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NADP-malate dehydrogenase of sweet sorghum improves salt tolerance of Arabidopsis thaliana Yuanyuan Guo, Yushuang Song, Hongxiang Zheng, Yi Zhang, Jianrong Guo, and Na Sui J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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Journal of Agricultural and Food Chemistry

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NADP-malate dehydrogenase of sweet sorghum improves salt tolerance of Arabidopsis thaliana

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Yuanyuan Guo+, Yushuang Song+, Hongxiang Zheng, Yi Zhang, Jianrong Guo, Na Sui*

3

Shandong Provincial Key Laboratory of Plant Stress, College of Life Science, Shandong Normal

4

University, Jinan, 250014, China.

5 6

+

Yuanyuan Guo and Yushuang Song have contributed equally to this work.

7 8

*Corresponding author.

9

Dr. Na Sui

10

Shandong Provincial Key Laboratory of Plant Stress

11

College of Life Science

12

Shandong Normal University

13

Jinan

14

Shandong

15

China

16

E-mail: [email protected]

17

ACS Paragon Plus Environment


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Abstract

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Sweet sorghum is a C4 crop that shows high salt tolerance and high yield. NADP-malate dehydrogenase

20

(NADP-ME) is a crucial enzyme of the C4 pathway. The regulatory mechanism of NADP-ME remains

21

unclear. In this study, we isolated SbNADP-ME from sweet sorghum. The open reading frame of

22

SbNADP-ME is 1911 bp and 637 amino acid residues. Quantitative real-time PCR analysis showed that

23

SbNADP-ME transcription in sweet sorghum was enhanced by salt stress. The SbNADP-ME transcript

24

level was highest under exposure to 150 mM NaCl. Arabidopsis overexpressing SbNADP-ME showed

25

increased germination rate and root length under NaCl treatments. At the seedling stage, physiological

26

photosynthesis parameters, chlorophyll content, PSII photochemical efficiency, and PSI oxidoreductive

27

activity in the wild type decreased more severely than in the overexpression lines, but less than in

28

T-DNA insertion mutants, under salt stress. Overexpression of SbNADP-ME in Arabidopsis may also

29

increase osmotic adjustment and scavenging activity on DPPH and decrease membrane peroxidation.

30

These results suggest that SbNADP-ME overexpression in Arabidopsis increases salt tolerance and

31

alleviates PSII and PSI photoinhibition under salt stress by improving photosynthetic capacity.

32

Key words: SbNADP-ME; photosynthesis; salt stress; sweet sorghum

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Introduction

34

Sweet sorghum (Sorghum bicolor (L.) Moench) is an annual C4 crop that shows a rapid growth rate and

35

high efficiency of biomass accumulation. It is used for ethanol production

36

humans and livestock. Amongst widely grown crop species, sorghum shows one of the highest degrees

37

of stress tolerance. 3,4 Soil salinity is an important global ecological problem. Salinity is not only a major

38

factor contributing to environmental deterioration but is also a major abiotic stress in plant agriculture

39

worldwide. 5,6 It is estimated that 6% of the world’s land and 30% of the world’s irrigated areas already

40

suffer from salinity problems. 7,8 Plant growth is a complex and tightly regulated physiological process;

41

with regard to aboveground plant parts, salt stress induces damage mainly on the photosynthetic

42

apparatus. Photosynthesis is crucial for the survival of plants. Salt stress suppresses photosynthesis and


1,2

and as a food source for

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9,10,11

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cell growth.

44

diffusion through the stomata and the mesophyll 12,13 or altering photosynthetic metabolism. 14 Salt stress

45

can severely disrupt the leaf photosynthetic machinery and chloroplast structure. 2

46

The effects may be direct by decreasing CO2 availability caused by limitation of CO2

Salt stress may inhibit photosynthesis by inducing photoinhibition. Photosystem II (PSII) 15

47

photochemical efficiency of light-adapted leaves is modified by salt stress.

48

reaction centers is decreased by salt stress.

49

under salt stress, which might be attributed to damage to the PSII oxidation side in the oxygen-evolving

50

complex. Alternatively, on the PSII reductive side, electron transport from the primary quinone acceptor

51

(QA) to the secondary quinone acceptor (QB) may be blocked.

52

cucumber seedlings showed that PSII activity was inhibited mainly on the receptor side, in which

53

electron transport from QA to QB was blocked. 19

16,17

The activity of PSII

PSII photochemical efficiency may also be inhibited

18

For example, previous research on

54

Malate dehydrogenase (ME) is widely distributed among eukaryotes and prokaryotes. It is a highly

55

active enzyme and one of the crucial enzymes involved in the metabolism of malic acid in the body. The

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enzyme catalyzes the decarboxylation of malic acid to pyruvate and CO2, while NADP+ is reduced to

57

NADPH.

58

namely NAD-dependent ME (NAD-ME) and NADP-dependent ME (NADP-ME).

59

sorghum is primarily of the NADP-ME type, and NAD-ME activity is not observed.

60

be divided into photosynthetic NADP-ME and non-photosynthetic NADP-ME types according to the

61

specific enzyme function.

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identified in plants. 24 Immunophenotyping results show that this protein is located in the chloroplast of

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vascular sheath cells and is regulated by light. 25

20

MEs can be divided into two major protein types on the basis of their cofactor preference,

23

21

Activity of ME in 22

NADP-ME can

The NADP-ME of maize was the first photosynthetic NADP-ME to be

64

NADP-ME is a critical enzyme of the C4 pathway. The main role of this enzyme is to catalyze

65

oxidative decarboxylation of malic acid to provide CO2 for the photosynthetic carbon fixation of the

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Rubisco enzyme, which is closely associated with photosynthesis.

67

mechanism of the crucial enzymes involved in the C4 pathway remain unclear. Therefore, it is important


26

At present, the regulatory


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to study the mechanism of NADP-ME activity in the C4 pathway. Previous studies have shown that

69

NADP-ME expression in tobacco may lead to changes in stomatal function and plant water use

70

efficiency.

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up-regulated and increased the drought and low temperature tolerance of the plant. 28 NADP-ME activity

72

is also increased in response to drought stress and damage to the photosynthetic apparatus is reduced in

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wheat.

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activity may be enhanced in Arabidopsis (Arabidopsis thaliana), which can affect plant development,

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stress tolerance, and specific diurnal and nocturnal cellular processes.

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OsNADP-ME4 gene of rice enhances the tolerance to salt and drought stresses of transgenic Arabidopsis.

77

32

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previous study we analyzed the transcriptomes of salt-sensitive and salt-tolerant sweet sorghum inbred

79

lines by high-throughput Illumina RNA-sequencing (RNA-seq).

80

treatment, the differentially expressed genes were mainly concentrated in three metabolic pathways,

81

namely light energy capture and absorption, photosynthesis, and carbon fixation and sucrose metabolism.

82

The expression level of NADP-ME in the salt-tolerant inbred line M-81E was extremely high, whereas

83

the expression level of NADP-ME in the salt-sensitive inbred line Roma was unchanged. These findings

84

indicate that NADP-ME may play an important role in carbon fixation and salt-resistance processes. 33

29

23,27

Under drought and low temperature stress, the expression of NADP-ME of wheat was

The expression of NADP-ME may also be induced by salt stress.

28,30

31

Cytosolic NADP-ME2

Over-expression of the

However, the function and regulatory mechanism of NADP-ME in sweet sorghum are unknown. In a

33

The results showed that under salt

85

However, little is known about the regulatory function of sweet sorghum NADP-ME in

86

salt-resistance processes. In this experiment, we isolated the SbNADP-ME gene from sweet sorghum,

87

transformed it into Arabidopsis and investigated the function of SbNADP-ME during salt stress. Results

88

of this study might provide important information for understanding the mechanism between

89

photosynthetic efficiency and salt tolerance.

90

Materials and Methods

91

Plant material, cultivation and treatment


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Seeds of the sweet sorghum inbred line M-81E was used in the study. Sweet sorghum (Sorghum bicolor (L.) Moench) is a variant of sorghum. Dry seeds were stored in a refrigerator at 4°C before use.

