cDNA library for mining functional genes in Sedum alfredii Hance

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cDNA library for mining functional genes in Sedum alfredii Hance related to cadmium tolerance and characterization of the roles of a novel SaCTP2 gene in enhancing cadmium hyperaccumulation Mingying Liu, Xuelian He, Tongyu Feng, renying zhuo, Wenmin Qiu, Xiaojiao Han, Guirong Qiao, and Dayi Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b03237 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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Environmental Science & Technology

1

cDNA library for mining functional genes in Sedum alfredii Hance

2

related to cadmium tolerance and characterization of the roles of a

3

novel SaCTP2 gene in enhancing cadmium hyperaccumulation

4

Mingying Liu1,2,3,*, Xuelian He1,2, Tongyu Feng1,2, Renying Zhuo1,2,*, Wenmin Qiu1,2,

5

Xiaojiao Han1,2, Guirong Qiao1,2, Dayi Zhang4,*

6

1. State Key Laboratory of Tree Genetics and Breeding, Xiangshan Road, Beijing

7

100091, P.R. China

8

2. Key Laboratory of Tree Breeding of Zhejiang Province, Research Institute of

9

Subtropical of Forestry, Chinese Academy of Forestry, Hangzhou 311400, P.R. China

10

3. School of Basic Medical Sciences, Zhejiang Chinese Medical University,

11

Hangzhou 310053, P.R. China

12

4. School of Environment, Tsinghua University, Beijing 100084, P.R. China

13

*Corresponding authors:

14

Dr Mingying Liu

15

School of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou

16

310053, P.R. China; Tel, +86-(0)571-63311860; E-mail: [email protected]

17

Prof Renying Zhuo

18

Key Laboratory of Tree Breeding of Zhejiang Province, Research Institute of

19

Subtropical of Forestry, Chinese Academy of Forestry, Hangzhou 311400, P.R.

20

China; Tel, +86-(0)571-63311860; E-mail: [email protected]

21

Dr Dayi Zhang

22

School of Environment, Tsinghua University, Beijing 100084, P.R. China; Tel,

23

+86-(0)10-62773232; E-mail: [email protected]

ACS Paragon Plus Environment


24

Table of Contents

25 26

For TOC only


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27

Abstract

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Heavy metal contamination presents serious threats to living organisms. Functional

29

genes related to cadmium (Cd) hypertolerance or hyperaccumulation must be

30

explored to enhance phytoremediation. Sedum alfredii Hance is a Zn/Cd

31

cohyperaccumulator exhibiting abundant genes associated with Cd hypertolerance.

32

Here, we developed a method for screening genes related to Cd tolerance by

33

expressing a cDNA-library for S. alfredii Hance. Yeast functional complementation

34

validated 42 of 48 full-length genes involved in Cd tolerance, and the majority of

35

them were strongly induced in roots and exhibited diverse expression profiles across

36

tissues. Coexpression network analysis suggested that 15 hub genes were connected

37

with genes involved in metabolic process, response to stimuli, metal transporter and

38

antioxidant activity. The functions of a novel SaCTP2 gene were validated by

39

heterogeneous expression in Arabidopsis, responsible for retarding chlorophyll

40

content decreases, maintaining membrane integrity, promoting reactive oxygen

41

species (ROS) scavenger activities and reducing ROS levels. Our findings suggest a

42

highly complex network of genes related to Cd hypertolerance in S. alfredii Hance,

43

accomplished via the antioxidant system, defense gene induction and the calcium

44

signaling pathway. The proposed cDNA-library method is an effective approach for

45

mining candidate genes associated with Cd hypertolerance to develop gene-modified

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plants for use in phytoremediation.

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Key words: Sedum alfredii Hance, cDNA library, cadmium, hypertolerance,

48

hyperaccumulation, yeast functional complementation

49 50



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1. Introduction

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Heavy metals are categorized as either essential elements with clear biological

53

functions (e.g., Fe, Mo, Mn, Zn, Ni, Cu and Co) or nonessential elements without

54

known roles in living organisms (e.g., As, Ag, Hg, Sb, Cd and Pb) 1. They are

55

potentially toxic depending on their bioavailable concentrations and receptor

56

exposure-sensitivity

57

valence state of ions (e.g., Hg, Pb and Cd) are deemed pose a greater threat because

58

they can be taken up and utilized by plants via the existing mineral uptake channels 3,

59

4.

60

has aroused attention worldwide 5. Phytoremediation is an approach utilizing naturally

61

occurring or genetically engineered plants to remove contaminants from polluted soils

62

and waters

63

plumbizincicola

64

decontaminating metal-polluted soils. The practical application of hyperaccumulators

65

suffers from their relatively low biomass and slow growth rates, restricting the

66

feasibility of their use in phytoremediation engineering 13, 14. The genetic engineering

67

of high-biomass plants with genes related to metal tolerance and accumulation

68

sourced from hyperaccumulators is an alternative for phytoremediation applications 8,

69

15.

70

tolerance in hyperaccumulators and to mine genes responsible for the uptake,

71

accumulation, volatilization and detoxification of metals as valuable biological

72

resources.

73

The hyperaccumulating ecotype (HE) of Sedum alfredii Hance is a native

74

non-Brassicaceae Zn/Cd cohyperaccumulator inhabiting the deserted Pb/Zn mining

75

area of Quzhou in Zhejiang Province, China 10, 16. S. alfredii Hance can accumulate up

76

to 6,500 μg/g (dry weight, DW) of Cd and 29,000 μg/g (DW) of Zn in its stems

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without displaying significant toxicity symptoms, and the Cd concentration can reach

78

9000 μg/g (DW) in leaves

79

system to protect the plants from the deleterious effects of excess toxic metals. The

80

detoxification system is highly dependent on the expression of key genes related to

81

metal hypertolerance. Previous studies have identified several genes related to Cd

82

sequestration, detoxification and tolerance

83

approaches, such as transcriptomics and proteomics, have been routinely applied to

2, 3.

Among these metals, those resembling nutrients in the

Accordingly, the question of how to efficiently mitigate heavy metal contamination

6-9,

and hyperaccumulating plants such as Sedum alfredii 11,

Noccaea spp. and Arabidopsis halleri

12

10,

Sedum

have been employed for

It is therefore necessary to elucidate the mechanisms of metal accumulation and

10, 16,

indicating the existence of a powerful detoxification

17-22.

Additionally, some high-throughput


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84

decipher

the

regulatory

networks

85

hyperaccumulation of S. alfredii Hance, providing a more precise map of the global

86

network in response to Cd stress

87

small RNAs and the degradome elucidated the regulatory roles of miRNAs and their

88

targets in the HE of S. alfredii, revealing 39 pairs of miRNA targets displaying

89

negatively correlated expression profiles 23. Transcriptomic comparisons between two

90

contrasting ecotypes of S. alfredii identified 57 conserved and 18 divergent

91

orthologous genes, and the latter group mainly participated in the processes of signal

92

transduction, transcription regulation, the stress response and protein metabolism

93

Nevertheless, there is a lack of experimental evidence validating the actual functions

94

of enzymes encoded by these identified genes in the tolerance of Cd stress. The

95

mechanisms of Cd hyperaccumulation and hypertolerance are still obscure, and the

96

characterization of vital genes related to these traits is of great urgency.

97

Conventional approaches confirming the evident functions of genes have relied on the

98

generation of loss-of-function and gain-of-function mutant resources but suffer from

99

inherent drawbacks. For example, a gene family knockout mutation might only result

23-25.

involved

in

the

hypertolerance

and

An integrated analysis of the transcriptome,

26.

