Accepted Manuscript

Spermidine-mediated hydrogen peroxide signaling enhances the antioxidant capacityof salt-stressed cucumber roots

Jianqiang Wu, Sheng Shu, Chengcheng Li, Jin Sun, Shirong Guo

PII: S0981-9428(18)30202-X

DOI: 10.1016/j.plaphy.2018.05.002

Reference: PLAPHY 5249

To appear in: Plant Physiology and Biochemistry

Received Date: 9 January 2018

Revised Date: 22 April 2018

Accepted Date: 2 May 2018

Please cite this article as: J. Wu, S. Shu, C. Li, J. Sun, S. Guo, Spermidine-mediated hydrogen peroxidesignaling enhances the antioxidant capacity of salt-stressed cucumber roots, Plant Physiology etBiochemistry (2018), doi: 10.1016/j.plaphy.2018.05.002.

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Spermidine-mediated hydrogen peroxide signaling enhances the 1

antioxidant capacity of salt-stressed cucumber roots 2

Jianqiang Wua, Sheng Shua,b, Chengcheng Lia, Jin Suna,b, Shirong Guoa,b,* 3

a Key Laboratory of Southern Vegetable Crop Genetic Improvement in Ministry of 4

Agriculture, College of Horticulture, Nanjing Agricultural University, Nanjing 5

210095, China 6

b Suqian Academy of Protected Horticulture, Nanjing Agricultural University, Suqian 7

223800, China 8

9

Abstract 10

Hydrogen peroxide (H2O2) is a key signaling molecule that mediates a variety of 11

physiological processes and defense responses against abiotic stress in higher plants. 12

In this study, our aims are to clarify the role of H2O2 accumulation induced by the 13

exogenous application of spermidine (Spd) to cucumber (Cucumis sativus) seedlings 14

in regulating the antioxidant capacity of roots under salt stress. The results showed 15

that Spd caused a significant increase in endogenous polyamines and H2O2 levels, and 16

peaked at 2 h after salt stress. Spd-induced H2O2 accumulation was blocked under salt 17

stress by pretreatment with a H2O2 scavenger and respective inhibitors of cell wall 18

peroxidase (CWPOD; EC: 1.11.1.7), polyamine oxidase (PAO; EC: 1.5.3.11) and 19

NADPH oxidase (NOX; EC: 1.6.3.1); among these three inhibitors, the largest 20

decrease was found in response to the addition of the inhibitor of polyamine oxidase. 21

Abbreviations: PAs, polyamines; H2O2, hydrogen peroxide; Put, putrescine; Spd, spermidine; Spm, spermine; ODC, ornithine decarboxylase; ADC, arginine decarboxylase; SAM, S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase; DAO, copper-containing amine oxidase; PAO, polyamine oxidase; NADPH oxidases, NOX; CWPOD, cell wall peroxidases; MGBG, methylglyoxal bis-guanylhydrazone; 1,8-DO, 1,8-diaminooctane; MDA, malondialdehyde; O2

•−, superoxide anion; DPI, diphenyleneiodonium chloride; SHAM, salicyhydroxamic; DMTU, dimethylthiourea; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase. * Corresponding author. Email address: srguo@njau.edu.cn.

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In addition, we observed that exogenous Spd could increase the activities of the 22

enzymes superoxide dismutase (SOD; EC: 1.15.1.1), peroxidase (POD; EC: 1.11.1.7) 23

and catalase (CAT; EC: 1.11.1.6) as well as the expression of their genes in 24

salt-stressed roots, and the effects were inhibited by H2O2 scavengers and polyamine 25

oxidase inhibitors. These results suggested that, by regulating endogenous 26

PAs-mediated H2O2 signaling in roots, Spd could enhance antioxidant enzyme 27

activities and reduce oxidative damage; the main source of H2O2 was polyamine 28

oxidation, which was associated with improved tolerance and root growth recovery of 29

cucumber under salt stress. 30

31

Key words: Spd; cucumber; salt stress; H2O2 signaling; antioxidant capacity; gene 32

expression 33

34

1. Introduction 35

Salinity is one of the most common environmental stresses and affects more than 6% 36

of the world’s total land area [1, 2]. High salinity reduces the water potential of the 37

external environment and causes functional changes in cell membranes, which results 38

in plants being unable to take up enough water and causes increased 39

electrolyte leakage. Moreover, the ability of a plant to take up other ions is negatively 40

affected by salt stress, thus inhibiting plant growth [1, 3]. Therefore, considering the 41

increasing population and food demand, enhancing stress tolerance and alleviating 42

stress damage are necessary for crop yield improvements [4]. 43

In recent years, there have been numerous reports about the roles of polyamines 44