94

Arabidopsis accession Col-0 was selected as the wild-type (WT) control. The SALK_064163

95

(nadp-me4), CS855818 (nadp-me4-1), SALK_036898 (nadp-me1), SALK_073818C (nadp-me2),

96

CS833585 (nadp-me2-1), and SALK_139336C (nadp-me3) were obtained from the Arabidopsis

97

Biological Resource Center (Columbus, OH, USA). SALK_064163 is a mutant of At1G79750

98

(AtNADP-ME4), CS855818 is a mutant of At1G79750, SALK_036898 is a mutant of At2G19900

99

(AtNADP-ME1), SALK_073818C is a mutant of At5G11670 (AtNADP-ME2), CS833585 is a mutant of

100

At5G11670, and SALK_139336C is a mutant of At5G25880 (AtNADP-ME3). The homozygosity of

101

each mutant for the T-DNA insertion was verified by PCR.

102

Sweet sorghum seeds of uniform size and no imperfections were selected, and were soaked with

103

water for 10 h. Plants grown under 28°C/20°C (day/night) with a 14 h/10 h (light/dark) photoperiod, and

104

light intensity of about 320 ± 50 μmol m−2 s−1. The relative humidity was 58%–64% and 48%–54%

105

during the day and night, respectively. Plump seeds were selected and sown in plastic pots containing

106

river sand. Each pot was planted with seven individual seedlings. Seedlings were watered with water

107

first, and then watered with half-strength Hoagland nutrient solution after two leaves had developed.

108

When three leaves had formed, the seedlings were watered with full-strength Hoagland nutrient solution.

109

At the four-leaf stage, some seedlings were used to isolate NADP-ME gene and the remaining seedlings

110

were treated with 0, 50, 100, 150, or 200 mM NaCl for 48 h. Leaves from the treated seedlings were

111

stored at −80°C for determination of the expression pattern of the NADP-ME gene. Three replicates were

112

included for each treatment.

113

Seeds of Arabidopsis Col-0 and each mutant were sterilized, washed and sown on half-strength

114

Murashige and Skoog (1/2 MS) medium supplemented with 0, 50, 100, or 150 mM NaCl and stratified

115

for three days at 4°C. After stratification, the seeds were transferred to a culture room and incubated at

116

25°C/20°C (day/night) under a 14 h/10 h (light/dark) photoperiod and light intensity of 150 μmol m−2 s−1.



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The root length of the germinated seeds was measured after germination for 7 days. To study the effect

118

of 100 mM NaCl treatment on the root length of Arabidopsis, we cultured seedlings of Arabidopsis

119

Col-0 and each mutant on 1/2 MS medium lacking supplementary NaCl for 3 days, and then seedlings of

120

similar growth were transferred to 1/2 MS medium supplemented with 50 mM NaCl for further culture.

121

After 3 days, the seedlings were transferred to 1/2 MS medium supplemented with 100 mM NaCl. The

122

primary root length of the seedlings was measured after culture for 3 days.

123

For adult-stage experiments, the Arabidopsis seeds were plated on 1/2 MS medium. After

124

stratification at 4°C for 3 days, the plates were transferred to a growth room. The seedlings were then

125

planted in nutrient soil after 10 days and watered with 1/2 MS solution. After 12 days, plants were

126

treated with 0, 50 or 100 mM NaCl. Physiological parameters, such as photosynthetic parameters,

127

chlorophyll content, chlorophyll fluorescence, and PSI activity, were determined.

128

Cloning the sequence of SbNADP-ME

129

Total RNA was isolated from leaves of sweet sorghum using the Total Plant RNA Extraction Kit

130

(Karroten 1103) in accordance with the manufacturer’s instructions. The full-length NADP-ME gene

131

was

132

(http://ensembl.gramene.org/Sorghum_bicolor/Info/Index). We obtained the sequence of SbNADP-ME

133

using

134

(5′-CAGCGATAACTACAACATTGC-3′).

135

Bioinformatic analysis of SbNADP-ME

determined

the

primers

from

the

ME-Q-5′

Sorghum

bicolor

reference

(5′-CTCTCTCTCTCTCTCTCTCCA-3′)

and

genome

ME-Q-3′

136

The BLASTp and SMART online tools, and the DNAstar, MegAlign, DNAman, and FASTTREE

137

software were used for homology analysis, phylogenetic tree construction, prediction of functional

138

domains and functional classification, and for the analysis of the phylogenetic relationships of the amino

139

acid sequences of SbNADP-ME with NADP-ME genes from other plant species.

140

The expression analysis of SbNADP-ME in sweet sorghum under salt stress

141

To evaluate the expression profiles of SbNADP-ME in leaves, NADP-ME-overexpressing


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142

Arabidopsis lines grown under salt stress conditions were investigated. Quantitative real-time PCR

143

(qPCR) was performed using the primers ME-5 (5′-GGCTTCCATCAATGAGAG-3′) and ME-3

144

(5′-AGTCCAGGTATATGCTTGT-3′) to amplify SbNADP-ME. Amplification of the ACTIN gene was

145

used as an internal control. The internal primer pairs of Actin-F (5′-TGGCATCTCTCAGCACATTCC-3′)

146

and

147

(5′-AAGCTGGGGTTTTATGAATGG-3′) and Actin 3 (5′-TTGTCACACACAAGTGCATCAT-3′),

148

were designed in accordance with the ACTIN nucleotide sequences of sweet sorghum and Arabidopsis,

149

respectively.

150

Plasmid construction and Agrobacterium-mediated transformation of Arabidopsis

Actin-R

(5′-AATGGCTCTCTCGGCTTGC-3′).

and

Actin

5

151

The nucleotide sequence of SbNADP-ME was inserted into the vector pCAMBIA3300 to generate

152

the construct pCAMBIA3300-NADP-ME. The construct was transformed into Arabidopsis using the

153

Agrobacterium-mediated inflorescences infected transformation method. Transgenic plants were

154

identified by PCR with a gene-specific 35S forward primer (5′-GACGCACAATCCCACTATCC-3′) and

155

NADP-ME reverse primer (5′-CAGCGATAACTACAACATTGC-3′) after the first screening with

156

kanamycin. Nineteen individual transgenic lines were obtained. Lines of T10 and T24 from the T3

157

generation were used for further analysis.

158

Detection of the NADP-ME T-DNA insertional Arabidopsis mutants and double mutant

159

To detect homozygous plants, the following gene-specific primers were used for nadp-me4:

160

MEL064163

(5′-AGGGTTAGGAGATCTTGGATG-3′)

161

(5′-CTCCACGTATAGGCCTCTTC-3′);

162

(5′-TCGAAGGTGGGAGGGTTGAG-3′) and ME855818 (5′-CCATCATAGCCATATACTTCT-3′); for

163

nadp-me1:

164

(5′-GCATACCTTCCTCTTTCTTG-3′);

165

(5′-TGGCTATCACTGTACTTAGAC-3′) and ME073818C (5′-TGGTATTTCTGACGTCTACG-3′); for

166

nadp-me2-1:

were

were

MEL036898

MEL833585

for

and nadp-me4-1:

(5′-TCAACGGTAGAGACGGTATGT-3′) for

ME064163

nadp-me2:

and

were

(5′-GCAACTGGCCAGGAATATG-3′)


MEL855818

ME036898 MEL073818C

and

ME833585


for

167

(5′-ACGGTAGTTTCTGTACACA-3′);

168

(5′-CGGAAGAAGATTTGGCTTGT-3′)

169

Plants generating no PCR products with the gene-specific primers were then evaluated using the

170

gene-specific forward primer and the T-DNA insertion left border specific primer LBb1

171

(5′-GCGTGGACCGCTTGCTGCAACT-3′). The nadp-me2 4 double mutant was obtained by crossing

172

the nadp-me2 and nadp-me4 mutants.

173

Analysis of seed germination and root length of Arabidopsis

and

nadp-me3:

Page 8 of 32

ME139336C

were

MEL139336C

(5′-ACGGAAGTTTCTGTAGACA-3′).

174

Seed germination rate was calculated after 1, 3, and 7 days as follows: Germination percentage =

175

(Germinated seed number/germinated total number) × 100. Germination potential is an index of seed

176

germination rate and germination uniformity. Germination potential (%) was calculated as follows:

177

Germination potential = (Germinated seed number at germination peak/Test seed number) × 100. 2 The

178

root length of the different lines was measured after 7 days. The root lengths were measured using a

179

ruler. Three replicates were performed for each treatment.