24.

100

in marginal differences from the wild type as a result of gene redundancy

101

common problem related to activation-tagged mutagenesis is the nonspecific

102

activation of genes by a transcriptional enhancer, and the upregulated transcription of

103

several genes results in complex phenotypes in some cases, making the identification

104

of target genes responsible for the observed mutant phenotypes challenging

105

circumvent these problems and systematically analyze gain-of-function mutations,

106

ectopic expression of a full-length cDNA library is an alternative for obtaining

107

information from the mRNA of a particular tissue or organism.

108

The construction of a full-length cDNA library for mature mRNAs facilitates gene

109

function characterization and enables manipulation of gene expression in

110

heterologous systems by generating tagged versions of a native protein 28, 29. Ichikawa

111

developed a novel gain-of-function tool referred to as the FOX (Full-length cDNA

112

Overexpressing) hunting system by exploiting an fl-cDNA collection from

113

Arabidopsis

114

Agrobacterium transformation and analyze gene functions in rice 31. A cDNA library

115

from tobacco roots acclimated to Cd using a Cd-sensitive yeast mutant (Δycf1) was

116

employed to successfully screen and identify a series of candidate genes involved in

30

27.

A

To

to heterologously express rice fl-cDNAs in Arabidopsis via



117

the response to Cd stress 32. A similar study revealed 53 transgenic yeast clones with

118

increased salt tolerance by expressing a cDNA library from high-salt-treated Atriplex

119

canescens 33. However, there have been no previous studies in which a cDNA library

120

was constructed for S. alfredii Hance to screen genes related to Cd hypertolerance and

121

hyperaccumulation.

122

In the present study, we constructed a cDNA library for the S. alfredii Hance response

123

to Cd stress, and we characterized forty-eight genes linked with Cd tolerance and

124

validated the functions of the SaCTP2 gene in Cd hypertolerance and

125

hyperaccumulation through heterologous expression in Arabidopsis. Our work

126

attempts to offer a plausible and efficient tool for the batch mining of functional genes

127

associated with the Cd response and to provide insights into the mechanism of Cd

128

hypertolerance, which is a highly complex process associated with ROS balance,

129

cellular hemostasis, antioxidant defense and the calcium signaling pathway. The

130

characterized Cd-responding or detoxification genes in S. alfredii Hance are

131

applicable in the breeding of genetically modified plants for Cd phytoremediation.

132 133

2. Materials and methods

134

2.1. Plant materials and Cd stress treatment for cDNA library construction

135

Seedlings of the HE of S. alfredii Hance were vegetatively propagated and cultured

136

hydroponically for root growth at 25 °C under long days (16 hr light/8 hr dark each

137

day) in an artificial climate chamber for 2 weeks. To extract mRNA from S. alfredii

138

Hance under Cd stress and construct the full-length cDNA library, uniform, healthy

139

rooted seedlings were cultivated in Hoagland-Arnon solution supplemented with

140

CdCl2 (400 μM) for 24 hr. Three tissues (whole roots; the middle part of stems; young

141

leaves, Figure S1C) were then collected separately and immediately frozen in liquid

142

nitrogen for RNA extraction. Wild-type Arabidopsis thaliana plants (Col-0) were

143

grown in another artificial chamber under a 16 hr light/8 hr dark regime at 22 °C for

144

40 days before transformation.

145

2.2. RNA extraction and S. alfredii Hance cDNA library construction

146

Total RNA was extracted from the tissues of S. alfredii Hance after 24 hr of exposure

147

to Cd using the Total RNA Purification Kit (Norgan Biotek Corp., Thorold, ON,


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Canada) and treated with RNase-free DNase I (NEB BioLabs, Ipswich, MA, USA) to

149

remove genomic DNA. The quality of RNA was assessed by agarose gel

150

electrophoresis, and the RNA concentration was quantified with a NanoDrop2000

151

spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Double-stranded

152

cDNA (dsDNA) was synthesized using the SMART™ cDNA Library Construction

153

Kit (Clontech Laboratories Inc., CA, USA) with minor modifications (for details, see

154

Electronic Supporting Information, ESI), then checked by agarose gel electrophoresis,

155

subjected to proteinase K digestion, digested with SfiI (New England Biolabs Inc.,

156

USA), and size fractionated with CHROMA SPIN-400 columns (Clontech

157

Laboratories Inc., USA). It was then ligated into the pYES2-SfiI vector (details see

158

ESI) overnight (approximately 12 hr) at 16 °C, which was subsequently transferred

159

into E. coli JM109 cells by electroporation. Successful transformants were selected on

160

Luria-Bertani (LB) agar plates supplemented with 100 µg/mL ampicillin as the cDNA

161

library.

162

2.3. Yeast transformation, screening and annotation of the S. alfredii Hance

163

cDNA library

164

We employed the Cd-sensitive mutant yeast strain Saccharomyces cerevisiae Δycf1

165

(BY4741; MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; YDR135c::kanMX4; Y04069)

166

as the heterologous host expressing the cDNA library of S. alfredii Hance. Briefly, the

167

plasmids extracted from the cDNA library were transformed into Δycf1 by the lithium

168

acetate method

169

half-strength synthetic dextrose agar plates lacking uracil (SG-Ura) and supplemented

170

with CdCl2 (40 µΜ) and were cultured for 3-5 days at 28 °C. The surviving clones

171

were picked and cultured in 10 mL of liquid SG-Ura medium with shaking at 200 rpm

172

at 28 °C. After centrifugation, the cell pellets were treated with lyticase (50 U/µL,

173

Sigma-Aldrich, St. Louis, MO, USA) and sorbitol (1.0 M) to obtain protoplasts,

174

which were further lysed with NaOH (200 mM) and sodium dodecyl sulfate (SDS, 10

175

g/L). Subsequently, the plasmids were extracted and analyzed by PCR with the

176

universal primer pair T7 and pYES2-R (Table S1) to confirm the inserts in the

177

pYES2-SfiI vector. Yeast cells containing the empty vector (Δycf1_EV) were used as a

178

negative control. In total, 127 plasmids were sequenced with an automatic sequencing

179

machine (ABI, Columbia, MD, USA). All the sequences were filtered by SeqScanner

180

to remove the leading vector, tailing and poor-quality sequences, and repeated

34.

Yeast transformants were selected on 2% galactose-containing



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transcripts. The qualified sequences were subjected to BlastX analysis against the

182

nonredundant protein database of the National Center for Biotechnology Information

183

(NCBI). The full-length sequences were submitted to Simple Modular Architecture

184

Research Tool (SMART) and the protein families (Pfam) database for the annotation

185

of domain structure.

186

The functions of the full-length sequences were reevaluated by reconstructing the

187

exact open reading frame (ORF) of the cDNA amplified with the primers (Table S1)

188

in the yeast expression vector pYES2.1/V5-His-TOPO (Invitrogen, Carlsbad CA,

189

USA). The ligation products were transformed into competent TOP10 One Shot® E.

190

coli (Invitrogen, Carlsbad CA, USA), and the positive clones observed on LB agar

191

plates supplemented with 100 μg/mL ampicillin were sequenced by using the GAL1

192

Forward and V5 C-term Reverse universal primer pair (Table S1). The recombinant

193

plasmids were then transformed into Δycf1 to evaluate their tolerance to Cd. Briefly,

194

the transgenic yeast lines expressing candidate full-length cDNAs were grown in

195

SG-Ura medium overnight at 28 °C until the optical density at 600 nm (OD600)

196

reached 1.0, followed by serial dilution and spotting on SG-Ura agar plates in the

197

absence or presence of CdCl2 (40 µM) and incubation at 28 °C for 3 days before

198

taking photographs.

