Supporting information
Title: Hydrogen sulfide alleviates zinc toxicity by reducing zinc uptake and regulating genes
expression of antioxidative enzymes and metallothioneins in roots of the cadmium/zinc
hyperaccumulator Solanum nigrum L.
Authors: XIANG LIU
1, §
, JUAN CHEN
1, §
, GUANG-HUI WANG
2, §
, WEN-HUA WANG 3,
ZHI-JUN SHEN 1, MEI-RONG LUO 1, GUI-FENG GAO 1, MARTIN SIMON 1, KABIR GHOTO
1
and HAI-LEI ZHENG 1,*
1
Key Laboratory for Subtropical Wetland Ecosystem Research of MOE, College of the Environment and
Ecology, Xiamen University, Xiamen, Fujian 361102, P.R. China
2
College of life sciences, Xiamen University, Xiamen, Fujian 361102, P.R. China
3
Fujian Key Laboratory of Subtropical Plant Physiology and Biochemistry, Fujian Institute of Subtropical
Botany, Xiamen, Fujian 361005, P.R. China
§
These authors contributed equally to this work
1
Supplementary Methods: Amplification of ZRT and MTP partial cDNAs. Since there are no
available ZRT and MTP cDNA sequences in the NCBI database, we cloned partial cDNA sequences.
Using ZRT sequences from Arabidopsis thaliana (NM_112111.3), Solanum lycopersicum
(NM_001247420.1),
Solanum
tuberosum
(XM_006345363.1)
and
Solanum
pennellii
(XM_004244292.1), degenerate primers were designed to different conserved regions by Primer
Premier 5.0 program. For the amplification of ZRT cDNA partial fragments, the upstream primer
(5’-GGDGAYAGCATYTTATTGATYGTTG-3’)
and
the
downstream
primer
(5’-ATCATCACMACVGCAATNATHC-3’) were used. The pair of primers were also listed in
Supporting information, Table S2. Reverse transcription was carried out with total RNA and oligo-dT18
primers using M-MLV reverse transcriptase (TaKaRa, Dalian, China), according to the manufacturer’s
instructions. PCR was performed with pre-denaturalization at 94 °C for 5 min, and then thermocycling
was performed at 30 cycles of 94 °C for 30 s, annealing for 30 s, 72 °C for 60 s, and an additional
polymerization step at 72 °C for 10 min in a thermocycler (Biorad, USA). PCR product was separated
by electrophoresis on a 1 % agarose gel stained with ethidium bromide, and purified by using the DNA
gel extraction kit (Sangon Biotech, China). The products were cloned into the pMD-19T vector (Takara,
Dalian, China) and then transformed into E. coli DH5α. Recombinant plasmids were sequenced by the
Invitrogen Sequencing Facility.
Using MTP sequences from Solanum tuberosum (XM_006359474.1), Solanum lycopersicum
(XM_004242656.1), Solanum pennellii (HG975446.1) and Nicotiana tabacum (AB201240.1),
degenerate primers were designed to different conserved regions by Primer Premier 5.0 program. For
the
amplification
of
MTP
cDNA
partial
(5’-TAAATGTHCADGGGGCTTATCTTC-3’)
and
2
fragments,
the
the
upstream
downstream
primer
primer
(5’-CTATYTGAATVGTNACRTGRCT-3’) were used. The pair of primers were also listed in
supporting information, Table S2. The remaining steps are similar as ZRT cloning as mentioned above.
3
Supplementary Figure S1.
Supplementary Figure S1. Effect of NaHS pretreatment on root fresh weight (A) and shoot fresh
weight (B) of three week-old Solanum nigrum seedlings. Seedlings were pretreated with different
concentration of NaHS (0, 50, 100, 200, 400 and 800 μM) for 12 h and then exposed to 0 μM ZnCl2 for
another 5 days. Error bars are SE (n = 30). The columns labeled with different letters are significantly
different at P < 0.05.
4
Supplementary Figure S2.