180

Determination of photosynthetic characteristics

181

The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci)

182

and transpiration rate (Tr) were measured by portable TPS-2 portable photosynthesis system (PP

183

Systems, Amesbury, MA, USA). The measurements were conducted from 10:00 to 12:00 under light

184

intensity of 1000 μmol m−2 s−1. When the measurement was carried out outdoors, the seedlings were

185

allowed to adapt to light for 30 min and the 15 treatments were repeated.

186

Chlorophyll content analysis

187

Chlorophyll (Chl) content was determined following the method we described previously. (17)

188

Arabidopsis leaves (0.2 g fresh weight [FW]) were extracted for 48 h in 80% acetone in the dark.

189

Chlorophyll content was calculated by determining the absorption at 645 and 663 nm with a TU-1810

190

ultraviolet-spectrophotometer. The contents of Chl a and Chl b were calculated as follows: Ca (mg/L) =

191

12.7A663 − 2.69A645; Cb (mg/L) = 22.9A645 − 4.68A663.


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192 193


Determination of chlorophyll fluorescence Chlorophyll fluorescence was detected using a portable fluorometer (FMS2, Hansatech, King’s 34

194

Lynn, UK) using the method of Kooten and Snel.

195

centers open was determined with modulated light. Maximal fluorescence (Fm) with all reaction centers

196

closed was determined by adapting the leaves in darkness for more than 15 min with saturating light of

197

8000 μmol m−2 s−1. The leaf was then illuminated by an actinic light of 500 μmol m−2 s−1. Steady-state

198

fluorescence (Fs) was recorded when the leaf attained steady-state photosynthesis (a time of 300 s

199

illumination is standard for induction of steady-state fluorescence Fs). Maximal fluorescence in the

200

light-adapted state (Fm′) was determined with additional saturating light of 8000 μmol m−2 s−1. PSII

201

maximal photochemical efficiency (Fv/Fm) was expressed as: Fv/Fm = (Fm – Fo)/Fm. PSII actual

202

photochemical efficiency (ΦPSII) was expressed as: ΦPSII = (Fm′ – Fs)/Fm′. Non-photochemical

203

quenching (NPQ) was calculated as: NPQ = Fm/Fm′ – 1. Photochemical quenching (qp) was calculated as

204

qp = (Fm′ − Fs)/(Fm − Fo′). 35

205

Determination of the oxidoreductive activity of PSI

206

Minimal fluorescence (Fo) with all PSII reaction

The oxidoreductive activity of PSI (△I/Io) was expressed by determining the absorption at 820 nm 36

Before measurement of △I/Io in vivo,

207

with a Plant Efficiency Analyzer (PEA Senior, Hansatech).

208

leaves were adapted in the dark for about 30 min. The first reliable measurement time point for

209

fluorescence change was at 20 μs, and the first measurement time point for transmission change was at

210

400 μs. The time constant used for the transmission measurements was 100 μs. The light intensity used

211

for the transmission measurements was 3000 μmol m−2 s−1 and was produced by four 650 nm

212

light-emitting diodes (LEDs). The far-red source was a QDDH73520 LED (Quantum Devices Inc.,

213

Barneveld, WI, USA) filtered at 720 ± 5 nm. The modulated (33.3 kHz) far-red measuring light was

214

provided by an OD820 LED (Opto Diode Corp., Newbury Park, CA, USA) and filtered at 830 ± 20 nm.

215

Command execution, such as turning on and off the LEDs, took approximately 250 μs. Commands for

216

activating the red light and starting the measurement were synchronized to correct for the delay; for the



Page 10 of 32

217

far-red light, there was a 250 μs delay between turning on the far-red light and initiation of

218

measurement.

219

Fresh mass and dry mass of seedlings

220

The plant material was rinsed in distilled water. Water on the plant was then absorbed by tissue

221

paper. The seedlings were weighed to determine the fresh mass (FM). The dry mass (DM) was measured

222

after drying the fresh material at 70°C for 4 days. For each treatment, three replicates were performed.

223

Quantitative real-time PCR analysis of salt-responsive genes

224

We evaluated the expression profiles of nine salt-responsive genes (KIN1, RD29B, RD22, P5CS1,

225

GSTU5, SOS1, NHX1, SOD, and APX) in WT, transgenic Arabidopsis plants, and T-DNA mutant

226

Arabidopsis lines grown under salt stress. The relative transcript level of each gene was determined by

227

(qPCR) in Arabidopsis seedlings that were treated with 100 mM NaCl for 48 h. The qPCR analysis was

228

performed with the primers as follows: KIN1: S: 5′-AAGAATGCCTTCCAAGCCGGTCAG-3′ and A:

229

5′-TACACTCTTTCCCGCCTGTTGTGC-3′; RD29B: S: 5′-AGAAGGAATGGTGGGGAAAG-3′ and A:

230

5′-CAACTCACTTCCACCGGAAT-3′; RD22: S: 5′-ATAATCTTTTGACTTTCGATTTTACCG-3′ and

231

A: 5′-CTTGGACGTTGGTACTTTTCTCG-3′; AtP5CS1: S: 5′-TAGCACCCGAAGAGCCCCAT-3′ and

232

A:

233

S:5′-ATGGCTGAGAAAGAAGAAGTGAAGC-3′

234

5′-TTAAGAAGATCTCACTCTCTCTGCC-3′; SOS1: S: 5′-TTCATCATCCTCACAATGGCTCTAA-3′

235

and A: 5′-CCCTCATCAAGCATCTCCCAGTA-3′; NHX1: S: 5′-GGTCTGATAAGTGCGTATG-3′ and

236

A: 5′-GCTCTCCGTTACATTGTG-3′; SOD: S:

237

5′-TAGGACCAGTCAGAGGAAT-3′; and APX: S:

238

5′-GCCACCAGTAACTTCAACME-3′. Amplification of the ACTIN gene was used as an internal

239

control, and the internal primers of Actin 5 (5′-AAGCTGGGGTTTTATGAATGG-3′) and Actin 3

240

(5′-TTGTCACACACAAGTGCATCAT-3′) were designed in accordance with the ACTIN nucleotide

241

sequence of sweet sorghum and Arabidopsis, respectively.

5′-TTTCAGTTCCAACGCCAGTAGA-3′;

AtGSTU5: and

A:

5′-GTATCTCAACAGGACCACAT-3′ and A: 5′-GTATCCACATTGCTCTTAGG-3′ and A:


Page 11 of 32

242


Determination of proline content

243

For proline determination, a standard curve was first developed. Leaf samples (0.5 g) were cut and

244

mixed in a centrifuge tube to which 5 mL of 3% (w/v) aqueous sulfosalicylic acid was added. After

245

incubation at 100oC for 10 min, the solution was centrifuged at 3000 rpm for 5 min to obtain the proline

246

extract. Finally, 4 ml toluene was added to the reaction mixture and absorbance at 520 nm was measured.

247

The proline content was calculated as follows: proline content (μg g−1 FW) = [(μg proline ml−1 × ml

248

toluene)/[(g sample)/5].

249

Assay of scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals

250

Leaf sample (0.5 g) was ground into powder, and 25 mL deionized water was added. After

251

incubation at 100oC for 1 h, 0.5 mmol l−1 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to the

252

mixture and placed in the dark at 37oC for 20 min. The absorption was determined at 514 nm. The

253

parameter EC50 represents the effective concentration at which 50% of the DPPH radicals were

254

scavenged.

255

Measurement of malondialdehyde content

256

Leaf sample (0.4 g) was ground into pulp, and 5 ml of 0.5% (w/v) trichloroacetic acid (TCA) was

257

added to the tube. The mixture was incubated at 100oC for 10 min, and then was placed into ice-cold

258

water for 1 h. After centrifugation at 3000 rpm for 15 min, an equal volume of (w/v) 0.6% TBA was

259

added to the supernatant. The absorbance was measured at 532 and 600 nm. The malondialdehyde

260

(MDA) content was calculated as follows: MDA content (μmol g−1 FW) = (OD532 − OD600) ×

261

supernatant volume (ml)/155 × sample (g).

262

Statistical analysis

263

Data were transformed (arcsine) prior to the statistical analysis to ensure homogeneity of variance.

264

All analyses were performed with SPSS Version 16.0 (SPSS, Chicago, IL, USA). Multiple comparisons

265

between different conditions were performed using Duncan’s multiple range test at the 0.05 significance

266

level. Figures were drawn by origin data analysis with Sigma Plot 10.0 (SPSS).