(PAs) in regulating the response of higher plants to salt stress. PAs are small 45

aliphatic nitrogenous compounds that widely exist in prokaryotes and eukaryotes. The 46

most abundant PAs in plants are putrescine (Put), spermidine (Spd) and spermine 47

(Spm), which exist in free, conjugated and bound forms [5], respectively. Put is 48

synthesized from the conversion of ornithine by ornithine decarboxylase (ODC) or 49

from arginine by arginine decarboxylase (ADC). Put is then converted to Spd by Spd 50

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synthase, and Spd subsequently produces Spm by Spm synthase. The two reactions 51

require aminopropyl provided by decarboxylated S-adenosylmethionine, which is 52

synthesized from S-adenosylmethionine (SAM) by S-adenosylmethionine 53

decarboxylase (SAMDC). The key enzymes of polyamine metabolism are the 54

copper-containing amine oxidase (DAO; EC: 1.4.3.22) and the FAD-containing 55

polyamine oxidase (PAO; EC: 1.5.3.11), which catalyze the oxidation of Put and 56

higher polyamines (Spd and Spm), respectively [5, 6]. 57

Endogenous PA accumulation under stress is considered to be an important aspect 58

of plant responses to stress, and in many cases, Spd is the PA that is most closely 59

associated with salinity tolerance [7]. Owing to their polycationic nature, PAs can 60

bind with phospholipid compounds, which contributes to the stability of the plasma 61

membrane under salt stress [8]. In addition to this kind of direct protective effect, PAs 62

can also enhance stress tolerance via complex signaling systems [9-11], including 63

H2O2 [12], which functions as an important signaling molecule for mediating stress 64

responses [13]. Li et al. [14] found that Put’s protection of hulless barely against 65

UV-B stress occurs via H2S- and H2O2-mediated signaling pathways. In addition, Guo 66

et al. [15] suggested that cold tolerance of Medicago sativa provided by up-regulating 67

polyamine oxidation is mediated by ABA, H2O2 and NO. Additionally, interactions 68

among H2O2, NO and Ca2+ are associated with tolerance to copper stress in Ulva 69

compressa [16]. In addition to PAO, there is evidence for H2O2 production in plants 70

via two other enzymes: nicotinamide adenine dinucleotide phosphate (NADPH) 71

oxidase (NOX; EC: 1.6.3.1) [17] and cell wall peroxidase (CWPOD; EC: 1.11.1.7) 72

[18]. However, different plants- even different parts of the same plant-may exhibit 73

different regulatory mechanisms. Reports of comparisons among these three pathways 74

are limited, and the relationship between PAs and H2O2 in cucumber under salt stress 75

has not yet been addressed. 76

In this study, we examined whether H2O2 is involved in Spd-induced antioxidant 77

defense in cucumber roots under salt stress and investigated the sources of H2O2 78

production, in order to better understand the alleviating effects of PAs on salt stress 79

damage to cucumber seedlings from the perspective of signal tansduction. 80

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81

2. Materials and methods 82

2.1. Plant materials and treatments 83

Cucumber (Cucumis sativus L. cv. Jinyou No. 4, obtained from Tianjin Kernel 84

Cucumber Research Institute, China) seeds were germinated in the dark for 24 h at 85

28±1 �. The germinated seeds were then sown in plastic trays (41×41×5cm) covered 86

with quartz sand and cultured in a greenhouse at Nanjing Agricultural University, 87

where the environmental conditions consisted of 28±1 �/19±1 � (day/night) and a 88

relative humidity of 60-75 %. One-quarter-strength Hoagland nutrient solution was 89

supplied to the seedlings when their cotyledons had expanded, and the nutrient 90

solution concentration was increased to half strength after the expansion of the first 91

true leaf. Relatively uniform seedlings with two fully expanded true leaves were 92

transplanted to plastic containers filled with half-strength Hoagland solution (pH 93