Supplementary Figure S2. Root fresh weight (A), shoot fresh weight (B), H 2S content in root (C) or
leaf (D) and total L-cysteine desulfhydrase (L-DES) activities in root (E) or leaf (F) of three week-old
Solanum nigrum seedlings. Seedlings were treated with different concentration of NaHS (0, 50, 100,
200 and 400 μM) and 400 μM ZnCl2 together for 5 days. Error bars are SE (n = 30 in Fig. A and B, n =
3 in Fig. C, D, E and F). The columns labeled with different letters are significantly difference at P <
0.05. CK, untreated control; Zn, 400 μM ZnCl2 treatment; 50S+Zn, 50 μM NaHS together with 400 μM
ZnCl2 treatment; 100S+Zn, 100 μM NaHS together with 400 μM ZnCl2 treatment; 200S+Zn, 200 μM
NaHS together with 400 μM ZnCl2 treatment; 400S+Zn, 400 μM NaHS together with 400 μM ZnCl2
treatment.
5
Supplementary Figure S3.
(A)
(B)
Supplementary Figure S3. Sequence alignments (A) and homology tree (B) of Solanum nigrum type 2
metallothionein (MT) mRNA, complete cds. MT-encoding cDNAs available in the NCBI database.
6
MT2a (accession: EU760481.1), MT2b (accession: EU760482.1), MT2c (accession: EU760483.1) and
MT2d (accession: EU760484.1). Sequences were aligned by the DNAMAN 7 program.
7
Supplementary Figure S4.
(A)
(B)
Supplementary Figure S4. Sequence alignments (A), homology tree (B) between Solanum nigrum
type 2 metallothionein (MT) and barley MT type 1 or 2 protein. MT protein sequences are available in
the NCBI database. MT2a (GenBank: ACF10395.1), MT2b (GenBank: ACF10396.1), MT2c (GenBank:
ACF10397.1), MT2d (GenBank: ACF10398.1), HvMT1a (GenBank: CAD54078.1) and HvMT2a
8
(GenBank: AEX32990.1). Sequences were aligned by the DNAMAN 7 program.
9
Supplementary Figure S5.
(A)
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
TCATAACAATGGCAATGACAACAACTCCAAACAACACAGC
TCATCACAACGGCAATTATCCCAACACCTAACAAAACAGC
tcat acaa ggcaat a
caac cc aacaa acagc
40
40
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
CAAAAACTTATAACGAGGCTCATCAACATGAACTTTCTTG
CAAAAACTTGAAATGGGCTTTGTCAATCAAGACCATTTTC
caaaaactt aa g g t tcaa
ac t tt
80
80
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
TTTGGTCGATACCCTTTTGCCAACAAATGGTTCACAGATA
TGAGGCTTGTATCCCTTAGAAAGCAAATGATTAATAGAAA
t gg
ta cc tt g a caaatg tt a aga a
120
120
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
CATACACAAATACCCCACACGCCAAGCTCATCGACAATGC
CATATATAAAAACTCCACAAGCCAATCCCATTGATATTGC
cata a aaa ac ccaca gccaa c cat ga a tgc
160
160
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
GAATATCCAATCCGCGATGGACCCTTGAGTCGTTGCATCG
AAAGATCCAGTCTGCAACAACCCCTTGAGCTGTGGCATCG
aa atcca tc gc a
cccttgag gt gcatcg
200
200
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
ATGACTATCCCGATGGCAACACCAATTGGGCTCGAGATGG
ATTATGATACCGATGGCAACACCAATTGGACTGGAGGTCG
at a at ccgatggcaacaccaattgg ct gag t g
240
240
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
CGAATGCGAAAGAATAAGTGATTGAGGAGAATAAAGGACG
CAAAGGCAAAAGCATAGGCCACGCAAGATAGTAAAGGGCG
c aa gc aaag ata g a
a ga a taaagg cg
280
280
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
GTCAGGGATCATACGGAGAAGAGCTATCCCCATTGCAATC
GTTTGGGATCATCCTAAGTAGAGCTATTCCCATTGCAATG