267

Results

268

Sequence analysis of SbNADP-ME

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269

The SbNADP-ME of sweet sorghum contained two structural domains (Figure S1A) and a complete

270

open reading frame of 1911 bp consisting of 637 amino acids with a molecular mass of 15.7 kDa (Figure

271

S1B). The highly conserved structural and functional domains were located between 161 and 342 amino

272

acids. The presence of the Malic_M structural domain in the N-terminal region indicated that the

273

NADP-ME protein was a member of the malic acid enzyme family (Figure S1A). To investigate

274

evolutionary relationships among plant NADP-ME genes, a phylogenetic tree of the conserved cyclin

275

box domains was constructed using the neighbor-joining method. The sweet sorghum NADP-ME

276

showed the highest identities with NADP-ME genes from Imperata cylindrica and Zea mays (Figure 1).

277

Expression of SbNADP-ME in sweet sorghum

278

The accumulation of SbNADP-ME mRNA in sweet sorghum seedlings was determined by qPCR

279

analysis. Sweet sorghum seedlings were treated with 0, 50, 100, 150, and 200 mM NaCl. As shown in

280

Figure 2A, the relative expression level of SbNADP-ME increased at first, attained its maximum level at

281

150 mM NaCl treatment, and thereafter decreased. The results revealed that 150 mM NaCl concentration

282

represented mild salt stress for the sweet sorghum line M-81E.

283

Expression of SbNADP-ME in Arabidopsis overexpression lines

284

To understand the role of NADP-ME in the plant response to salt stress, SbNADP-ME was

285

transformed into Arabidopsis (Figure S2A, B). After treatment with 0, 50, or 100 mM NaCl, the relative

286

transcript level of SbNADP-ME in the Arabidopsis overexpression lines T10 and T24 increased

287

significantly (Figure 2B).

288

Screening of homozygous mutants of Arabidopsis

289

To screen the homozygous mutants of nadp-me4 (At1G79750), nadp-me4-1 (At1G79750),

290

nadp-me1 (At2G19900), nadp-me2 (At5G11670), nadp-me2-1 (At5G11670) and nadp-me3 (At5G25880)

291

genomic locus, we amplified and sequenced the nadp-me fragments (Figure S3).


Page 13 of 32

292


Seed germination on saline soil is a frequent condition for terrestrial plants. Therefore, it is of 37

293

practical and theoretical significance to study the effects of salt stress on seed germination.

In the

294

present study, no differences in germination percentage were observed between the WT and mutant

295

Arabidopsis lines under the control condition. Under treatment with NaCl, the germination percentage,

296

germination potential, and root length of nadp-me2 and nadp-me4 were significantly lower than those of

297

the WT (Figure S4). Thus, the nadp-me2 and nadp-me4 mutants were more sensitive to salt treatment

298

than the WT. Therefore, these two salt-sensitive mutants were selected for subsequent experiments.

299

Germination percentage and root length in different Arabidopsis lines under salt stress

300

No significant difference in germination percentage and root length of the WT, Arabidopsis

301

overexpression lines, and the mutants was observed under the control condition (Figure 3A, D, E). The

302

germination percentage and root length of the WT, overexpression lines, and the mutants were all

303

inhibited by salt stress. The degree of inhibition exhibited by the mutants was greater than that of the WT

304

and overexpression lines, especially in the 100 and 150 mM NaCl treatments (Figure 3B, C). After

305

germination for 7 days, the germination percentage of the WT, T10, T24, nadp-me4, and nadp-me2

306

under 100 mM NaCl treatment was 85.6%, 89.7%, 88.3%, 80.1%, and 80.4%, respectively. Under 150

307

mM NaCl treatment, the germination percentage of the WT, T10, T24, nadp-me4, and nadp-me2 was

308

50.3%, 58.4%, 56.3%, 11.1%, and 13.3%, respectively (Figure 3D). Under 100 mM NaCl treatment, the

309

root length of the WT, T10, T24, nadp-me4, and nadp-me2 was decreased by 44.4%, 37.1%, 37.7%,

310

64.5%, and 60.8%, respectively. Under 150 mM NaCl treatment, the root length of the WT, T10, T24,

311

nadp-me4, and nadp-me2 decreased 87.7%, 74.6%, 75.1%, 98.6%, and 97.7%, respectively (Figure 3E).

312

These results indicated that overexpression of SbNADP-ME enhanced germination of Arabidopsis under

313

salt stress.

314

Effect of salt stress on photosynthetic parameters at the seedling stage

315

No significant difference in the net photosynthetic rate (Pn), intercellular CO2 concentration (Ci),

316

transpiration rate (Tr), and stomatal conductance (Gs) of WT, T10, T24, nadp-me4, nadp-me2 and



Page 14 of 32

317

nadp-me2 4 was observed in the absence of NaCl treatment (Table 1). Under treatment with 100 mM

318

NaCl, Pn, Ci, Tr, and Gs of T10 and T24 were significantly higher than those of the WT, nadp-me4,

319

nadp-me2, and nadp-me2 4 (Table 1). Under 100 mM NaCl treatment, the Pn of the WT, T10, T24,

320

nadp-me4, nadp-me2, and nadp-me2 4 decreased by 44.8%, 20.2%, 21.3%, 54.1%, 55.1%, and 63.3%,

321

respectively (Table 1). The Ci of the WT, T10, T24, nadp-me4, nadp-me2, and nadp-me2 4 decreased by

322

15.8%, 10.5%, 10.0%, 20.9%, 21.4%, and 26.7%, respectively (Table 1). The Tr of the WT, T10, T24,

323


324

respectively (Table 1). The Gs of the WT, T10, T24, nadp-me4, nadp-me2, and nadp-me2 4 decreased by

325

39.3%, 24.5%, 28.3%, 49.3%, 50.3%, and 62.7%, respectively (Table 1). Thus, the photosynthetic

326

capacity of T10 and T24 was enhanced by overexpression of SbNADP-ME under salt stress.

327

Effect of salt stress on chlorophyll content in Arabidopsis lines

328

The content of Chl a and b decreased significantly under 100 mM NaCl treatment (Table 1). The

329

Chl a content of the WT, T10, T24, nadp-me4, nadp-me2, and nadp-me2 4 decreased by 21.4%, 15.3%,

330

13.9%, 26.3%, 28.2%, and 34.1%, respectively (Table 1). The Chl b content of the WT, T10, T24,

331


332

respectively (Table 1).

333

PSI and PSII activity in Arabidopsis lines under salt stress

334

No significant differences in Fo, Fv/Fm, 1−qP, NPQ, ΦPSII, and ΔI/Io among the WT, T10, T24,

335

nadp-me4, nadp-me2, and nadp-me2 4 were observed under 0 mM NaCl treatment (Table 1). However,

336

under salt treatment, the Fo, 1−qp, and NPQ of the WT, T10, T24, nadp-me4, nadp-me2, and nadp-me2 4

337

were significantly increased compared with those of the controls (Table 1), whereas Fv/Fm, ΦPSII, and

338

ΔI/Io decreased under salt stress (Table 1). Under 100 mM NaCl treatment, the Fo of the WT, T10, T24,

339

nadp-me4, nadp-me2, and nadp-me2 4 increased by 23.1%, 6.3%, 11.5%, 72.0%, 57.8%, and 81.7%,

340

respectively; the 1−qp of the WT, T10, T24, nadp-me4, nadp-me2, and nadp-me2 4 increased by 33.7%,

341

13.1%, 16.9%, 41.3%, 35.6%, and 50.0%, respectively; the NPQ of the WT, T10, T24, nadp-me4,


Page 15 of 32


342

nadp-me2, and nadp-me2 4 increased by 63.5%, 30.7%, 42.9%, 100.6%, 99.3%, and 112.0%,

343

respectively. The ΦPSII in the WT, T10, T24, nadp-me4, nadp-me2, and nadp-me2 4 decreased by

344

14.9%, 7.0%, 5.1%, 28.1%, 29.3%, and 34.6%, respectively. The Fv/Fm of Arabidopsis overexpression

345

lines was not significantly affected by salt stress, but it decreased by 6.1%, 13.1%, 12.8%, and 23.5% in

346

the WT, nadp-me4, nadp-me2, and nadp-me2 4, respectively. The PSI activity of the WT, T10, T24,

347


348

respectively.