6.5±0.1, electrical conductivity of 2.0-2.2 dS·m-1) that was aerated by an air pump. 94

When the third leaves were fully expanded, the cucumber seedlings were subjected 95

to different treatments: half-strength Hoagland solution containing 75 mM NaCl, 0.1 96

mM Spd, 0.2 mM methylglyoxal bis-guanylhydrazone (MGBG, an inhibitor of 97

SAMDC), 0.2 mM 1,8-diaminooctane (1,8-DO, an inhibitor of PAO), 20 µM 98

diphenyleneiodonium chloride (DPI, an inhibitor of NADPH oxidase), 5 mM 99

salicyhydroxamic acid (SHAM, an inhibitor of cell wall peroxidase), or 5 mM 100

dimethylthiourea (DMTU, a H2O2 scavenger) alone or in combination. The above 101

concentrations were determined based on the results of preliminary experiments. For 102

treatments containing inhibitors or scavengers, the plants were pretreated with those 103

chemicals for 12 h before adding NaCl or Spd to the Hoagland solution. The roots of 104

the control and treated seedlings were sampled after different treatment time points 105

(for the length of time of the different treatments with chemicals, please see each 106

figure legend), immediately frozen in liquid nitrogen and stored at -80 � until 107

subsequent analyses. 108

109

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2.2. Measurement of root growth 110

Except for the fresh and dry weights, all the other growth parameters were 111

measured with a scanner (Expression 1680; Epson, Sydney, Australia) and image 112

analysis software (WinRHIZO; Regent Instruments, Montreal, Canada). For the 113

determination of fresh weight, the cucumber roots were first washed with distilled 114

water, after which the excess water was wiped off followed by weighing of the roots. 115

The roots were then oven dried at 75 � in a forced-air oven to obtain their dry weight 116

after being dried at 115 � for 15 min. 117

118

2.3. Quantification of endogenous polyamines 119

Polyamines were extracted and determined according to the method of Shu et al. 120

[19], with small modifications. First is the polyamines extraction: homogenates were 121

obtained by grinding the harvested root samples in 1.6 mL of cold 5% (w/v) 122

perchloric acid (PCA) with a precooled mortar and pestle. After incubation for 1 h on 123

ice, the homogenates were centrifuged at 12000 g and 4 � for 20 min. The 124

supernatant was then used to determine the free and conjugated polyamines, and the 125

pellet was used to determine the bound polyamines. For free polyamines, 1.4 mL of 126

NaOH (2 M) and 15 µL of benzoyl chloride were added to 0.7 mL of supernatant, 127

after which the solution was first vortexed for 30s and then incubated at 37 � for 30 128

min. To terminate the reaction, the resulting solution and 2 mL of saturated NaCl 129

solution were mixed together. Two milliliters of diethyl ether was then added to the 130

mixed solution for extracting benzoyl polyamines, which were centrifuged for 5 min 131

at 3000 g and 4 �. Finally, 1 mL of diethyl ether phase was pipetted and evaporated to 132

dryness, after which 100 µL of 64 % (v/v) methanol was added to redissolve the 133

benzoyl polyamines. A high- performance liquid chromatography (HPLC) 1200 series 134

system (Agilent Technologies, Santa Clara, CA) with a C18 reversed-phase column 135

(4.6 by 250 mm, 5 µm Kromasil) and a two solvent system including a methanol 136

gradient (36%-64%, v/v) at a flow rate of 0.8 mL·min-1 was used for determining the 137

polyamine content. For the conjugated polyamines, 0.7 mL of the supernatant was 138

hydrolyzed at 110 °C for 18 h in flame-sealed glass ampoules after the supernatant 139