199

2.4. Expression profiles of screened genes under Cd stress

200

To assess the expression profiles of the screened genes in response to Cd stress in S.

201

alfredii Hance, RNA was extracted from three tissues of S. alfredii seedlings (whole

202

roots; the middle part of stems; young leaves) subjected to Cd stress (400 μM) for 1,

203

12 or 24 hr. Untreated S. alfredii seedlings were used as a negative control. The

204

reverse transcription reactions were performed using the Superscript III First-Strand

205

Synthesis system, followed by RNase H treatment (Invitrogen, Carlsbad, CA, USA).

206

The primers (Table S1) for the target genes were designed using the online Primer3

207

program (http://frodo.wi.mit.edu/primer3/). Beta-tubulin (TUB) was selected as the

208

reference 35. Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

209

was performed in 96-well plates using a SYBR PrimeScriptTM Kit (TaKaRa, Dalian,

210

China) in a 7300 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The

211

amplification procedure and data analysis followed a previous study

212

reactions were carried out in triplicate.


36.

All the

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213

2.5. Construction of the coexpression network

214

On the basis of comparative transcriptome sequencing under Cd stress and a

215

coexpression regulatory network generated through weighted gene coexpression

216

network analysis (WGCNA)

217

interconnections were defined as hub genes potentially associated with the Cd

218

response. All the screened transcripts belonging to these hub genes were analyzed for

219

their correlation with other annotated genes. The Pearson correlation coefficients of

220

the FPKM (fragments per kilobase of exon per million reads mapped) values for each

221

gene pair were calculated using R software

222

coefficients above 0.40 were classified by their annotations. All correlations were

223

visualized using Cytoscape v3.6.1 and analyzed using the NetworkAnalyzer plugin in

224

Cytoscape.

225

2.6. Heterologous expression of the SaCTP2 gene in Arabidopsis

226

An uncharacterized gene from the cDNA library, SaCTP2, was selected due to

227

showing the strongest ability to alleviate the Cd sensitivity of transformed yeasts

228

among all 5 uncharacterized genes. This gene was transformed into Arabidopsis to

229

validate its roles in Cd tolerance and accumulation. First, a preliminary bioinformatic

230

analysis including a BlastX analysis against the NCBI nonredundant protein database

231

and phylogenetic clustering was performed. All the homologous proteins (Table S2,

232

ESI) were subjected to phylogenetic and conserved domain analysis. The

233

phylogenetic tree was constructed by using MEGA7.0

234

iTOL tool (http://itol.embl.de)

235

with the Pfam database and depicted with Illustrator for Biological Sequences (IBS)

236

software 40.

237

For the ectopic expression of the SaCTP2 gene in Arabidopsis to validate its roles in

238

Cd tolerance and accumulation, the ORF of the SaCTP2 gene was amplified by PCR

239

using High-Fidelity KOD-Plus DNA Polymerase (Toyobo, Japan) with the specific

240

primer set listed in Table S1. The purified PCR products were cloned into the

241

Gateway entry vector pENTR/D-Topo (Invitrogen, Carlsbad, USA), and positive

242

plasmids with the correct direction and sequence were recombined in pH2GW7.0 to

243

generate the plant overexpression vector pH2GW7.0-SaCTP2. The vector was then

244

transformed into Agrobacterium tumefaciens strain EHA105, and the positive strains

23,

39.

numerous coexpressed genes with strong

37.

All the edges with correlation

38

and visualized by using the

The conserved domain distribution was predicted



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245

were used to transfect A. thaliana ecotype Columbia plants via the floral dip method

246

41.

247

confirmed by PCR using the first-strand cDNA synthesized from the total RNA

248

extracted from the young leaves of transgenic and WT plants with gene-specific

249

primers

250

5’-TCAAGCGACTTGAATTG-3’). The PCR program was as follows: denaturation

251

at 94 °C for 5 min, 30 cycles of amplification (94 °C for 30 s, 55 °C for 30 s, and

252

72 °C for 60 s), and a final cycle of 72 °C for 7 min. Homozygous lines (T3

253

generation) characterized by nonsegregation were used for Cd stress treatment.

254

2.7. Physiological and chemical analyses of SaCTP2 transgenic lines under

255

Surviving transformants selected with hygromycin (Hyg, 20 µg/mL) were further

(SaCTP2O-F,

5’-ATGGCTTCCGGCACGTTC-3’;

SaCTP2O-R,

Cd stress

256

The Cd treatment of juvenile and adult Arabidopsis plants was performed separately

257

to elucidate the growth and physiological differences between WT and SaCTP2

258

transgenic plants under Cd stress. For juvenile seedling treatment, seeds of wild-type

259

(WT) A. thaliana and three homozygous A. thaliana lines expressing the SaCTP2

260

gene (OE-1, OE-2 and OE-3) were germinated and grown vertically in half-strength

261

Murashige and Skoog (MS) medium with or without CdCl2 (50 µM) for 10 days after

262

stratification (4 °C for 48 hr in dark). Approximately 15-20 individuals were then

263

collected from each treatment to measure their root length, fresh weight, dry weight

264

and metal contents. All the treatments were carried out in triplicate.

265

For the Cd treatment of adult Arabidopsis seedlings, four-leaf-stage wild-type (WT)

266

and SaCTP2-expressing Arabidopsis seedlings (OE-1, OE-2 and OE-3) were exposed

267

to Hoagland-Arnon solution supplemented with or without CdCl2 (30 µM) for 7 days.

268

Healthy young leaves of the WT and SaCTP2-expressing Arabidopsis seedlings were

269

sampled on day 0 before Cd exposure and day 7 after Cd treatment. Electrolyte

270

leakage (%) was calculated on the basis of conductivity measurements

271

assessment of enzyme activities (U/g protein), 1.0 g of leaf tissue was harvested from

272

Arabidopsis seedlings and homogenized in 8 mL of sodium phosphate buffer (PBS,

273

50 mM, pH=7.8) using a prechilled mortar and pestle, followed by centrifugation at

274

10,000 ×g for 15 min at 4 °C. The supernatant was then used to measure O2•- levels

275

and the activities of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD,

276

EC1.11.1.7) and catalase (CAT, EC 1.11.1.6)

43, 44.

42.

For the

The contents of malondialdehyde


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(MDA) and H2O2 were determined as described previously 45.

278

The accumulation of H2O2 and O2∙- (μmol/g FW) in rosette leaves was visualized by

279

diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) staining as

280

described previously

281

separately with DAB and NBT and destaining was conducted overnight in absolute

282

ethanol. Chlorophyll contents (mg/g FW) were measured as described by Arnon with

283

minor modifications 46. Approximately 0.1 g of leaf tissue was incubated in 5 mL of a

284

mixture of ethanol and acetone (1:2, v/v) for 48 hr in the dark. The absorbance at 665

285

and 649 nm (OD665 and OD649) was measured using a DU 800 UV/Vis

286

spectrophotometer (Beckman Coulter, California, CA, USA) following Equations (1)

287

and (2).

22.

Staining was processed by overnight treatment of leaves

288

Chla = (12.72 × OD665 ―5.59 × OD649) × V × N/(1000 × W) (1)

289

Chlb = (22.88 × OD649 ―4.67 × OD665) × V × N/(1000 × W) (2)

290

where V, N and W refer to the volume of the reaction system (5 mL), the dilution

291

factor (1.0) and the fresh weight (FW) of leaves (g), respectively.