349

Effect of salt stress on fresh and dry mass at the seedling stage

350

Fresh and dry mass of the WT, Arabidopsis overexpression lines, and the mutants significantly

351

decreased under salt treatment. Under 100 mM NaCl treatment, the FM of the WT, T10, T24, nadp-me4,

352

nadp-me2, and nadp-me2 4 decreased 30.9%, 19.0%, 15.2%, 49.1%, 46.2%, and 58.3%, respectively

353

(Table 1). The DM of the WT, T10, T24, nadp-me4, nadp-me2, and nadp-me2 4 decreased 38.2%, 25.6%,

354

12.9%, 51.7%, 48.6%, and 57.7%, respectively (Table 1).

355

Changes in proline and MDA content, and scavenging ability on DPPH radicals under salt stress

356

No significant difference in proline content was observed in the WT, Arabidopsis overexpression

357

lines, and the mutants under the control condition (Figure 4A). Under treatment with 100 mM NaCl,

358

proline content of these plants increased. The Arabidopsis overexpression lines contained a higher

359

proline content, followed by the WT, and the nadp-me2 4 double mutant contained the lowest proline

360

content.

361

Under the control condition, the WT, transgenic lines, and mutants all showed higher scavenging

362

abilities on DPPH radicals (Figure 4B), among which no significant difference was observed. Under

363

treatment with NaCl, the scavenging abilities on DPPH radicals decreased in the WT, transgenic plants,

364

and mutants. However, the scavenging ability remained higher in the Arabidopsis overexpression lines

365

than in the WT and mutants. The scavenging ability on DPPH radicals was lowest in the double mutant



Page 16 of 32

366

nadp-me2 4. The Arabidopsis overexpression lines showed obviously better DPPH scavenging abilities

367

under salt stress than the other plants.

368

Malondialdehyde is the product of membrane peroxidation. Thus, the MDA content may reflect the

369

degree of membrane damage. Under the control condition, the MDA content was low in the WT,

370

transgenic plants, and mutants (Figure 5). After treatment with 100 mM NaCl, the MDA content of the

371

WT, T10, T24, nadp-me4, nadp-me2, and nadp-me2 4 increased 74.1%, 33.23%, 39.4%, 158.2%,

372

145.9%, and 199.7, respectively.

373

Expression level of salt-responsive genes in Arabidopsis

374

To detect whether selected stress-related genes were affected by SbNADP-ME, we determined the

375

relative transcript levels of KIN1, RD29B, RD22, P5CS1, GSTU5, SOS1, NHX1, SOD, and APX in the

376

WT, Arabidopsis overexpression lines and mutants under 100 mM NaCl treatment for 48 h (Figure 6).

377

KIN1, RD29B, and RD22 were salt-related marker genes. P5CS1 is a gene associated with osmotic

378

substance synthesis. GSTU5, SOD, and APX are genes associated with oxidation. SOS1 and NHX1 are

379

genes associated with ion transport across membrane. The transcription of all of the genes was promoted

380

by overexpression of SbNADP-ME and was inhibited by deletion of SbNADP-ME.

381

Discussion

382

Sweet sorghum is primarily grown to produce sugar for syrup and is often used in animal feed. 38 It

383

is also a suitable crop for growing on saline and alkali land with high yield. Interestingly, previous

384

research has shown that the brix of salt-tolerant sweet sorghum remains stable or is even increased by

385

salt stress. The brix of salt-sensitive species, however, decreases under salt stress.

386

the main source of carbon and energy in the sink tissues is sucrose. The accumulation of stalk sugar in

387

sweet sorghum depends on the synthesis and accumulation of photosynthetic products.

388

function of NADP-ME is to catalyze oxidative decarboxylation of malic acid to provide CO2 for the

389

photosynthetic carbon fixation of Rubisco enzyme. 26 Many studies have shown that salt stress results in

390

the expression of NADP-ME, which is resistant to salt damage. 28 In a previous study we showed that the


39

In sweet sorghum,

33

The main

Page 17 of 32


391

expression of the gene encoding NADP-ME is extremely enhanced by salt stress in the salt-tolerant

392

sweet sorghum line M-81E.

393

line M-81E, which encoded a protein of 637 amino acids (Figure S1A). Amino acid sequence analysis

394

revealed that the protein contained the conserved structural (Figure S1B) and functional domains and

395

showed the highest homology with NADP-ME proteins from Imperata cylindrica and Zea mays (Figure

396

1). Analysis of SbNADP-ME transcripts in the leaves of M-81E under different salt treatments showed

397

that the highest transcript level was attained under 150 mM NaCl treatment (Figure 2A). This finding

398

suggested that the transcription of SbNADP-ME in line M-81E may be induced by NaCl treatment. We

399

screened and identified Arabidopsis overexpression lines and salt-sensitive mutants (Figure S2A, B and

400

S3). nadp-me4 is a mutant of At1G79750 and nadp-me2 is a mutant of At5G11670. NADP-ME encoded

401

by At1G79750 is localized in the chloroplast, and is expressed throughout the plant as well as during

402

embryogenesis and germination. NADP-ME encoded by At5G11670 may be involved in malic acid

403

metabolism and may play a role in oxidization in the pentose phosphate pathway by cytoplasmic

404

enzymes. At present, most studies of these two genes have focused on plant development and

405

photosynthetic pathways, and little is known on their salt tolerance.

33

Therefore, in this study, we isolated the cDNA of SbNADP-ME from the

406

The germination of seeds is a prerequisite for the normal growth and development of plants in

407

saline-alkali soils and is also the most critical stage in a plant’s life. 37 In the present study, we observed

408

that the germination percentage, germination potential, and root length of the WT, Arabidopsis

409

overexpression lines, and the T-DNA mutants were inhibited under 50, 100, and 150 mM NaCl treatment

410

(Figure S4). The degree of inhibition in the WT was significantly higher than that of the overexpression

411

lines, but lower than that of the mutants (Figure 3). These results suggested that overexpression of

412

SbNADP-ME may increase plant salt tolerance to a certain extent at the seed germination stage.

413

The level of SbNADP-ME transcripts in the leaves of Arabidopsis overexpression lines was

414

enhanced with the increase in NaCl concentration and attained the highest level under 100 mM NaCl in

415

the seedling stage (Figure 2B). Furthermore, the photosynthetic physiological index, chlorophyll content,



Page 18 of 32

416

fluorescence parameters, and seedling biomass of the WT and overexpression lines were also determined

417

under 100 mM NaCl treatment (Table 1). Under salt treatment, a plant regulates the stomata to ensure

418

high photosynthetic efficiency, which is an anti-stress mechanism of the plant.

419

enzyme in the C4 pathway, which catalyzes the photosynthetic efficiency of plants by catalyzing the

420

decarboxylation of malic acid to provide CO2 for the photosynthetic carbon fixation of Rubisco enzyme.

421

41

422

photosynthesis.

423

related to salt tolerance.

424

salt stress and the rice NADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.

425

present experiment indicated that overexpression of SbNADP-ME in Arabidopsis enhanced salt tolerance

426

by increasing the photosynthetic efficiency. In addition, the photosynthetic efficiency of the nadp-me4

427

and nadp-me2 mutants was lower than that of the WT Arabidopsis. The higher photosynthetic efficiency

428

of overexpression lines (Table 1) alleviated PSII and PSII photoinhibition (Table 1) during salt stress. In

429

the current study, seed germination, seedling root length, photosynthetic parameters, chlorophyll content,

430

and PSI and PSII activity of the nadp-me4, nadp-me2, and nadp-me2 4 mutants decreased significantly

431

under salt stress compared with the WT and Arabidopsis overexpression lines, indicating that

432

At1G79750 and At5G11670 may be involved in salt tolerance. The decrease in these parameters was

433

most severe in the double mutant nadp-me2 4, which indicated a partial redundancy effect may be at

434

play between AtNADP-ME4 and AtNADP-ME2 in Arabidopsis.

435

40

NADP-ME is a key

Under drought stress, wheat showed increased activity of NADP-ME and reduced damage to 29

Salt induces the expression of NADP-ME in leaves of Aloe vera L., which is closely 42

NADP-ME gene expression and protein activity in rice were up-regulated by 30

The

During seedling development, photosynthesis is a critical factor in survival. 43,49 The photosynthetic 7,43

436

parameters of Pn, Ci, Tr and Gs reflect the plant photosynthetic capability.

