Growth of WT and Os08g01480 expressing Arabidopsis lines (L1, L2 and L3)
in ½ MS plates in control conditions. (A) Pictorial representation of WT and transgenic
lines after 11 d of germination. (B) Root length comparison of WT and transgenic lines after
11 d of germination. All values are the mean of triplicates (±SD).Values marked with similar
letters are not significantly (Duncan’s test: p<0.05) different. (C) Pictorial representation of
comparative analysis of germination percentage of WT and transgenic lines after 5 d of
germination. (D) Percentage germination (recorded after radical emergence) comparison of
WT and transgenic lines. All values are the mean of triplicates (±SD).Values marked with
similar
letters
are
not
significantly
(Duncan’s
test:
p<0.05)
different.
50
A
c
Metal Content (µg mg-1 DW)
45
35
30
ab
c
40
b
a
a
a
a
a
a
a
a
WT
L1
25
L2
20
L3
15
ab
a a
10
a
5
0
As(III)
As(V)
Cd
Cr(VI)
B
2.5
2
1.5
1
0.5
0
39
40
41
42
43
44
45
46
47
48
WT
L1
L2
L3
4
3.5
3
2.5
2
1.5
1
0.5
0
3.5
Relative Expression
Level (AtABCC2/Actin)
3
Relative Expression
Level (AtABCC1/Actin)
Relative Expression
Level (AtPHT1/Actin)
3.5
3
2.5
2
1.5
1
0.5
0
WT
L1
L2
L3
WT
L1
L2
L3
Figure E. Heavy metal accumulation and relative expression analysis (A) Heavy metal
accumulation was measured in whole seedling as described in materials and method. Seeds of
Arabidopsis thaliana (WT) and three transgenic lines (L1, L2 and L3) were grown on ½ MS
media plates supplemented with 5 µM As(III), 100 µM As(V), 50 µM Cd and 50 µM Cr(VI).
All values are the mean of triplicates (±SD).Values marked with similar letters are not
significantly (Duncan’s test: p<0.05) different. (B) Expression analysis of AtPHT1, AtABCC1
and AtABCC2 in Arabidopsis. qRT-PCR analysis was carried out in RNA isolated from
seedlings of Arabidopsis thaliana (WT) and three transgenic lines (L1, L2 and L3) were
grown on ½ MS media plates.
Fv/Fm
1
qL
0.8
Y(II)
0.6
0.4
WT NT
0.2
qP
Y(NPQ)
0
L1 NT
L2 NT
L3 NT
qN
Y(NO)
NPQ
49
50
51
52
53
54
55
Figure F. Spider plot of chlorophyll fluorescence in WT and three independent
transgenic lines (L1, L2 and L3) in control conditions. NT represents for no treatment.
Spider plot represents relative changes of mean values of selected fluorescence parameters of
maximum photosynthetic efficiency (Fv/Fm), photosynthetic yield Y(II), nonphotochemical
quenching (NPQ), regulated energy dissipation Y (NPQ), nonregulated heat dissipation Y
(NO) and coefficient of photochemical quenching and non photochemical quenching (qN).
A
qL
qP
Fv/Fm
3.5
3
2.5
2
1.5
1
0.5
0
qN
Fv/Fm
3.5
3
2.5
2
1.5
1
0.5
0
B
Y(II)
qL
WT NT
WT As(III)
Y(NPQ)
qP
WT As(V)
L1 Cd
qN
Y(NO)
NPQ
Fv/Fm
2
D
Y(II)
qL
1
0.5
L2 As(III)
Y(NPQ)
0
L2 As(V)
1.5
Y(II)
1
L2 NT
qP
L3 NT
0.5
qP
L3 As(III)
Y(NPQ)
0
L2 Cr(VI)
qN
57
58
59
60
61
62
63
Y(NO)
NPQ
L3 As(V)
L3 Cr(VI)
L2 Cd
56
L1 As(V)
L1 Cr(VI)
Y(NO)
1.5
L1 As(III)
Y(NPQ)
WT Cd
Fv/Fm
2
qL
L1 NT
WT Cr(VI)
NPQ
C
Y(II)
L3 Cd
qN
Y(NO)
NPQ
Figure G. Spider plot of chlorophyll fluorescence in (A) WT plant (Col-0), (B) Transgenic
line (L1), (C) Transgenic line (L2), (D) Transgenic line (L3) during heavy metal stress 100
µM As(III), 200 µM As(V), 200 µM Cd and 400 µM Cr(VI). NT represents for No
Treatment. Spider plot represents relative changes of mean values of selected fluorescence
parameters of maximum photosynthetic efficiency (Fv/Fm), photosynthetic yield Y(II),
nonphotochemical quenching (NPQ), regulated heat dissipation Y (NPQ), and nonregulated
heat dissipation Y (NO). Level of significance considered as P≤0.05.