292

Cd-treated seedlings (WT and SaCTP2 transgenic plants) were first resorbed using

293

ethylenediamine tetraacetic acid (EDTA, 10 mM) for 30 min and washed thoroughly

294

with distilled water. The roots and stems were then separated, dried at 105 °C for 30

295

min and kept at 70 °C until reaching a constant weight. These samples were next

296

digested with a concentrated acid mixture of HNO3, HClO4 and H2SO4 (4:1:0.5,

297

v:v:v) at 250 °C for 8 h. Zn or Cd concentrations (mg/kg DW or μg/plant) in the

298

digested solution were determined with an inductively coupled plasma-mass

299

spectrometer (ICP-MS; NexION 300; PerkinElmer). All the treatments were

300

performed in triplicate, and each replicate consisted of twelve Arabidopsis seedlings.

301

2.8. Statistical analysis

302

The presented data are the mean ± standard deviation (SD) from all the replicates. All

303

statistical analyses were carried out by using SPSS v17.0 software (SPSS Inc.,

304

Chicago, IL, USA). Significant differences between the WT and three transgenic lines

305

of A. thaliana was determined with Student’s t-tests, and significant differences are



306

indicated by different small letters in the figures.

307 308

3. Results

309

3.1 Functional screening of the cDNA library of S. alfredii Hance with a

310

Cd-sensitive yeast mutant

311

In total, 127 individual yeast clones that survived on SG-Ura medium supplemented

312

with Cd (40 μM, 2% galactose) were obtained. Excluding those clones with

313

noncoding RNA or poor quality or nonreferenced sequences, there were 92

314

recombinant plasmids with cDNA insertions from S. alfredii Hance, including 48

315

full-length cDNAs (Table 1) and 44 partial cDNA sequences (not shown). All 48

316

full-length genes were annotated with BLAST and functionally categorized according

317

to their homologs (Figure 1A and Table 1). Among these genes, only one gene was

318

associated with stress signal perception (SaPERK3), which encodes a proline-rich

319

receptor-like protein kinase. Regarding signal transduction, the two genes involved in

320

calcium signaling are SaCaM and SaCIPK8. All the remaining 45 genes are

321

associated with detoxification, including metal homeostasis (9, SaPCS, SaHIPP,

322

SaMTPl1, SaMTPl2, SaMT3, SaCoPA, SaNTR, SaVIT and SaCys), transcription

323

regulation (3, SaHSF, SaERF and SaWRKY), the epigenetic response (3, SaH2A,

324

SaRPL24 and SaRPS29), ROS homeostasis (11, SaPRX, SaTRX, SaGST, SaFA2H,

325

SaF3H, SaUSPl, SaSAP, SaPOD, SaNQO, SaAPX and SaAPX1), cellular pH and

326

osmotic homeostasis (3, SaGRP, SaPPlase and SaAQP), and stress tolerance and

327

adaption (16, SaCTP1-5, SaGELP, SaMS1, SaCtns, SaChi, SaMYR, SaTLP3, SaDRP,

328

SaAIR6B, SaLHC6A, SaPCCR and SaDRT100). It is worth mentioning that 5 novel

329

genes were previously uncharacterized, and these genes were designated as SaCTP

330

(Cadmium Tolerance Protein) genes, which might be unique to S. alfredii Hance.

331

After reconstruction in the yeast expression vector pYES2.1/V5-His-TOPO, the

332

functions of the 48 full-length transcripts in tolerating Cd were validated and are

333

illustrated in Figure 2. Forty-two yeast cell lines expressing candidate genes exhibited

334

significantly better growth than the control (Δycf1_EV), particularly for those related

335

to metal homeostasis (SaPCS, SaHIPP, SaMTPl1, SaMTPl2 and SaMT3), ROS

336

homeostasis (SaPRX, SaGST, SaF3H, SaUSPl, SaSAP, SaPOD, SaNQO, SaAPX and


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SaAPXl), cellular osmotic homeostasis (SaGRP and SaAQP) and transcription

338

regulation (SaHSF and SaERF). Two of the five novel genes (SaCTP1 and SaCTP2)

339

remarkably alleviated the Cd sensitivity of transformed yeasts.

340

3.2 Candidate genes responding to Cd stress as predicted by the coexpression

341

network

342

The reconstructed gene coexpression network identified 15 out of the 48 full-length

343

genes as hub genes strongly affected by Cd stress, exhibiting 867 nodes and 2,361

344

connections (Figure 1B). They were associated with signal perception (SaCaM),

345

transcription regulation (SaWRKY), ROS homeostasis (SaPRX and SaFA2H), metal

346

homeostasis (SaCys, SaMTPl1, SaMT3, SaHIPP and SaNTR), and stress and tolerance

347

adaption (SaGRP, SaLHC6A, SaAIR6B, SaCTP2 and SaCTP3). These hub genes were

348

mainly linked with metabolic processes (GO:0008152), cellular processes

349

(GO:0009987), biological regulation (GO:0065007) and five other categories

350

(response to stimuli, transcription factor, transporter activity, antioxidant activity and

351

molecular function regulator). SaPRX (471 edges) and SaAIR6B (431 edges)

352

presented the highest degrees. Two novel SaCTP2 and SaCTP3 genes also behaved as

353

hub genes, with 144 and 27 edges, respectively.

354

3.3 Responses of candidate genes to Cd stress

355

The inducible expression of candidate genes under Cd stress resulted in diverse

356

profiles according to RT-qPCR assays (Figure S1A, ESI). For most genes, obvious

357

positive induction was observed in roots, with strong increases across the three time

358

points. In contrast, the expression of almost all genes was downregulated in stems,

359

and elevated expression appeared for a small proportion of the genes in leaves after 12

360

hr and 24 hr of exposure. In roots, most of the genes showing strong, instant induction

361

by Cd stress belonged to the categories associated with stress tolerance and adaption

362

(SaCTP1, SaCTP2, SaCTP3, SaLHC6A and SaDRT100), and the others were

363

associated with ROS homeostasis (SaF3H), metal homeostasis (SaNTR1), cellular pH

364

and osmotic homeostasis (SaAQP), and stress tolerance and adaption (SaMS1). Some

365

genes associated with metal homeostasis (SaVIT1), transcription regulation (SaERF),

366

ROS homeostasis (SaAPXl and SaTRX), and stress tolerance and adaption (SaGELP)

367

showed stepwise elevation. In stems, all genes were downregulated except for four

368

genes that were induced at 1 hr (SaERF, SaCys, SaLHC6A and SaAQP) and two genes



369

that were induced at 6 hr (SaCtns and SaCIPK8).

370

In leaves, only three genes related to stress tolerance and adaption (SaCTP3,

371

SaLHC6A and SaDRT100) exhibited moderate elevation in the early stage (1 hr),

372

whereas 12 genes that were strongly upregulated after 12 and 24 hr were associated

373

with signal perception (SaPERK3), metal homeostasis (SaHIPP and SaVIT1),

374

transcription regulation (SaHSF and SaWRKY), the epigenetic response (SaRPS29 and

375

SaH2A), ROS homeostasis (SaF3H and SaNQO), cellular pH and osmotic

376

homeostasis (SaGRP), and stress tolerance and adaption (SaCTP1 and SaAIR6B). It is

377

worth mentioning that most of the hub genes (Figure 1B) were not significantly

378

induced postexposure to Cd stress.