437

environments stress by stomatal closure, which reduces CO2 availability in the chloroplasts,

438

progressively decreasing photosynthesis and photosynthetic capacity. In the present study, upon

439

exposure of Arabidopsis to salt stress, Pn, Ci, Tr and Gs declined (Table 1). Photosynthetic parameters

440

decreased to lesser degrees in the Arabidopsis overexpression lines and more severely in the mutants


Plants respond to

Page 19 of 32


441

than those in the WT (Table 1). For more than 10 years researchers in laboratories around the world have

442

attempted to improve photosynthesis and crop yield by introducing a single-cell C4-cycle into C3 plants

443

using a transgenic approach.

444

the key enzymes of the C4 cycle in rice, potato, and tobacco. The overexpression of C4-cycle enzymes in

445

transgenic C3 plants has been shown to improve C3 photosynthesis. 31,44 The present results also showed

446

that the overexpression of the C4 sweet sorghum NADP-ME improved salt tolerance of the C3 plant

447

Arabidopsis by influencing photosynthesis.

44

In the meantime, there has been substantial progress in overexpressing

448

Chlorophyll is an important indicator of plant photosynthetic capacity, but also one of the main

449

physiological indicators of plant salt tolerance. Under salt stress conditions, the plant chlorophyll content

450

is usually reduced.

451

transfer in photosynthesis, regulating light absorption, transition, and distribution. 17 Chlorophyll content

452

can reflect the photosynthesis capability to some extent. Under exposure to salt stress, the Chl a and b

453

content declined in Arabidopsis. In addition, the Chl content decreased less in the Arabidopsis

454

overexpression lines and more strongly in the mutants than that of the WT (Table 1). Higher Chl content

455

can result in higher photochemical efficiency of PSII in NADP-ME overexpression lines (Table 1). This

456

finding revealed that light energy absorbed by the light-harvesting complex was higher in the

457

overexpression lines and lower in the mutants relative to that in the WT, which resulted in the different

458

degrees of PSII photoinhibition.

459

45

Chlorophyll a molecules are critical components for light-harvesting and electron

Changes in Fo depend on the dominant factor between energy dissipation and damage to PSII; 46

460

inactivation or damage to PSII causes the increase in Fo.

We showed that Fo increased in the WT,

461

overexpression lines, and the mutants (Table 1). However, Fo increased to a lesser extent in the

462

overexpression lines compared with that in the WT and the mutants. The photoinhibition of PSII is

463

closely associated with the redox state of QA to some extent under stress conditions. 47,50 The parameter

464

1−qp is usually used to estimate the relative redox state of QA in vivo. 48 The present results showed that

465

1−qp of the WT, overexpression lines, and the mutants increased under 100 mM NaCl, and the increase

466

was highest in the mutants (Table 1). This finding shows that the extent of reduction in QA in ACS Paragon Plus Environment


Page 20 of 32

467

overexpression lines was lower under salt stress. The increase in 1−qp was accompanied by an increase

468

in NPQ (Table 1), which increased to the greatest degree in the mutants. We observed that ΦPSII of

469

overexpression lines decreased to a lesser extent than that of the WT and the mutants (Table 1), which

470

suggested that the overexpression lines showed higher photosynthetic capacity. The PSI oxidoreductive

471

activity (ΔI/Io) also decreased in the WT, overexpression lines, and the mutants, but the decrease was

472

less severe in the overexpression lines relative to that in the WT and the mutants (Table 1). The

473

significant decline in ΔI/Io in the mutants might be attributable not only to the limitation of electron

474

acceptors but also probably to damage to the PSI components. These results suggest that the extent of

475

PSII and PSI photoinhibition is decreased by overexpression of SbNADP-ME, and as a result, the FM

476

and DM are higher (Table 1).

477

Plants may accumulate compatible osmolytes under environmental stress. Proline is an important

478

osmolyte during tolerance to abiotic stress because it can maintain redox balance and protect protein

479

structures. In the current study, proline content in the WT, transgenic plants, and mutant lines increased

480

under salt stress (Figure 4A). The increase was highest in transgenic plants. This finding indicated that

481

overexpression of SbNADP-ME may increase osmotic adjustment in Arabidopsis. The DPPH radical is a

482

stable nitrogen-based free radical. The present results showed that the scavenging abilities on DPPH

483

radicals of the WT, transgenic plants, and mutant lines decreased under salt stress (Figure 4B), but the

484

scavenging ability decreased the least in transgenic plants. This finding suggests that overexpression of

485

SbNADP-ME can increase scavenging activity on DPPH under salt stress. Environmental stress can lead

486

to accumulation of reactive oxygen species, which causes oxidative damage to cells and generation of

487

MDA as an end product of membrane peroxidation. In the present study, MDA content increased under

488

salt stress (Figure 5). The MDA content increased to the least degree in the transgenic plants. This

489

finding suggests that overexpression of SbNADP-ME may decrease the extent of membrane

490

peroxidation.

491

In this study, we also detected the expression of stress responsive genes (Figure 6). We found that


Page 21 of 32


492

SOS1, responsible for ion efflux on plasma membrane, NHX1, responsible for ion influx on vacuole

493

membrane, were both up-regulated in the overexpressed lines and down-regulated in the mutant lines.

494

This suggests that overexpression of SbNADP-ME could help to keep less Na+. The expression of P5CS1,

495

GSTU5, SOD and APX were also promoted by overexpression of SbNADP-ME and inhibited by the

496

deletion of SbNADP-ME, which showed that SbNADP-ME might play a positive regulatory role in

497

peroxide stress and reduce ROS damage by overexpression in Arabidopsis. These results suggest that

498

SbNADP-ME might regulate osmotic stress and ionic stress related pathways by changing the expression

499

of some associated genes.

500

In conclusion, we demonstrated that the expression of NADP-ME in the sweet sorghum line M-81E

501

was activated by salt stress. Overexpression of SbNADP-ME increased the photosynthetic capacity in

502

Arabidopsis under salt stress. The increase in photosynthetic efficiency may protect the photosynthetic

503

apparatus and maintain membrane function under salt treatment. These findings provide information

504

valuable for bioengineering of plant fitness and present insight into the molecular mechanisms

505

underlying photosynthetic efficiency and salt tolerance.

506

Authors’ contributions

507

NS and YYG wrote this manuscript; YYG, YSS, HXZ and JRG performed experiments; YSS and YZ

508

collected data and carried out all analyses; NS conceptualized the idea and revised the manuscript.

509

Acknowledgements

510

We are grateful for financial support from Shandong Natural Science Foundation (ZR2016JL028), Major

511

Program of Shandong Provincial Natural Science Foundation (2017C03), the NSFC (National Natural

512

Science Research Foundation of China) (31300205).

513

Thanks professor Xinqi Gao of Shandong Agricultural University for helping us make double knock out

514

mutant of NADP-ME.

515

We thank Robert McKenzie, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for

516

editing the English text of a draft of this manuscript.

517 ACS Paragon Plus Environment


518

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11. Sui, N.; Tian, S.; Wang, W., et al. Overexpression of Glycerol-3-Phosphate Acyltransferase from Suaeda salsa Improves Salt Tolerance in Arabidopsis Frontiers in plant science 2017, 8: 1-14 12. Flexas, J.; Bota, J.; Loreto, F., et al. Diffusive and metabolic limitations to photosynthesis under drought and salinity in C3 plants Plant Biology 2004, 6(03): 269-279 13. Flexas, J.; DIAZ-ESPEJO, A.; GalmES, J., et al. Rapid variations of mesophyll conductance in response to changes in CO2 concentration around leaves Plant, Cell & Environment 2007, 30(10): 1284-1298 14. Lawlor, D. W.; Cornic, G. Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants Plant, Cell & Environment 2002, 25(2): 275-294 15. Baker, N. R. A possible role for photosystem II in environmental perturbations of photosynthesis Physiologia Plantarum 1991, 81(4): 563-570 16. Murata, N.; Takahashi, S.; Nishiyama, Y., et al. Photoinhibition of photosystem II under environmental stress Biochimica et Biophysica Acta (BBA)-Bioenergetics 2007, 1767(6): 414-421 17. Sui, N.; Han, G. Salt-induced photoinhibition of PSII is alleviated in halophyte Thellungiella halophila by increases of unsaturated fatty acids in membrane lipids Acta physiologiae plantarum 2014, 36(4): 983-992 18. Nishiyama, Y.; Allakhverdiev, S. I.; Murata, N. A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II Biochimica et Biophysica Acta (BBA)-Bioenergetics 2006, 1757(7): 742-749 19. Shu, S.; Sun, J.; Guo, S., et al. Effects of Exogenous Putrescine on PSⅡ Photochemistry and Ion Distribution of Cucumber Seedlings under Salt Stress Acta Horticulturae Sinica 2010, 7: 007 20. Bologna, F. P.; Andreo, C. S.; Drincovich, M. F. Escherichia coli malic enzymes: two isoforms with substantial differences in kinetic properties, metabolic regulation, and structure Journal of bacteriology 2007, 189(16): 5937-5946 21. Fukuda, W.; Sari Ismail, Y.; Fukui, T., et al. Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 Archaea 2005, 1(5): 293-301 22. Majeran, W.; van Wijk, K. J. Cell-type-specific differentiation of chloroplasts in C4 plants Trends in plant science 2009, 14(2): 100-109 23. Drincovich, M. F.; Casati, P.; Andreo, C. S. NADP-malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways Febs Letters 2001, 490(1-2): 1-6