gt gggatcat c ag agagctat cccattgcaat
320
320
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
GCTGCAAATATCTTATGGAGTGTTATTGTCCAGAGAGCTC
GCAGCAAATATCTTGTGTAAACATACAGTCCAAAGAGCTC
gc gcaaatatctt tg a
ta gtcca agagctc
360
360
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
TCCAAGCATCTGATTTAGTCTCTGAAATACCAATAGCTAT
TCCAAGCATCGGCTTGTGAATCTGCAACACCAATGGCTAT
tccaagcatc g tt g tctg aa accaat gctat
400
400
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
GCCTTCGAAGACAGAGTGAAAACAGAGAGCTACGATCAAT
TCCCTCAAAGACAGAATGGAAACATAGCGCAACAATCAAT
cc tc aagacaga tg aaaca ag gc ac atcaat
440
440
AtZIP2_NM_104468.2.seq
SnZRT.seq
Consensus
AGAATGCTATCTC
AAAATGCTATCAC
a aatgctatc c
453
453
(B)
10
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
TAAATGTACATGGGGCTTATCTTCATGTACTAGGAGATTC
TAAATGTTCAGGGGGCTTATCTTCATGTACTAGGAGATTC
taaatgt ca ggggcttatcttcatgtactaggagattc
40
40
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
AATTCAGAGCATAGGCGTCATGATTGGGGGAGCTATTATA
TATTCAGAGCATAGGGGTCATGATTGGAGGAGCTATCATA
attcagagcatagg gtcatgattgg ggagctat ata
80
80
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
TGGTATAAACCAGAGTGGAAAATCATTGATCTAATTTGCA
TGGTATAAACCAGAATGGAAAATCATTGATCTAATTTGCA
tggtataaaccaga tggaaaatcattgatctaatttgca
120
120
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
CACTCATTTTCTCCGTTGTTGTTCTTGCCACAACCATCAG
CTCTCATTTTCTCTGTCATTGTTCTTGCCACAACCATTAG
c ctcattttctc gt ttgttcttgccacaaccat ag
160
160
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
GATGATTCGGAGTATTCTTGAAGTATTAATGGAGAGCACA
GATGATTCGGAGTATTCTTGAAGTATTAATGGAGAGCACA
gatgattcggagtattcttgaagtattaatggagagcaca
200
200
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
CCGAGAGAAATTGATGCAACATGGCTTGAGAAGGGGCTAT
CCGAGAGAAATTGATGCAACAAGGCTTGAGAAGGGGCTTT
ccgagagaaattgatgcaaca ggcttgagaaggggct t
240
240
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
GTGAGATGGAGGAGGTCGTTGCAATCCACGAATTGCACAT
GCGAGATGGAGGAGGTTGTTGCAATCCATGAATTGCACAT
g gagatggaggaggt gttgcaatcca gaattgcacat
280
280
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
ATGGGCTATTACAGTCGGGAAAGTACTCTTGGCTTGCCAT
TTGGGCGATTACAGTCGGGAAAGTACTCTTGGCTTGCCAT
tgggc attacagtcgggaaagtactcttggcttgccat
320
320
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
GTCAAGATTAAGCCTGATGCTGATGCTGACATGGTGCTGG
GTCAAGATTAAACCTGATGCCGATGCTGACATGGTGCTGG
gtcaagattaa cctgatgc gatgctgacatggtgctgg
360
360
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
ATAAGGTGGTCGATTATATTAGAAGGGAATATAACATTAG
ATAAGGTTGTCGATTATATTAGGAGGGAATATAACATTAG
ataaggt gtcgattatattag agggaatataacattag
400
400
Sn_MTP.seq
St_MTP1_XM_006359474.1.seq
Consensus
CCACGTTACCATTCAAATAG
CCACGTAACCATTCAAATAG
ccacgt accattcaaatag
420
420
Supplementary Figure S5. Multiple alignment analysis of partial ZRT (A) and MTP (B) S. nigrum
cDNA sequences by DNAMAN 7 program. Sequence alignment revealed that cloned ZRT shared
approximately 73% identity with the ZIP2 sequences reported in Arabidopsis thaliana (Accession:
NM_104468.2), while MTP shared 94% identity with the MTP1 sequences reported in Solanum
tuberosum (Accession: XM_006359474.1). Conserved regions and sites are marked.