379

The tissue expression profiles of 48 genes are illustrated in Figure S1B. Ten genes

380

that were highly expressed in roots were only associated with calcium signaling

381

(SaCaM), signal perception (SaPERK3), metal homeostasis (SaMTPl1), ROS

382

homeostasis (SaUSPl and SaAPX), epigenetic response (SaRPS29 and SaRPL24), and

383

stress tolerance and adaption (SaCtns, SaCTP3 and SaDRP). Eleven genes that

384

showed high expression only in stems were related to metal homeostasis (SaCoPA,

385

SaHIPP, SaNTR1, SaVIT1), ROS homeostasis (SaTRX, SaF3H and SaFA2H), and

386

stress tolerance and adaption (SaMS1, SaChi). Almost all genes that were only highly

387

expressed in leaves were associated with stress tolerance and adaption, including

388

SaCTP2, SaCTP4, SaGELP, SaDRT100, SaLHC6A, SaRCCR and SaTLP3. Among all

389

genes, only the SaCTP3 gene was strongly induced and highly expressed in roots

390

postexposure to Cd.

391

3.4 Transgenic SaCTP2-expressing Arabidopsis lines displayed Cd tolerance

392

and accumulation

393

The SaCTP2 gene has a full-length of 300 bp and encodes a protein of 99 amino acids

394

with an approximate molecular mass of 11.08 kDa (Figure 3A). Conserved domain

395

analysis showed the presence of a FCS-like C2-C2 zinc-finger domain (FLZ,

396

pfam04570) from residue 25 to residue 64 (Figure 3B), characterized by the

397

consensus cysteine-signature sequence acting as a protein-protein interaction module

398

47, 48.

399

third cysteine are indicated by asterisks in Figure 3A. Phylogenic analysis showed

400

that SaCTP2 clustered with a hypothetical protein (GenBank accession number:

Conserved residues such as phenyl alanine and a serine residue associated with a


Page 14 of 47

Page 15 of 47


401

RAL53989.1) from Cuscuta australis in the family Convolvulaceae (Figure 3C).

402

The results from the coexpression network suggested that the novel SaCTP2 gene

403

interacted with other genes related to the metabolic process, cellular process and

404

response to stimulus categories (Figure 1B), alleviating the Cd sensitivity of

405

transformed yeasts (Figure 2). It was also significantly induced postexposure to Cd

406

(Figure S1) and, thus, was selected and transformed in Arabidopsis to validate its

407

roles in Cd tolerance. In the absence of Cd, the WT and SaCTP2-expressing

408

Arabidopsis lines (OE-1, OE-2 and OE-3) did not show significant differences after

409

10 days of cultivation (Figure 4A). The biomass of 10 seedlings was 54.4 mg (FW)

410

for WT, 54.6 mg (FW) for OE-1, 55.0 mg (FW) for OE-2 and 56.2 mg (FW) for

411

OE-3. The WT, OE-1, OE-2 and OE-3 Arabidopsis lines exhibited average root

412

lengths of 3.43, 3.53, 3.47 and 3.48 cm, respectively. Postexposure to Cd, all the

413

seedlings showed retarded growth (Figure 4A). The transgenic lines (Figure 4B)

414

presented stronger tolerance to Cd stress, exhibiting significantly longer root lengths

415

(2.17-2.28 cm for OE-1, OE-2 and OE-3) and higher biomass (35.9-38.8 mg in FW

416

for 10 seedlings of OE-1, OE-2 and OE-3) than the WT plants (1.42 cm in length and

417

22.3 mg in FW, respectively).

418

The in vivo visualization of H2O2 and O2∙- levels in leaves postexposure to Cd is

419

illustrated in Figure 4C-4F. In the absence of Cd, there was no significant difference

420

between WT and SaCTP2-overexpressing Arabidopsis. After treatment with Cd, the

421

SaCTP2-overexpressing Arabidopsis lines displayed remarkably lower staining

422

intensities compared to WT, indicating less accumulation of H2O2 and O2∙- (Figure 4C

423

and 4E). These results were obtained from the direct measurement of H2O2 and O2∙-

424

contents in leaves. In the absence of Cd, H2O2 contents were comparable in WT and

425

the three transgenic Arabidopsis lines (Figure 4D). Postexposure to Cd for 7 days,

426

H2O2 contents increased by 214% in WT, to levels much higher than those in the

427

SaCTP2-overexpressing Arabidopsis lines (129% on average, Figure 4D). Similarly,

428

the O2∙- contents of SaCTP2-overexpressing Arabidopsis plants increased by only

429

64-78%, but that of WT increased by 162% postexposure to Cd (Figure 4F). More

430

evidence was obtained by examining the activities of ROS scavengers, which showed

431

negligible differences between WT and transgenic plants under nonstressed

432

conditions. SOD and POD activities increased 90.3-118.3% and 354.6-430.9%,

433

respectively, after 7-day exposure to Cd in the three transgenic lines, to levels



Page 16 of 47

434

significantly higher than those in WT (36.4% and 134.76%, Figure 5A and 5B).

435

Surprisingly, CAT activities behaved in the opposite manner, decreasing more in the

436

three transgenic lines (38.4-44.9%) than in WT (26.9%, Figure 5C).

437

In the absence of Cd, representative physiological indices of WT and

438

SaCTP2-overexpressing Arabidopsis lines such as chlorophyll contents, MDA levels

439

and electrolyte leakages showed no significant differences (Figure 5D-5F).

440

Postexposure to Cd, the total chlorophyll contents decreased by 50.6% in WT plants,

441

whereas the decline was only 13.6-37.2% in the three SaCTP2-overexpressing

442

Arabidopsis lines (Figure 5D). Similarly, MDA levels in the SaCTP2-overexpressing

443

Arabidopsis lines increased by 31.7-54.5%, was much lower than the increase in WT

444

(122.9%,

445

SaCTP2-overexpressing Arabidopsis lines ranged from 1.9% to 55.8%, which was

446

lower than that in WT (92.1%, Figure 5F).

447

Additionally, SaCTP2-expressing Arabidopsis lines (OE-1, OE-2 and OE-3)

448

displayed 46.3% higher Cd contents in their stems and leaves than WT, although there

449

was no significant difference (p>0.05) in root Cd contents between WT and the

450

SaCTP2-expressing Arabidopsis lines (Figure 6A). The Cd translocation factor (TF)

451

was 0.687 for the SaCTP2 transgenic seedlings, which was 49.5% higher than that for

452

WT (0.459). As the biomass increased, the SaCTP2-expressing Arabidopsis lines took

453

up and stored more Cd in their roots (96.1% higher) or stems/leaves (342.0% higher)

454

(Figure 6B). In contrast, seedlings of the SaCTP2-expressing Arabidopsis lines

455

exhibited a 22.1% lower Zn content in their roots but a 14.5% higher Zn content in

456

their stems/leaves in comparison with WT (Figure 6C). Zn accumulation was

457

therefore significantly lower than Cd accumulation: approximately 56.8% higher in

458

the roots and 244.7% higher in the stems/leaves of the SaCTP2-expressing

459

Arabidopsis lines (Figure 6D). These results suggested that overexpression of

460

SaCTP2 preferentially increased the uptake and accumulation of Cd in Arabidopsis.