A
qL
Fv/Fm
4
Fv/Fm
4
3
B
qL
Y(II)
WT NT
2
qP
Y(NPQ)
L1 NT
qP
WT After 0 ˚C
L1 After 37 ˚C
1
WT 10 D After 37 ˚C
0
Y(II)
2
WT After 37 ˚C
1
3
L1 10 D After 37 ˚C
Y(NPQ)
0
L1 14 d After Drought
WT 14 d After Drought
L1 Salt
WT Salt
qN
qN
Y(NO)
Y(NO)
NPQ
NPQ
C
qL
Fv/Fm
3
qL
L2 NT
1
qP
Fv/Fm
4
D
Y(II)
2
Y(NPQ)
0
3
L2 After 37 ˚C
2
L2 10 D After 37 ˚C
1
L2 After 0 ˚C
qP
Y(II)
Y(NO)
NPQ
L3 NT
L3 After 37 ˚C
L3 10 D After 37 ˚C
Y(NPQ)
0
L3 After 0 ˚C
L2 15 d After 0 ˚C
L3 15 d After 0 ˚C
L2 14 d After Drought
L3 14 d After Drought
L2 Salt
qN
L1 After 0 ˚C
L1 15 d After 0 ˚C
WT 15 d After 0 ˚C
L3 Salt
qN
Y(NO)
NPQ
64
65
66
67
68
69
70
Figure H. Spider plot of chlorophyll fluorescence in (A) WT plant (Col-0), (B) Transgenic
line (L1), (C) Transgenic line (L2), (D) Transgenic line (L3) NT represents for No Treatment.
Spider plot represents relative changes of mean values of selected fluorescence parameters of
maximum photosynthetic efficiency (Fv/Fm), photosynthetic yield Y(II), nonphotochemical
quenching (NPQ), regulated energy dissipation Y (NPQ), and nonregulated heat dissipation
Y (NO). Level of significance considered as P≤0.05.
S -5
S -4
S -3
S -2
S -1
P -6
P -5
P2
P3
P -4
P1
P -3
M3
P -2
M1
M2
P -1
STIGMA
OVARY
An1
Mei1
B
SAM
YL
R
ML
A
5
Relative transcript level
(Os08g01480/Actin)
C
4
3
2
1
0
71
72
73
74
75
76
Figure I. Analysis of expression profile of Os08g01480. (A) Expression profile of
Os08g01480 during different developmental stages of rice. (B) Expression profile of
Os08g01480 during different stages of anther development. (C) qRT-PCR analysis of
Os08g01480 during different developmental stages of rice.
HindIII
A
LB
Pnos
nptII
PROOs08g01480
Tnos
λHE
BamHI
L1
L2
uidA
Tnos
RB
L3
B
500bp
77
78
79
80
81
Figure J. Construct preparation and selection of transgenic lines. (A) Schematic
representation of T-DNA of plant expression construct carrying Os08g01480 promoter in pBI
121vector, used for Arabidopsis transformation. (B) Genomic DNA PCR to confirm presence
of 500 bp Os08g01480 promoter in transgenic lines.
82
83
84
85
86
87
Figure K. Promoter activity in Arabidopsis line expressing ProOs08g01480:uidA.