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and 5 mL of 6 M HCl were mixed together and then evaporated at 70 °C. The residue 140

was then resuspended in 5 % PCA. The next steps are same with the second step of 141

polyamines extraction and the steps after that. For the bound polyamines, the pellet 142

was washed four times with 5 % PCA and centrifuged for 5 min at 3000 g. Then the 143

supernatant was discarded and the pellet was suspended in 5 mL of 6 M HCl, 144

following by the steps in accordance with conjugated polyamines determination. 145

Total polyamine content was calculated by summing the three forms of each 146

polyamine. 147

148

2.4. Determination of H2O2 level 149

The method described by Su et al. [20] with modifications was used to analyze 150

H2O2 level. The root samples were homogenized in a precooled mortar by the 151

addition of 2 mL of cold acetone, after which the homogenate was centrifuged at 152

10000 g for 15 min; the resulting supernatant constituted the H2O2 extract. Fifty 153

microliters of 20 % TiCl4 solution and 0.1 mL of strong ammonia were added to 0.5 154

mL of the H2O2 extract, and the solution was mixed by vortexing to produce a 155

peroxide-Ti complex; the supernatant was discarded after the mixed solution was 156

centrifuged at 3000 g for 5 min. The pellet was then washed four times by vortexig in 157

2 mL of cold acetone followed by centrifugation. Afterward, 3 mL of 2 M H2SO4 was 158

added to dissolve the precipitate and the resulting solution was used for 159

spectrophotometric analysis at 415 nm. 160

161

2.5. Determination of malondialdehyde (MDA) content 162

The roots were ground in 5 % trichloroacetic acid (TCA) and the homogenates 163

were centrifuged at 4000 g for 10 min. Two milliliters of 0.67 % thiobarbituric acid 164

(TBA) was then added to 2 mL of the supernatant, and the resulting solution was 165

mixed together. The mixture was heated in a boiling water bath for 30 min and then 166

centrifuged at 3000 g for 15 min after cooling. The absorbance of the supernatant 167

was read at 450, 532 and 600 nm, and the MDA concentration was calculated using 168

the method of Dhindsa et al. [21]. 169

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170

2.6. Determination of the O2•− generation rate 171

The O2•− generation rate was determined by the method of Elstner and Heupel [22]. 172

The homogenate was obtained by grinding root samples in a cold mortar that 173

contained phosphate buffer (0.05 M, pH 7.8) followed by centrifugation at 1000 g 174

for 10 min and a second centrifugation at 15000g for 20 min. The supernatant, 0.05 175

M phosphate buffer, and 1 mM hydroxylamine hydrochloride were then mixed 176

together, after which the solution was incubated at 25 � for 1 h. Seventeen 177

millimolar sulfanilic acid and α- naphthylamine were then added to the mixture, 178

which was subsequently incubated at 25 � for 20 min. The absorbance at 530 nm 179

was ultimately recorded, and the O2•− generation rate was calculated using a 180

NaNO2-based standard curve. 181

182

2.7. Polyamine oxidase activity assays 183

The roots were ground in 1.6 mL of phosphate buffer (0.1 M, pH 6.5) on ice, after 184

which the homogenate was collected. After centrifugation at 10000 g for 20 min, the 185

supernatant was used to determine the PAO activity. The reaction mixture contained 186

0.1 M phosphate buffer, 250 U/ML horseradish peroxidase, 0.5 mM 187

4-aminoantipyrine, 2 mM N,N-dimethylaniline and 0.2 mL of enzyme extract in total 188

volume of 3 mL as described by Mhaske et al. [23]. The reaction was started by 189

adding 0.1 mL of 20 mM Spd or Spm followed by incubation at 25 � for 30 min. 190

The increase in absorbance at 550 nm was recorded, and one unit of enzyme activity 191

was expressed as 0.001 ∆OD550·min-1. 192

193

2.8. Cell wall peroxidase activity assays 194

The preparation of cell walls and the extraction of peroxidase (POD; EC: 1.11.1.7) 195

that was ionically bound to the cell wall was performed as described by Lin and Kao 196

[24]. The roots were homogenized in 2 mL of cold phosphate buffer (0.2 M. pH 6.0) 197

using a mortar and pestle. The supernatant was then removed after centrifugation at 198