Figure

5E),

and

the

increase

in

electrolyte

leakage

in

461 462

4. Discussion

463

The 48 genes identified from the full-length cDNA library were involved in signal

464

perception (SaPERK3), signal transduction (SaCaM, SaCIPK8, SaPRX and SaTRX)

465

and detoxification (metal homeostasis, ROS homeostasis, etc., Figure 1A). Compared


Page 17 of 47


466

to a previously reported transcriptome database 23, our coexpression network analysis

467

identified 15 hub genes as key candidates influencing Cd hypertolerance and

468

hyperaccumulation in S. alfredii Hance (Figure 1B). In addition to metabolic and

469

cellular processes, these hub genes are also linked to other genes associated with

470

metal transport, transcription regulation, the response to stimuli and antioxidant

471

processes. Accordingly, the genetic functions related to Cd tolerance in S. alfredii

472

Hance form a complex network. Among the identified genes, 13 hub genes have been

473

reported previously, and the two novel genes (SaCTP2 and SaCTP3) are linked to Cd

474

tolerance for the first time in the present study. Their roles in Cd tolerance were

475

further validated by expression in Cd-sensitive yeast cells (Figure 2), and they might

476

trigger upstream biological processes for activating detoxification and defense

477

reactions under Cd stress.

478

Regarding signal perception, the SaPERK gene encodes a subclass of the plant

479

receptor-like kinases that act as sensors/receptors with extracellular domains on the

480

cell wall

481

abiotic/biotic stresses and activate associated cellular responses

482

cell wall-associated kinases for invertase activity and cell growth) 51-53. However, the

483

SaPERK gene failed to complement the yeast Cd-sensitive mutant (Δycf1), and its role

484

in sensing extracellular Cd and perceiving Cd stress signals must be questioned.

485

Two genes associated with signal transduction are related to calcium signaling

486

(SaCaM and SaCIPK8). Ca deficiency has been reported to trigger highly efficient

487

phloem remobilization of Cd in S. alfredii Hance and to subsequently increase Cd

488

accumulation in leaves

489

extensively studied Ca2+ sensors and is proposed as an integrator of different stress

490

signaling pathways modulating a number of stress-associated proteins

491

encodes a plant-specific Ca2+ sensor, calcineurin B-like (CBL)-interacting protein

492

kinase (CIPK), that modulates the expression and activity of downstream stress

493

effectors 56. Although previous studies have indicated key roles of mitogen-activated

494

protein kinase (MAPK) members

495

signal transduction, these proteins and systems were not identified in the present

496

study. Our findings broaden our knowledge and suggest that calcium signaling

497

crosstalk provides additional versatility to stress-associated signal transduction

498

pathways by regulating the activities of several kinases, modulating proteins

49.

These sensors/receptors monitor extracellular changes postexposure to

54.

50

(e.g., Arabidopsis

SaCaM encodes calmodulin, which is one of the most

57, 58

and hormone signaling systems


55.

59

SaCIPK

in metal


Page 18 of 47

499

associated with defense responses, perceiving the signals from upstream receptors,

500

and ultimately helping plants to maintain cellular homeostasis.

501

Other genes associated with detoxification encode proteins that chelate heavy metals

502

(SaPCS, SaMT3 and SaHIPP), scavenge ROS (SaPRX, SaTRX, SaAPX, SaPOD,

503

SaGST and SaUSP), modulate transcription (SaERF, SaWRKY and SaRLP24) or

504

participate in other response processes (SaMTR, SaDRT100, SaPCCR and SaDRP).

505

These genes showed diverse expression profiles in response to Cd stress and were

506

induced in the roots in particular (Figure S1), indicating their functional diversity and

507

involvement in diverse biological processes. Previous studies have reported

508

interdependency between cellular redox imbalance and metal toxicity, implying the

509

disruption of equilibrium between ROS generation and detoxification by metal stress,

510

which induces oxidative stress and causes cell damage 60, 61. Generally, the main roles

511

of these genes are to maintain metal homeostasis or executing antioxidant activities to

512

alleviate ROS injuries 62, 63.

513

Among the genes associated with metal homeostasis, the SaHIPP gene was

514

moderately upregulated in roots and highly induced in leaves (Figure S1). This gene

515

encodes heavy metal-associated isoprenylated plant proteins (HIPPs) that serve as

516

metallochaperones, which are composed of a metal-binding domain and a C-terminal

517

isoprenylation motif and play a role in metal homeostasis and act as regulatory

518

elements in the transcriptional response to cold and drought

519

linked to the biosynthesis of metal chelates (metallothioneins, SaMT3) and

520

phytochelatins (SaPCS), indicating the key roles of Cd chelation proteins in Cd

521

tolerance. Plant chelators such as phytochelatins, glutathione and metallothioneins can

522

bind Cd and ultimately sequester it into vacuoles

523

NRT1/PTR FAMILY (NPF) proteins capable of transporting nitrate, di/tri-peptide,

524

plant hormones (e.g., indole-3-acetic acid, abscisic acid, gibberellin) and secondary

525

metabolites (e.g., glucosinolates)

526

roles in K+ translocation from roots to shoots and is involved in coordinating the

527

K+/NO3- distribution

528

subgroup of NPF from Arabidopsis and serves as a constitutively expressed

529

transporter with a significant contribution to the NO3- translocation response to

530

salinity

531

gene to Cd hypertolerance in S. alfredii Hance on the basis of ectopic expression in

70.

69.

67, 68.

66.

64, 65.

Other genes were

The SaNTR1 gene encodes

The AtNRT1.5 gene is reported to play crucial

NPF2.3, a member of the nitrate excretion transporter

For the first time, our findings hinted at the contribution of the SaNTR1


Page 19 of 47


532

Cd-sensitive yeast, which can probably be explained by the transport of peptides,

533

glucosinolate defense compounds or plant hormones.

534

Metal stress can induce oxidative injuries

535

antioxidant systems for antioxidant defense and cellular homeostasis maintenance.

536

The antioxidant defense enzymes include peroxidase, thioredoxin, glutathione

537

S-transferase, peroxiredoxin and ascorbate peroxidase

538

encodes peroxiredoxin (PRX), which is a robust peroxide-decomposing enzyme 60, 74,

539

and PRX expression contributes to increasing ROS levels to repair damaged

540

macromolecules, thus enhancing the antioxidant system and reducing heavy metal

541

concentrations in cytoplasmic compartments

542

encoding flavanone 3-hydroxylase (F3H), an important enzyme catalyzing flavonol

543

synthesis. Flavonoids are important secondary metabolites in plants and are associated

544

with a wide range of biological functions, such as defense protection. A tomato

545

F3H-like protein has been shown to improve chilling tolerance 61. Other genes related

546

to cellular homeostasis are also responsible for stress responses, including heat shock

547

proteins (HSPs), regulatory transcription factors, structural proteins and DNA repair

548

proteins76, 77. SaAQP encodes aquaporins, a group of membrane-intrinsic proteins that

549

transport water along with some small neutral solutes and ions and is important in the

550

responses to a wide range of environmental stresses 78, 79. SaDRT100, encoding DNA

551

repair and tolerance proteins, is linked to genetic stability

552

protein-encoding gene (SaGRP) is related to the cellular stress response 81. However,

553

none of these genes were previously linked to Cd tolerance in S. alfredii Hance, and

554

our study suggested that numerous enzymes associated with the antioxidant system

555

and cellular homeostasis are responsible for Cd tolerance in S. alfredii Hance.

556

Five novel genes (SaCTP1 to SaCTP5) were uncharacterized in the NCBI database

557

and exhibited Cd tolerance traits. To further validate their functions in Cd

558

hypertolerance and hyperaccumulation, heterologous expression of the SaCTP2 gene

559

was performed in Arabidopsis. According to the examined physiological indices

560

(weight, root length, chlorophyll contents, electrolyte leakage, MDA levels, ROS

561

levels and H2O2 contents, Figure 4 and 5), the activities of ROS-scavenging enzymes

562

(SOD and POD, Figure 5) and Cd accumulation (Figure 6), the SaCTP2-expressing

563

transgenic lines displayed significantly higher Cd tolerance and accumulation than

564

WT. These results confirmed the functions of the SaCTP2 genes in Cd

71

75.

and subsequently trigger complex

72, 73.