Representative images after GUS staining of (A) seedling (10 d old grown in ½ MS media)
(B) Leaf, (C) Shoot, (D) Root, (E) Flowers, and (F) Silique of mature plant show promoter
activity grown in pot for 15 d under normal conditions. (G) Relative expression of uid-A gene
in different tissues of transgenic line grown in pot for 10 d under normal conditions.
A
5 µM As(III)
50 µM As(V)
30 µM Cd
50 µM Cr(VI)
25 µM As(III)
100 µM As(V)
50 µM Cd
100 µMCr(VI)
5 µM As(III)
50 µM As(V)
30 µM Cd
50 µM Cr(VI)
25 µM As(III)
100 µM As(V)
50 µM Cd
100 µM Cr(VI)
B
88
89
90
91
92
93
Figure L. Promoter activity in flower and leaves of Arabidopsis line (L1) expressing
ProOs08g01480:uidA under different heavy metal stresses. Histochemical GUS staining
of (A) flowers and (B) leaves of transgenic mature plants after 15 d of growth in pots under
normal conditions supplemented with nutrient media containing different heavy metals
As(III) 5 and 25 µM, As(V) 50 and 100 µM, Cd 30 and 50 µM, Cr(VI) 50 and 100 µM.
A
5 µM As(III)
50 µM As(V)
30 µM Cd
50 µM Cr(VI)
25 µM As(III)
100 µM As(V)
50 µM Cd
100 µM Cr(VI)
B
94
95
96
97
98
99
5 µM As(III)
50 µM As(V)
30 µM Cd
50 µM Cr(VI)
25 µM As(III)
100 µM As(V)
50 µM Cd
100 µM Cr(VI)
Figure M. Promoter activity in shoots and roots of Arabidopsis line (L1) expressing
ProOs08g01480:uidA under different heavy metal stresses. Histochemical GUS staining
of (A) shoot and (B) roots of transgenic mature plants after 15 d of growth in pots under
normal conditions supplemented with nutrient media containing different heavy metals: 5 and
25 As(III) µM, 50 and 100 µM As(V), 30 and 50 µM Cd, 50 and 100 µM Cr(VI).
Table A. Details of rice microarray experiments from GEO database used in this
study.
S.
Series
Sample
Description
No. accession
No.
1.
GSE4471
Rice Azucena 0ppm As(V)
Gene expression data from rice
(Control)
roots (variety Azucena and Bala)
Rice Azucena 1ppm As(V)
grown in hydroponics for one
Rice Bala 0ppm As(V)
week with 1ppm and 0ppm
(Control)
As(V).
Rice Bala 1ppm As(V)
2.
GSE6893
Root, 7-day-old Seedling
The spatial and temporal gene
Mature Leaf (ML)
expression
in
various
Young Leaf (YL)
tissues/organs and developmental
SAM (Shoot apical meristem) stages of rice using microarray
Young inflorescence (P1, upto technology is used to identify the
3 cm)
genes differentially expressed
Inflorescence (P2, 3 - 5 cm)
during
various
stages
of
Inflorescence (P3, 5 - 10 cm)
reproductive development.
Inflorescence (P4, 10 - 15 cm)
Inflorescence (P5, 15 - 22 cm)
Inflorescence (P6, 22 - 30 cm)
Seed (S1, 0 - 2 dap)
Seed (S2, 3 - 4 dap)
Seed (S3, 5 - 10 dap)
Seed (S4, 11 - 20 dap)
Seed (S5, 21 - 29 dap)
(dap represents day after
pollination)
3.
GSE7951
Stigma
Rice
stigma-specific
gene
Ovary
expression
profiles
through
comparing
genome-wide
expression patterns of hand
dissected unpollinated stigma at
anthesis with seven tissues
including
seedling
shoot,
seedling root, mature anther,
ovary at anthesis, seeds of five
days after pollination, 10-day-old
embryo, 10-day-old endosperm
as well as suspension cultured
cells studied by using 57K
Affymetrix rice whole genome
array.
hypodermal Gene expression
cells forming developing anther.
4.