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24. Rothermel, B. A.; Nelson, T. Primary structure of the maize NADP-dependent malic enzyme Journal of Biological Chemistry 1989, 264(33): 19587-19592 25. Maurino, V. G.; Drincovich, M. F.; Casati, P., et al. NADP-malic enzyme: immunolocalization in different tissues 34 plant maize and the C3 plant wheat Journal of Experimental Botany 1997, 48(3): 799-811 26. Drincovich, M. F.; Lara, M. V.; Andreo, C. S., et al. C4 decarboxylases: different solutions for the same biochemical problem, the provision of CO 2 to Rubisco in the bundle sheath cells C4 Photosynthesis and Related CO2 Concentrating Mechanisms. Springer Netherlands 2010, 277-300 27. Laporte, M. M.; Shen, B.; Tarczynski, M. C. Engineering for drought avoidance: expression of maize NADP-malic enzyme in tobacco results in altered stomatal function Journal of Experimental Botany 2002, 53(369): 699-705 28. Fu, Z. Y.; Zhang, Z. B.; Hu, X. J., et al. Cloning, identification, expression analysis and phylogenetic relevance of two NADP-dependent malic enzyme genes from hexaploid wheat Comptes Rendus Biologies 2009, 332(7): 591-602 29. Hýsková, V. D.; Miedzińska, L.; Dobra, J.; Vankova, R.; Ryšlavá, H. Phosphoenolpyruvate carboxylase, NADP-malic enzyme, and pyruvate, phosphate dikinase are involved in the acclimation of Nicotiana tabacum L. to drought stress Journal of plant physiology 2014, 171(5): 19-25 30. Cheng, Y.; Long, M. A cytosolic NADP-malic enzyme gene from rice (Oryza sativa L.) confers salt tolerance in transgenic Arabidopsis Biotechnology Letters 2007, 29(7): 1129-1134 31. Badia, M. B.; Arias, C. L.; Tronconi, M. A., et al. Enhanced cytosolic NADP-ME2 activity in A. thaliana affects plant development, stress tolerance and specific diurnal and nocturnal cellular processes Plant Science 2015, 240: 193-203 32. Chen, L.; Tsugama, D.; Takano, T., et al. Rice (Oryza sativa L.) OsNADP-ME4 gene responds to adversity stresses Cell Biology and Biophysics 2015, 4: 1-7 33. Sui, N.; Yang, Z.; Liu, M., et al. Identification and transcriptomic profiling of genes involved in increasing sugar content during salt stress in sweet sorghum leaves BMC genomics 2015, 16(1): 534 34. Kooten, O.; Snel, J. F. The use of chlorophyll fluorescence nomenclature in plant stress physiology Photosynth Res 1990, 25:147-150 35. Schreiber, U.; Bilger, W.; Neubauer, C. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis Ecophysiol. Photosynth. 1994, 100: 49-70 36. Schansker, G.; Srivastava, A.; Strasser, R. J. Characterization of the 820-nm transmission signal paralleling the chlorophyll a fluorescence rise (OJIP) in pea leaves Funct. Plant Biol. 2003, 7: 785-796 37. Misra, N.; Dwivedi, U. N. Genotypic difference in salinity tolerance of green gram cultivars Plant Science 2004, 166(5): 1135-1142 38. Almodares, A.; Hadi, M. R. Production of bioethanol from sweet sorghum: A review African Journal of Agricultural Research 2009, 4(9): 772-780 39. Vasilakoglou, I.; Dhima, K.; Karagiannidis, N.; Gatsis, T. Sweet sorghum productivity for biofuels under increased soil salinity and reduced irrigation Field Crop Res 2011, 120(1): 38-46 40. Gill, S. S.; Tuteja, N. Polyamines and abiotic stress tolerance in plants Plant signaling & behavior 2010, 5(1): 26-33 41. Edwards, G. E.; Huber, S. C. The C4 pathway The biochemistry of plants: a comprehensive treatise 2014, 8: 237-281 42. Sun, S. B.; Shen, Q. R.; Wan, J. M., et al. Induced expression of the gene for NADP-malic enzyme in leaves of Aloe vera L. under salt stress Acta Biochimica et Biochimica et Biophysica Sinaca-Chinese Edition 2003, 35(5): 423-429 43. Chaves, M. M.; Pereira, J. S.; Maroco, J., et al. How plants cope with water stress in the field? Photosynthesis and growth Annals of botany 2002, 89(7): 907-916 44. Häusler, R. E.; Hirsch, H. J.; Kreuzaler, F., et al. Overexpression of C4‐cycle enzymes in transgenic C3 plants: a biotechnological approach to improve C3‐photosynthesis Journal of Experimental Botany 2002, 53(369): 591-607 45. Fall, D.; Diouf, D.; Neyra, M., et al. Physiological and biochemical responses of acacia seyal (Del.) seedlings under salt stress conditions Journal of Plant Nutrition 2009, 32(7): 1122-1136 46. Chen, H. X.; Li, W. J.; An, S. Z., et al. Characterization of PSII photochemistry and thermostability in salt-treated Rumex leaves Journal of plant physiology 2004, 161(3): 257-264 47. Xu, C. C.; Jeon, Y. A.; Lee, C. H. Relative contributions of photochemical and non‐photochemical routes to excitation energy dissipation in rice and barley illuminated at a chilling temperature Physiologia Plantarum 1999, 107(4): 447-453



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48. Sui, N. Photoinhibition of Suaeda salsa to chilling stress under low irradiance is related to energy dissipation and water-water cycle Photosynthetica 2015, 53(2): 207-212 49. Lee, H. J.; Choi, J.; Lee, S. M., et al. Photosynthetic CO2 conversion to fatty acid ethyl esters (FAEEs) using engineered cyanobacteria Journal of agricultural and food chemistry 2017, 65(6): 1087-1092 50. Nain-Perez, A.; Barbosa, L. C.; Maltha, C. R., et al. Tailoring Natural Abenquines To Inhibit the Photosynthetic Electron Transport through Interaction with the D1 Protein in Photosystem II Journal of agricultural and food chemistry 2017, 65(51): 11304-11311

519


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520


Legends to figures Figure 1 Phylogenetic relationships of amino acid residue sequences of the conserved cyclin box domains of sweet sorghum SbNADP-ME and NADP-ME genes from Oryza brachyantha (Locus ID: 102719626),

Oryza

sativa

(acc.

no.

AB053295),

Brachypodium

distachyum

(acc.

no.BRADI_2g33450), Triticum aestivum (acc. no. AK455007.1), Imperata cylindrica (acc. no. FN397879.1), Zea mays (acc. no. GRMZM2G085019), Paspalum paniculatum (acc. no. AJ318587), Setaria viridis (acc. no. FN397881.1), Arabidopsis thaliana (acc. no. At1G79750), Cyrtococcum patens (acc. no. FN397866.1), Echinochloa crusgalli (acc. no. FJ603315.1). Figure 2 Relative transcript levels of SbNADP-ME in sweet sorghum (A) and in transgenic Arabidopsis (B) measured by qPCR. Total RNA was isolated from leaves of seedlings in culture. The expression levels were normalized to sweet sorghum actin. Sweet sorghum seedlings were treated with 0, 50, 100, 150, or 200 mM NaCl. The relative transcript levels of NADP-ME in the Arabidopsis overexpression lines T10 and T24 were determined at 0, 50, and 100 mM NaCl treatment. Values are means ± SD of five measurements for each of five plants (n = 5). Bars with different lower-case letters indicate a significant difference at the P ≤ 0.05 significance level. Figure 3 The phenotype of wild type (WT), transgenic overexpression plants, and T-DNA mutant lines of Arabidopsis treated with 0, 100, and 150 mM NaCl for 7 d (A, B, C). Effect of different levels of NaCl stress on germination percentage (D) and root length (E) of WT, transgenic overexpression plants, and T-DNA mutant lines of Arabidopsis after 7 d. Values are means ± SD of five measurements for each of five plants (n = 5). Bars with different lower-case letters indicate a significant difference at the P ≤ 0.05 significance level. Figure 4 Proline content (A) and scavenging ability (B) of wild type (WT), transgenic overexpression plants, and T-DNA mutant lines of Arabidopsis treated with 0 and 100 mM NaCl for 7 d. Values are means ± SD of five measurements for each of five plants (n = 5). Bars with different lower-case letters indicate a significant difference at the P ≤ 0.05 significance level.