13000 g and 4 � for 5 min. The pellet was subsequently washed three times with the 199

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abovementioned phosphate buffer and then collected as a cell wall fraction. 200

Afterward, the pellet was shaken in 1 M NaCl at 30 � for 2 h and then centrifuged at 201

13000 g for 5 min; and the supernatant was subsequently used to detect cell wall 202

POD activity by the guaiacol method. The reaction solution contained 0.2 M 203

phosphate buffer (pH 6.0), 3.5 M guaiacol and 30 % H2O2. The change in absorbance 204

at 470 nm within 40 s was measured by a spectrophotometer after 40 µL of enzyme 205

extract was added to 3 mL of reaction solution to initiate the reaction. One unit of 206

cell wall peroxidase activity was defined as an increase of 0.01 A470·min-1. 207

208

2.9. NADPH oxidase activity assay 209

The NADPH oxidase activity was measured in the root samples with plant 210

NADPH oxidase ELISA assay kit (MEIMIAN, China) according to the 211

manufacturer’s instruction. 212

213

2.10. Antioxidant enzyme activity assay 214

The roots were homogenized in 1.6 mL of 0.05 M cold phosphate buffer (pH 7.8) 215

and the supernatant obtained after the centrifugation of homogenate at 12000 g for 20 216

min was used to determine antioxidant enzyme activity. Superoxide dismutase (SOD; 217

EC: 1.15.1.1) activity was detected by inhibiting the photochemical reduction of 218

NBT [25]. Thirty microliters of enzyme extract was added to a reaction solution that 219

consisted of 0.05 M phosphate buffer, 14.5 mM methionine, 30 µM EDTA-Na2, 2.25 220

mM NBT, and 60 µM riboflavin, and the change in absorbance was recorded at 560 221

nm. One unit of SOD activity was defined as the amount of enzyme that caused 50% 222

inhibition of NBT reduction. The activity of peroxidase (POD) was measured based 223

on the aforementioned methods of assaying the cell wall peroxidase activity, with the 224

exception that the first step of cell wall preparation was omitted. For determination of 225

catalase (CAT; EC: 1.11.1.6) activity, 100 µL of the supernatant and the reaction 226

mixture (0.05 M phosphate buffer containing 30 % H2O2) were mixed together, and 227

CAT activity was calculated as the decrease in absorbance at 240 nm as described by 228

Li et al. [12]. 229

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230

2.11. H2O2 detection by fluorescence microscopy 231

H2O2 was detected by using the H2O2-sensitive, cell-permeable fluorescent probe 232

2’, 7’-dichlo-rodihydrofluorescein diacetate (H2DCFDA; Sigma, USA) according to 233

the methods of Shu et al. [26]. Briefly, the root tips were incubated in 25 µM 234

H2DCFDA dissolved in Tris-HCl (10 mM, pH 6.1) in darkness for 20 min. Afterward, 235

the root tips were rinsed twice with Tris-HCl to remove excessive dye and then 236

observed with a fluorescence microscope (Leica DM 2500; Leica, Germany). 237

238

2.12. Total RNA extraction and quantitative real-time PCR (qRT-PCR) analysis 239

Total RNA was extracted from the cucumber roots using an RNAsimple Total 240

RNA Kit (TIANGEN, China) following the manufacturer’s instructions. For all 241

samples, 1 µg of total RNA was reverse transcribed into cDNA using a SuperScript 242

First-strand Synthesis System for qRT-PCR in accordance with the manufacturer’s 243

instructions (Takara, Japan). The DNA sequences of the tested enzymes were 244

obtained by searching the NCBI database and the Cucumber Genome Database 245

(cucumber.genomics.org.cn), and gene-specific primers were designed with Beacon 246

Designer 7.9 (Premier Biosoft International, CA, USA). The sequences of primer 247

pairs used in this study were presented in Table 1. qRT-PCR was performed on a 248

StepOnePlus™ Real-Time PCR System (Applied Biosystems) with a SYBR® Premix 249

Ex Taq™ II kit (Takara). The reaction mixture contained 2 µL of diluted cDNA, 10 250