11
Supplementary Table S1. Zinc accumulation in Solanum nigrum. Seedlings were pretreated with 200
μM NaHS for 12 h and then followed by 400 μM ZnCl2 for 5 days. Different letters indicated
significantly different at P < 0.05. CK, untreated control; S, 200 μM NaHS pre-treatment; Zn, 400 μM
ZnCl2 treatment; S+Zn, 200 μM NaHS pretreatment followed by 400 μM ZnCl2 treatment.
Zn concentration (mg/g DW)
Treatment
Roots
Shoots
CK
1.16±0.08c
0.63±0.02c
200 μM NaHS (S)
1.18±0.04c
0.66±0.01c
400 μM ZnCl2 (Zn)
8.17±0.56a
5.44±0.05a
6.24±0.18b
5.10±0.07b
200 μM NaHS +
400 μM ZnCl2 (S + Zn)
12
Supplementary Table S2. Sequence of forward (F) and reverse (R) primers used for quantitative real-time PCR analysis
Gene
Accession
Cu/Zn-SOD
FJ402835
Sequences (5’ to 3’)
TACCGACAAGCAGATTCCTCTCACT
Product
Tm (℃)
Function
158 bp
53
qRT-PCR
193 bp
55
qRT-PCR
149 bp
54
qRT-PCR
130 bp
53
qRT-PCR
147 bp
53
qRT-PCR
191 bp
50
qRT-PCR
433 bp
46
qRT-PCR
362 bp
49
qRT-PCR
146 bp
49
qRT-PCR
168 bp
56
qRT-PCR
101 bp
51
qRT-PCR
159 bp
55
qRT-PCR
454 bp
52
Gene clone
161 bp
50
qRT-PCR
420 bp
52
Gene clone
121 bp
50
qRT-PCR
length
GGAGCCCAATAATACCACAAGCAATC
Fe-SOD
FJ402836
TGGGTTACACGCCGCTGCTCAC
TAGATTTGCCTCTTCATCCTTC
CAT2
FJ402838
GCGATGAAGAGGTGGATTATTT
GTATCTCTCCCCTGCCTGTTTGAAG
pAPX
FJ979919
CGTATGACGCTAAATCAAAGACTGG
GGATGTTTAGACTTCACTGCTTCGC
18SRNA
KC535782
AGTTGGTGGAGCGATTTGTCTGGTT
GACCTGTTATTGCCTCAAACTTCC
MT2ab
MT2b
EU760481.1/
GCTGTGGAGGATGCAAGAT
EU760482.1
CTTAGAGCAAGTGCAAGGGTTAC
EU760482.1
GGGATCCGATTATGTCTTG
ATTACCAGAAGCAGAGATGC
MT2c
EU760483.1
GATGTGGGATGTACCCTGAC
GTTACAAGCCCATGTCAACTTC
MT2cd
Nramp1
EU760483.1/
GATGTGGGATGTACCCTGAC
EU760484.1
GCAGTTTGATCCACATTTGC
GU575298.1
TGCCATTCCCCTTCTATGAGCCCAG
AGCGTATGTCCCAGTTATTGTCG
IRT
GE650052.1
TTCTTCGCAGTAACAACTCCAT
AATCCAACAGTTATTAACGCCCGTG
HMA4
JQ408670.1
ATGCTCACCTTTTCGTTG
CTGCTGCCTCCATGTAAT
ZIP
GGDGAYAGCATYTTATTGATYGTTG
ATCATCACMACVGCAATNATHC
ACGCAAGATAGTAAAGGGC
TTCCATTCTGTCTTTGAGG
MTP
TAAATGTHCADGGGGCTTATCTTC
CTATYTGAATVGTNACRTGRCT
TGTTCTTGCCACAACCATCA
AACGACCTCCTCCATCTCAC
13