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Figure 5 Malondialdehyde (MDA) content of wild type (WT), transgenic overexpression plants, and T-DNA mutant lines of Arabidopsis treated with 0 and 100 mM NaCl for 7 d. Values are means ± SD of five measurements for each of five plants (n = 5). Bars with different lower-case letters indicate a significant difference at the P ≤ 0.05 significance level. Figure 6 Relative transcript levels of salt-responsive marker genes and function genes in wild type (WT), transgenic overexpression plants, and T-DNA mutant lines of Arabidopsis under salt stress. Relative transcript levels of the genes were determined by qPCR in Arabidopsis seedlings treated with 100 mM NaCl for 48 h. Data are represented as the mean of three measurements ±SD. Supporting Information Available: Figure S1 Structural domain prediction of sweet sorghum SbNADP-ME protein (A); sequence analysis of sweet sorghum SbNADP-ME amino acid sequences (B). Figure S2 Identification of Arabidopsis overexpression lines. Total RNA was isolated from leaves of Arabidopsis seedlings in culture. Basta screening in overexpressing lines of Arabidopsis (A); genomic DNA PCR of overexpression lines (B). Figure S3 Identification of Arabidopsis T-DNA insertion mutant lines. The T-DNA insertion was confirmed using PCR with the indicated primer sets. M represents a 2000 bp marker. A, nadp-me4; B, nadp-me2; C, nadp-me4-1; D, nadp-me2-1; E, nadp-me1; F, nadp-me3. Figure S4 Effect of different NaCl stress on germination percentage, germination potential, and root length of wild type (WT) and T-DNA mutant lines of Arabidopsis. A, Germination percentage after 1 day; B, germination potential after 3 days; C, germination percentage after 7 days; D, root length after 7 days. Values are means ± SD of five measurements for each of five plants (n = 5). Bars with different lower-case letters indicate a significant difference at the P ≤ 0.05 significance level.


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Figure 1

Figure 2



Figure 3

Figure 4


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Figure 5

Figure 6

14 WT T10 T24 nadp-me4 nadp-me2 nadp-me2 4

Relative expression level

12 10 8 6 4 2 0 -2 -4 -6 KIN1

RD29B

RD22

P5CS1

GSTU5

SOS1


NHX1

SOD

APX


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Table 1 Effect of NaCl stress on the Pn, Ci, Tr, Gs, chlorophyll a, b contents, Fo, 1-qp, NPQ, ΦPSII, Fv/Fm, ΔI/Io, fresh mass and dry mass of WT, transgenic Arabidopsis plants and T-DNA mutant Arabidopsis lines for 7 days. Parameters 0 mM

WT 100 mM

0 mM

T10 100 mM

0 mM

T24 100 mM

Pn (μmol m-2 s-1)

2.9±0.17a

1.6±0.14c

3.0±0.12a

2.4±0.09b

3.0±0.12a

Ci (μmol mol-1)

559±8.33a

471±14.42c

576±10.41

516±15.28

a

b

nadp-me4

nadp-me2 100 mM

nadp-me2 4 0 mM 100 mM

0 mM

100 mM

0 mM

2.3±0.09b

3.0±0.21a

1.4±0.15d

2.9±0.16a

1.3±0.06d

3.0±0.24a

1.1±0.09e

570±8.19a

513±14.00b

553±3.46a

438±7.37d

549±6.43a

432±16.86d

562±5.41a

412±8.84e

Tr (mmol m-2 s-1)

1.2±0.02a

0.6±0.05d

1.3±0.04a

0.9±0.04b

1.2±0.03a

0.9±0.06bc

1.2±0.04a

0.5±0.04e

1.2±0.03a

0.5±0.05e

1.2±0.07a

0.4±0.05f

Gs (mmol m-2 s-1)

117±4.54a

71±1.73c

122±2.73a

92±2.65b

120±2.70a

86±3.00b

113±4.53a

57±3.06d

112±5.37a

56±4.62d

118±4.16a

44±3.18e

Chl a content (mg L-1 FW)

4.1±0.12a

3.2±0.23c

4.3±0.16a

3.6±0.15b

4.2±0.12a

3.6±0.12b

4.1±0.18a

3.0±0.15d

4.0±0.14a

2.9±0.23d

4.1±0.17a

2.7±0.18e

Chl b content (mg L-1 FW)

2.2±0.12a

1.9±0.11b

2.3±0.12a

2.1±0.11ab

2.2±0.13a

2.0±0.14ab

2.1±0.16a

1.7±0.17c

2.1±0.14a

1.8±0.13c

2.1±0.08a

1.6±0.15d

Fo

112±4.93f

138±4.04d

114±3.88f

121±3.45e

113±3.22f

126±3.44e

110±5.69f

190±6.25b

111±5.29f

175±6.56c

115±4.21f

209±7.19a

1-qp

0.31±0.00 5f 0.25±0.00 4f 0.53±0.00 5a 0.81±0.01 3a 8.3e-3±1. 33e-4a 0.94±0.02 7a 0.071±5.1 7e-3a

0.42±0.012c

0.31±0.01 1f 0.25±0.00 8f 0.54±0.01 0a 0.83±0.02 1a 8.3e-3±1. 40e-4a 0.96±0.05 3a 0.074±2.2 5e-3a

0.35±0.01 4e 0.33±0.00 8e 0.51±0.01 3b 0.81±0.00 7a 7.5e-3±4. 23e-4b 0.77±0.03 2b 0.055±4.0 0e-3c

0.32±0.01 3f 0.26±0.00 3f 0.54±0.01 4a 0.82±0.02 0a 8.3e-3±1. 79e-4a 0.97±0.03 8a 0.073±2.5 2e-3a

0.38±0.009d

0.31±0.012f

0.31±0.011f

0.42±0.010c

0.25±0.005f

0.25±0.004f

0.50±0.007b

0.51±0.014b

0.52±0.008a

0.52±0.015a

0.37±0.011d

0.79±0.011a

0.81±0.015a

0.81±0.017a

0.70±0.015c

8.1e-3±2.85 e-4a 0.91±0.010a

5.0e-3±2.99 e-4e 0.49±0.044d

0.34±0.01 8d 0.25±0.00 2f 0.52±0.01 0a 0.81±0.01 2a 8.2e-3±2. 43e-4a 0.93±0.01 9a 0.071±2.8 9e-3a

0.51±0.018a

0.37±0.010d

0.44±0.00 6b 0.51±0.00 9b 0.37±0.01 2d 0.70±0.00 7c 4.8e-3±1. 74e-4e 0.45±0.05 5d 0.033±2.5 2e-3e

NPQ Ф PSII Fv/Fm ΔI/Io Fresh mass (g) Dry mass (g)

0.41±0.014c 0.45±0.017c 0.76±0.025b 6.3e-3±1.49 e-4d 0.65±0.044c 0.044±3.12e -3d

b

7.2e-3±2.58 e-4bc 0.82±0.017b 0.064±4.58e -3b

8.2e-3±3.69 e-4a 0.89±0.027a 0.069±3.16e -3a

0.069±3.51e -3a

0.036±4.16e -3e

Values are means ±SD of five measurements for each of five plants (n = 5). Different lowercase letters show significant differences at the P≤0.05 level.


0.53±0.011a 0.34±0.017e 0.62±0.013d 4.2e-3±2.73 e-4f 0.43±0.026d e

0.030±1.77e -3ef

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TOC Graphic



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NADP-Malate Dehydrogenase of Sweet Sorghum ... - ACS Publications

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