µL of SYBR® Premix Ex Taq™ II (2×), 0.8 µL of 10 each specific primer (10 µM)，251

0.4 µL of ROX reference dye and 6 µL of ddH2O in 20 µL total volume. Reactions 252

were conducted in three biological replicates and the thermocycler conditions were: 253

95 for 5 min,℃ followed by 40 cycles of 95 for 15 s, 60 for 1 min, and℃ ℃ a final 254

extension of 15 s at 95 . Relative expression was calculated using the 2℃ −∆∆Ct method，255

where the relative mRNA expression level was normalized against actin (the internal 256

standard gene) and compared with the control. 257

258

2.13. Statistical analysis 259

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No fewer than five seedlings per treatment were used for sample collecting, and all 260

measurements were performed using at least three replicates. Each experiment 261

included at least three biological replicates that were independently tested. All the 262

data were statistically analyzed with the SPSS 17.0 software program (SPSS Inc., 263

Chicago, IL, USA) using Duncan’s multiple range test at the P < 0.05 level of 264

significance. 265

266

3. Results 267

3.1. The effects of exogenous spermidine on the root growth of cucumber seedlings 268

under salt stress 269

The mitigative effects of exogenous Spd on the root growth of cucumber seedlings 270

under salt stress were clarified (Table 1). According to the larger values of all growth 271

parameters, exogenous Spd could promote root growth, although no significant 272

difference was observed between the control plants and those supplied with Spd. 273

NaCl stress significantly reduced the root fresh and dry weights, surface area and 274

total root volume. Compared with those under NaCl treatment only, the root fresh 275

and dry weights under the combined treatment of NaCl and Spd increased 276

significantly. Other parameters increased albeit without significant differences, 277

suggesting that Spd could alleviate the inhibition of root growth caused by salt stress. 278

Table 1 Effects of exogenous spermidine on the root growth of cucumber seedlings under salt 279

stress for 7 days 280

Treatment Fresh

weight

(g·plant-1)

Dry weight

(g·plant-1)

Total root

length

(cm·plant-1)

Root surface

area

(cm2·plant-1)

Total root

volume

(cm3·plant-1)

Total

number of

root tips

(No.·plant-1)

Cont 2.00±0.14a 0.10±0.01a 180.51±13.99a 101.66±5.35a 4.14±0.29a 549±72a

Spd 2.01±0.13a 0.11±0.02a 185.09±20.06a 103.46±4.69a 4.16±0.70a 615±64a

NaCl 1.26±0.07b 0.06±0.01b 158.69±47.91a 76.35±5.96b 2.47±0.52b 459±52a

NaCl+Spd 1.77±0.14a 0.09±0.01a 162.00±15.13a 83.40±2.90b 3.62±0.44ab 526±55a

Each value is the mean ± SE of 6 independent experiments. Different letters indicate significant 281

differences between treatments (P < 0.05). Cont, seedlings cultured in normal nutrient solution; 282

Spd, seedlings cultured in nutrient solution containing 0.1 mM Spd; NaCl, seedlings cultured in 283

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nutrient solution containing 75 mM NaCl; NaCl+Spd, combination of Spd and NaCl treatments. 284

285

3.2. The effect of exogenously applied spermidine on the accumulation of 286

endogenous polyamines 287

288

289

Figure 1 Effects of Spd on endogenous polyamines levels (A: Put, B: Spd, C: Spm) in the roots 290

of salt-stressed cucumber. The values are the means ± SE of three independent experiments. The 291

bars represent the standard errors. The different letters indicate significant differences at P < 0.05 292

according to Duncan’s multiple range test. Except for treatment time, the treatment methods were 293

the same as those in the Table 1 notes; the dynamic change was measured in this figure. 294

As shown in Fig. 1, the total polyamine levels in the control plants and in those 295

supplied with Spd remained relatively stable. However, greater changes were 296

observed under salt stress, especially in the salt-stressed plants whose roots received 297

Spd; these plants displayed a more substantial increase in polyamine content. 298

Regardless of whether exogenous Spd was applied to the salinized nutrient solution, 299

the contents of the three polyamines increased significantly only at 1 h after NaCl 300

treatment (P < 0.05); this increase was caused mainly by the increase in free 301