For instance, SaPRX

SaF3H belongs to a gene family


80.

The glycine-rich


Page 20 of 47

565

hyperaccumulation. One explanation arising from protein sequence analysis suggested

566

that SaCTP2 is a 99-aa protein harboring a partial FLZ domain

567

putative heavy metal-binding motifs and contributes to the increased Cd tolerance

568

(Figure 3). Previous studies have reported the functions of the FLZ domain in

569

regulating heavy metal tolerance

570

which carries two AN1 zinc finger domains and a Cys2-His2 domain, show high

571

tolerance to toxic metals and exhibit increased Zn accumulation 83. Another possible

572

explanation is metabolic homeostasis, supported by a close correlation between the

573

FLZ domain and the SNF1-related protein kinase 1 (SnRK1) signaling cascade in

574

Arabidopsis, which triggers massive transcriptional reprogramming to enable plant

575

survival under low-energy conditions

576

could achieve metabolic homeostasis by protecting chlorophyll from Cd-induced

577

degradation, decreasing ROS levels, maintaining membrane integrity and protecting

578

the root architecture from damage, as evidenced by the functions of the SaREFl gene

579

observed in a previous study 19. The higher levels of Cd translocation factors recorded

580

in this study also suggested more Cd accumulation in stems and leaves, implying that

581

the SaCTP2 protein promoted the root-to-shoot translocation of Cd and altered the Cd

582

distribution in Arabidopsis. Taken together, the available findings indicate that the

583

SaCTP2 gene might act as a protein-protein interaction module and that the encoding

584

enzyme interacts with biomolecules involved in Cd uptake, transport and

585

detoxification. This gene trait is valuable for constructing transgenic plants for

586

application in phytoremediation at Cd-contaminated sites. Additional experimental

587

studies are suggested to validate the functions of other genes screened in the present

588

study, such as SaHIPP, SaF3H, SaAQP, SaDRT100, SaGRP and SaNTR1, which

589

could broaden our understanding of the mechanisms of Cd tolerance in S. alfredii

590

Hance.

591

In conclusion, our work revealed 48 genes that are directly associated with Cd

592

tolerance from a full-length cDNA library of S. alfredii Hance and validated their

593

functions in Cd-sensitive yeast cells. Coexpression network analysis of the response

594

to Cd stress suggested that the Cd hypertolerance of S. alfredii Hance is a highly

595

complex phenomenon associated with diverse gene functions carried out by the

596

antioxidant system, defense gene induction and calcium signaling pathway. Among

597

the 5 novel genes linked to Cd tolerance in S. alfredii Hance for the first time, the

82.

48

that includes

Transgenic plants overexpressing AtSAP13,

84-87.

SaCTP2 expression in transgenic plants


Page 21 of 47


598

functions of the SaCTP2 gene were further confirmed in transgenic Arabidopsis lines.

599

Our findings offer a novel high-throughput approach for cDNA library construction

600

for screening, validating and understanding the genes that confer metal hypertolerance

601

traits and provide guidance for the breeding of plant varieties for metal

602

phytoremediation.

603 604

Supporting Information

605

Details of dsDNA synthesis; construction of the pYES2-SfiI yeast expression vector;

606

list of primers used in this study; homologous genes of SaCTP2 in the phylogenic tree

607

and domain analysis based on the Pfam database; heatmap of the expression profiles

608

of 48 screened genes in different tissues of S. alfredii Hance under Cd stress; heatmap

609

of the tissue-specific expression patterns of 48 screened genes of S. alfredii Hance

610

without Cd stress; sketch of S. alfredii Hance individuals and the sampled root, stem

611

and leaf tissues.

612 613

Acknowledgments

614

This work was supported by the National Natural Science Foundation of China (No.

615

31870647) and the National Key Technology R&D Program of China (No.

616

2016YFD0800801). DZ also acknowledges the support of the Chinese Government's

617

Thousand Talents Plan for Young Professionals.

618



619

5. References

620

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621

why do they do it? And what makes them so interesting? Plant Science 2011, 180 (2),

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Chen, L. Z.; Zhang, D. Y., Impacts of environmental factors on the whole microbial

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10. Yang, X. E.; Long, X. X.; Ye, H. B.; He, Z. L.; Calvert, D. V.; Stoffella, P. J.,

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Page 34 of 47

Page 35 of 47

998 999 1000


Table Table 1. Annotation of sequences coding for amino acids in the proteins (CDSs) of 48 candidate genes screened from the cDNA library of S. alfredii Hance. Gene (Accession No) SaCTP1 (MK044850) SaPCS (MK044851) SaCTP2 (MK044852) SaUSPl (MK044853) SaSAP (MK044854) SaCTP3 (MK044855) SaHSF (MK044856) SaGRP (MK044857) SaGELP (MK044858)

CDS Homologs (Accession No) Hypothetical protein DVH24_029503 in Malus domestica (RXH74782.1) Phytochelatin synthase in Boehmeria nivea (AHC98018.1) Uncharacterized protein LOC111464337 in Cucurbita moschata (XP_022964268.1) Universal stress protein PHOS32-like in Juglans regia (XP_018811680.1) Zinc finger A20 and AN1 domain-containing stress-associated protein 5-like in Camellia sinensis (XP_028079437.1) Uncharacterized protein LOC114266108 isoform X1 in Camellia sinensis (XP_028062803.1) Heat shock transcription factor in Trema orientale (PON68120.1) Glycine rich protein in Nicotiana tabacum (AAK57546) GDSL esterase/lipase in Gossypium arboretum (XP_017604964.1)

Amino acid sequence similarity 57.89%

Annotation

Reference

Unknown

-

89.00%

Cd2+ tolerance.

88

51.52%

Unknown

-

73.93%

Control of ROS homeostasis under anoxia

89

59.09%

Ubiquitin ligase, redox sensor, and regulation of gene expression during stress

90

60.27%

Unknown

-

45.31%

Regulation of stress tolerance

44

48.00%

Stress responses and signaling.

81

55.87%

Diverse physical functions in growth and stress responses.

91



SaRPS29 (MK044859) SaAIR6B (MK044860) SaPOD (MK044861) SaPERK3 (MK044862) SaPPlsae (MK044863) SaMYR (MK044864) SaDRT100 (MK044865)

40S ribosomal protein S29 in Cicer arietinum (XP_004491390.1) Auxin-responsive protein SAUR72-like in Nelumbo nucifera (XP_010259870.1) Cationic peroxidase 1 in Vitis vinifera (XP_002268412.1) Proline-rich receptor-like protein kinase PERK3 in Hevea brasiliensis (XP_021639397.1) Peptidyl-prolyl cis-trans isomerase in Actinidia chinensis var. chinensis (PSS29498.1) myb-related protein 308 in Prunus yedoensis var. nudiflora (PQQ14404.1) DNA damage-repair/toleration protein DRT100 in Sesamum indicum (XP_011087364.1)

96.43%

SaF3H (MK044866) SaMS1 (MK044867) SaH2A (MK044868) SaRPL24 (MK044869) SaCoPA (MK044870) SaHIPP

Flavanone 3-hydroxylase in Arabidopsis thaliana (NP_001190050.1) Mannan synthase 1 isoform in Vitis vinifera (XP_002277171.1) Histone H2A in Erythranthe guttata (XP_012848194.1)