GSE14304
Anther,
archesporial
stage (An1)
Anther, Pollen mother cells at
pre-meiotic s/g2 stage (Mei1)
Anther, Pollen mother cells at
meiotic leptotene stage (M1)
Anther, Pollen mother cells at
meiotic
zygotene-pachytene
stage (M2)
Anther, Pollen mother cells at
meiotic diplotene-tetrad stage
(M3)
Anther, Uni-nucleated
gametopyte stage (P1)
Anther, bi-cellular gametopyte
stage (P2)
Anther, tri-cellular mature
pollen stage (P3)
5.
GSE6901
Rice Seedlings
data
from
Seven-day-old light-grown rice
seedlings grown under controlled
conditions and those subjected to
various abiotic stress conditions
were used for RNA extraction
and hybridization on Affymetrix
microarrays. Three biological
replicates of each sample were
used for microarray analysis. For
salt treatment (SS), the rice
seedlings were transferred to a
beaker containing 200 mM NaCl
solution for 3 h. For desiccation
(DS), rice seedlings were dried
for 3 h between folds of tissue
paper at 28±1 degree C, in a
culture room. For cold treatment
(CS), the seedlings were kept at
4±1 degree C for 3 h. The
seedlings kept in water for 3 h, at
28±1 degree C, served as control
(Seedling).
Table B. List of primes
(GBF3) AT2G46270F
AAGTCGA GAGGGAACTC C AAC
(GBF3) AT2G46270R
AGAGAGTATAGCTCCAGATCC
(EDS1) AT3G48090F:
ATGT TTACCTTGAG CCTCGTTG
(EDS1) AT3G48090R:
TCCGAGGGACAATATCGAATC
(HY5) AT5G11260F:
AGCTCAGCAAGCAAGAGAGAG
(HY5) AT5G11260R:
AGCATCTGGTTCTCGTTCTG
(ER) AT2G26330F:
GCCA A CTGTCTCAGTCTCACTG
(ER) AT2G26330R:
AGCCAACTACCGCAAAGACCAG
(COI1) AT2G39940F
GAAACTGAAGACCATTGTACGC
(COI1) AT2G39940R
CTTGTTCATCTGCACCGCGTTC
(PAD4) AT3G52430F:
TCCATCCACGACCTCGTTCC
(PAD4) AT3G52430R:
TGAGTTGCTGTGGTGTTGAGG
(AOS) AT5G42650F:
AAGTCAAAGCCGGTGAAATG
(AOS) AT5G42650R:
ACAACACATGCCTCAAAAGC
(YUC4) AT5G11320F:
TGTAGATACAATGCTCTGCCTC
(YUC4) AT5G11320R:
CCATTTCAGTAGTGTCATGGC
(CYP707A1) AT4G19230F:
GGGAGGGAACAATGATCAACAC
(CYP707A1) AT4G19230R:
CTCGAGAATGTAGTAGCATCG
(BRI1) AT4G39400F:
AGTTACCGATGGATACGTTGTTG
(BRI1) AT4G39400R:
GAGATCTAACGTTAGCAACGAAGC
(CYP82C2) AT4G31970F:
CATGGGTTACGATTGTGCTG
(CYP82C2) AT4G31970R:
CACGGACATGCTTGAGCATCTG
(CYP72C1) AT1G17060F:
TGG ACTCACTGTCATGACTTAAC
(CYP72C1) AT1G17060R:
TTGAGCCAGGAATGTAGACG
AtPHT1F
CCTCAACTCTCCAGAGAAGTTC
AtPHT1R
TCTTAATCAGCTTGGCAGGAG
AtABCC1F
CCGCAGAAATCCTCTTGGTCTTGATG
AtABCC1R
GTGAATCATCACCGTTAGCTTCTCTGG
AtABCC2F
AGCGTGCCAAAGATGACTCACACCAC
AtABCC2R
TACTTATCACGAAGAACACAACAGGG
Os08G01480RTF
AGCCAACAATGGCATCCTAC
Os08G01480RTR
GCGGAGAAGCATTAGGTCAG
Os08G01480F1
TGACAGGTCTCACACACCAACCTC
Os08G01480R1
CCTGCGAGGTGTAGCCGAAGCATC
Os08G01480F2
AGACACTAGAGAGATTAGGCAG
Os08G01480R2
GAAGCATCTCCTTTATCACTGC
M13F
GTAAAACGACGGCCAGT
M13R
CAGGAAACAGCTATGAC
Os08g01480XbaI
AGACAGTCTAGAGATTAGGCAGCCATGGCTG
Os08g01480SacI
GAAGGAGCTCCTTTATCACTGCCTTTA