63.32%

60S ribosomal protein L24-like in Lactuca sativa (XP_023760011.1) Copper-transporting ATPase in Actinidia chinensis var. chinensis (PSR98747.1) Heavy metal-associated isoprenylated plant protein

95.65%

Page 36 of 47

Components of the protein synthesis machinery Auxin signaling pathway

92

Biotic and abiotic stress tolerance and senescence Sensors/receptors at the cell wall

94

Chaperones induced by intracellular acidification Modulation of ROS; accumulation and stress response Reparation and toleration of UV-B-induced DNA damage

95

Participation in the flavonoid biosynthesis pathway Synthesis of cell-wall polysaccharides

97

99

63.73%

Escort of histones and function in nucleosome structural configuration. Essential components of the protein synthesis machinery Transmembrane proteins transporting copper

61.36%

Heavy metal homeostasis and detoxification

52.17% 72.41% 50.97% 90.80% 66.43% 68.41%

75.59% 82.31%


93

49

96

80

98

92

100

64

Page 37 of 47


(MK044871) SaCtns (MK044872) SaERF (MK044873)

3-like in Nelumbo nucifera (XP_010253988.1) Cystinosin homolog in Prunus mume (XP_008230459.1) Ethylene-responsive transcription factor TINY-like in Juglans regia (XP_018823586.1)

SaCaM (MK044874)

Calmodulin-like in Brassica oleracea var. oleracea (XP_013631799.1)

91.20%

SaCTP4 (MK044875) SaNTR1 (MK044876) SaWRKY (MK044877) SaCys (MK044878) SaVIT (MK044879) SaNQO (MK044880) SaTRX (MK044881) SaFA2H (MK044882) SaChi

Uncharacterized LOC105110269 in Populus euphratica (XP_011003552.1) Nitrate transporter 1/peptide transporter family protein in Malus domestica (AYW00847.1) WRKY transcription factor 31 in Ziziphus jujube (XP_015877768.1) Cysteine synthase in Eucalyptus grandis (XP_010024285.1) Vacuolar iron transporter 1 in Nicotiana sylvestris (P_009787813.1) NADPH:quinone oxidoreductase-like in Nelumbo nucifera (XP_010276653.1) Thioredoxin M3 in Vitis vinifera (RVW49697.1)

86.62%

Dihydroceramide fatty acyl 2-hydroxylase FAH1-like in Herrania umbratical (XP_021298290.1) Endochitinase-like in Juglans regia (XP_018854709.1)

76.36%

Lysosomal cystine transporter

101

86.67%

Regulatory proteins controlling metabolism, growth and development; responses to environmental stimuli Major Ca2+ sensors with critical roles in interpreting encrypted Ca2+ signals Unknown

102

Transportion of a wide variety of substrates such as nitrate and plant hormones Response to biotic and abiotic stress

67

61.62% 64.97% 93.33%

103

-

104

105.

88.43%

Cysteine synthesis, the final step of assimilatory sulfate reduction Iron transport

73.43%

ROS balance

107

61.39%

Component of antioxidant system

81.01%

Fatty acid 2-hydroxylation

110

69.77%

Plant defense

111


106

108, 109


(MK044883) SaMTPl2 (MK044884) SaAPXl (MK044885) SaLHC6A (MK044886) SaCIPK8 (MK044887) SaMTPl1 (MK044888) SaAPXl (MK044889) SaMT3 (MK044890) SaGST (MK044891) SaCTP5 (MK044892) SaAQP (MK044893) SaRCCR (MK044894) SaDRP (MK044895) SaPRX

Page 38 of 47

Metallothionein 2 in Sedum alfredii (AIG51062.1)

99.10%

Responsible for iron hemostasis in cell

18

Putative L-ascorbate peroxidase 6 in Jatropha curcas (XP_012076559.1) Light harvest chlorophyll a-b binding protein 6 in Prunus persica (XP_007201239.1) CBL-interacting serine/threonine-protein kinase 8 isoform X2 in Theobroma cacao (XP_007018315.2) metallothionein-like protein, Sedum plumbizincicola, ANF89428.1 L-ascorbate peroxidase 2 in Nicotiana tabacum (ANF89428.1) Metallothionin 3 in Salvia miltiorrhiza (AEQ54919.1)

64.47%


109

87.9%

17

98.00%

Main light-harvesting pigment-protein complex of photosystem Plant-specific family of serine-threonine kinases which CBLs specifically target Responsible for iron hemostasis in cell

88.80%


112

55.56%

Iron hemostasis

18

Glutamine synthetase nodule isozyme in Fragaria vesca subsp. Vesca (XP_004288594.1) Uncharacterized protein in Vigna angularis (XP_017424345.1) Aquaporin TIP1-1 in Manihot esculenta (XP_021620289.1) Red chlorophyll catabolite reductase in Vigna angularis (XP_017428980.1) Disease resistance protein At4g14610 in Arabidopsis lyrata subsp. Lyrate (XP_017428980.1) Peroxiredoxin-2E-2 in Cynara cardunculus var.

89.30%

Response to plant nitrogen status and environmental cues Unknown

113

79

60.37%

Transport of water, small neutral solutes and ions Central reaction of chlorophyll breakdown

46.11%

Disease defense responses

115

78.72%


109

82.29%

85.71% 81.25%


56

18

-

114

Page 39 of 47


(MK044896) SaTLP3 (MK044897)

scolymus (XP_024978976.1) Tubby-like F-box protein 3 in Theobroma cacao (XP_017981743.1)

73.88%


Responses to stress

116


1002

Figure captions

1003

Figure 1. (A) Functions of 48 candidate genes related to signal perception, signal

1004

transduction and detoxification. (B) Coexpression network of 15 hub genes. Nodes

1005

indicate genes, and edges represent significant coexpression events between genes.

1006

Target genes involved in the same biological process are grouped together and

1007

distinguished with different colors.

1008

Figure 2. Validation of 48 candidate genes from S. alfredii Hance related to Cd

1009

tolerance by yeast complementation in the Cd-sensitive strain Δycf1. Yeast cells were

1010

spotted in four concentrations (OD600 = 1, 0.1, 0.01, 0.001). The negative control was

1011

Δycf1 carrying the empty vector pYES2 (Δycf1_EV). The plates were incubated for 3

1012

days at 30 °C, and the Cd exposure concentration was 40 µΜ.

1013

Figure 3. (A) Nucleotides of the SaCTP2 gene and deduced amino acid sequences of

1014

the SaCTP2 protein. (B) Conserved domain of the SaCTP2 protein predicted by the

1015

Pfam database. (C) Phylogenetic tree of the SaCTP2 gene constructed by MEGA7.0

1016

(details see Table S2 in ESI).

1017

Figure 4. Physiological indices of WT and SaCTP2-expressing Arabidopsis

1018

transgenic lines (OE-1, OE-2, and OE-3). (A) Phenotypic changes after being grown

1019

vertically for 10 days in half-strength Murashige and Skoog medium in the absence or

1020

presence of Cd. (B) Root length and fresh weight (FW). (C) Leaves with DAB

1021

staining. (D) H2O2 contents of leaves. (E) Leaves with NBT staining. (F) O2∙- contents

1022

of leaves. Bars represent the mean ± standard deviation (SD) from 15-20 individuals

1023

of each genotype. Different small letters indicate significant differences (p

cDNA library for mining functional genes in Sedum alfredii Hance

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