ProOs08g01480F1
GGAAAATGGAGGAGGTGACA
ProOs08g01480R1
GGTGCTGGTGCAAGAGTAGTAG
ProOs08g01480F2
GAGAAAGTGACATGACAAGAGGTG
ProOs08g01480R2
GGCACTAGCATCACAGCCATGGCTG
ProOs08g01480 BamHI
ATTGGATCCTAGCATCACAGCCATGGCTGCC
ProOs08G01480 HindIII
ATAGACGAAGCTTAAAGAGGTCCGT
OsActinF
GATGGATCCTCCAATCCAGACACTGTA
OsActinF
GTATTGTGTTGGACTCTGGTGATGGTGT
OsUbiqutinF
GACGGACGCACCCTGGCGAACTAC
OsUbiqutinF
TGCTGCCAATTACCATATACCACGAC
AtActinF
ATGACATGGAGAAGATCTGGCATCA
AtActinR
AGCCTGGATGGCAACATACATAGC
Table C. cis-regulatory elements present in promoter of Os08g01480
PLACE_ID
MOTIF
DESCRIPTION
DOFCORE
AAAG
Dof1 and Dof2 transcription factors are associated with
ZM
expression of multiple genes involved in carbon metabolism
in maize
GTGANTG
GTGA
10
WRKY71O
"GTGA motif" found in the promoter of the tobacco late
pollen gene g10 (pollen development)
TGAC
S
"A core of TGAC-containing W-box" of, e.g., Amy32b
promoter Binding site of rice WRKY71, a transcriptional
repressor of the gibberellin signaling (pathway; Parsley
WRKY proteins bind specifically to TGAC-containing W
box elements within the Pathogenesis-Related Class10 (PR10) genes (Defence related pathway)
POLLEN1L
AGAA
One of two co-dependent regulatory elements responsible
ELAT52
A
for
pollen
specific
activation
of
tomato
(Pollen
Development)
TATABOX
TTATT "TATA box"; TATA box found in the 5'upstream region of
5
T
pea glutamine synthetase gene
WBOXAT
TTGA
"W-box" found in promoter of Arabidopsis thaliana NPR1
NPR1
C
gene; Located between +70 and +79 in tandem; They were
recognized specifically by salicylic acid (SA)-induced
WRKY DNA bindingproteins (Defence,SAR)
CBFHV
RYCG
Binding site of barley CBF1, and also of barley CBF2; CBF
AC
DE
= C-repeat (CRT) binding factors; CBFs are also
known as DE
dehydration-responsive element (DRE)
binding proteins (DREBs) (Cold Response)
PREATPR
ACTC
"PRE (Pro- or hypoosmolarity-responsive element) found in
ODH
AT
the promoter region of
proline dehydrogenase (ProDH)
gene in Arabidopsis (stress response)
MYCCONS CANN
MYC recognition site found in the promoters of the
ENSUSAT
dehydration-responsive gene rd22 and many other genes in
TG
Arabidopsis; Binding site of ATMYC2 (stress response)
BOXLCOR
ACCW
Consensus of the putative "core" sequences of box-L-like
EDCPAL
WCC
sequences in carrot PAL1 promoter region; DCMYB1
bound to these sequences in vitro (Environmental Stress)
CCAATBO
CCAA
CONSTANS and the CCAAT Box Binding Complex Share
X1
T
a Functionally Important Domain and Interact to Regulate
Flowering of Arabidopsis (Flower development)
MYB2CON
YAAC
MYB recognition site found in the promoters of the
SENSUSA
KG
dehydration-responsive gene rd22 and many other genes in
T
Arabidopsis (ABA signaling in stress-response and seed
development).
100