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polyamine content (Fig. S1), and all of them showed a similar trend of an initial 302

increase followed by a decrease within 24 h of treatment. Compared with those in 303

the control treatment, the total Spd levels in the NaCl+Spd treatment increased by 304

39.8% at 1 h, which was 6.6- and 2.7-fold greater than the variation in the individual 305

Put and Spm contents (6.0% and 14.5%, respectively). Unlike the gradual increase in 306

Put and Spm contents, the range of change in the Spd content decreased after a 307

marked increase at 1 h (P < 0.05), and it remained a higher but relatively stable level 308

from 2 to 12 h. The polyamine contents ultimately increased again after decreasing 309

gradually between 8 h and 24 h due to an increase in conjugated and bound 310

polyamines at a later stage (Fig. S2, 3). The total Spd and Spm contents between the 311

NaCl and NaCl+Spd treatments both showed significant differences (P < 0.05), and 312

the largest increase was observed in the Spm content (58.5%). 313

314

3.3. Polyamines are involved in the production of H2O2 under salt stress 315

316

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Figure 2 Effects of MGBG on the H2O2 content in the roots of salt-stressed cucumber. A: The 317

time course change in H2O2 content under normal conditions (Cont), 0.1 mM Spd treatment (Spd), 318

75 mM NaCl treatment (NaCl) and 75 mM NaCl + 0.1 mM Spd treatment (NaCl + Spd); B, C: 319

The treatment methods of Cont, NaCl and NaCl + Spd were the same as above. MGBG, nutrient 320

solution containing 0.2 mM MGBG; NaCl + MGBG, combination of NaCl and MGBG 321

treatments; NaCl + Spd + MGBG, combination of NaCl, Spd and MGBG treatments. Seedlings 322

were pretreated with 0.2 mM MGBG for 12 h, after which they were treated for 2 h as described 323

above. The images were visualized using fluorescence microscopy after the root tips were 324

incubated in 25 µM H2DCFDA in darkness for 20 min. The values are the means ± SE of three 325

independent experiments. The different letters indicate significant differences at P < 0.05 326

according to Duncan’s multiple range test. 327

To investigate the effects of exogenously applied Spd on the H2O2 content in 328

cucumber roots, we examined the changes in H2O2 over time. Fig. 2A shows that 329

significant differences in the increasing range of H2O2 content at 2 h existed between 330

the control and stress conditions (P < 0.05). Compared with those in the control 331

treatment, the plants in the other three treatments exhibited a rapid increase in H2O2 332

content within 1 h of treatment, and this content varied similarly as did the total 333

polyamine contents (Fig. 1). The H2O2 content in the control plants treated with Spd 334

sharply decreased to control levels after peaking at 1 h. However, this pattern was 335

different from that of the plants under salt stress; the H2O2 content in the plants under 336

salt stress continued to increase from 2-fold to more than 20-fold the level of the 337

control plants at a faster rate and peaked at 2 h. However, the greater peak value 338

between the two treatments under salt stress was caused by exogenous Spd. MGBG, 339

an inhibitor of PA biosynthesis, was used to examine whether Spd could induce the 340

accumulation of H2O2 in salt-stressed cucumber roots, the results of which are shown 341

in Fig. 2B and 2C. Treatment with MGBG (an inhibitor of polyamine biosynthesis) 342

resulted in a significant decrease in H2O2 levels in cucumber seedlings exposed to 343

NaCl, while the application of exogenous Spd markedly restored the increase in 344

H2O2 accumulation that was inhibited by MGBG (Fig. 2B, C). 345

346

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3.4. PAO-induced H2O2 production is the main source of H2O2 accumulation in the 347

roots of cucumber under salt stress 348

349

350

Figure 3 Effects of MGBG on the enzyme activities (A-C) and gene expression levels (D-F) of 351

CWPOD (A, D), PAO (D, E) and NOX (C, F). The treatment methods of Cont, NaCl and NaCl + 352

Spd were the same as those above. MGBG, nutrient solution containing 0.2 mM MGBG; NaCl + 353

MGBG, combination of NaCl and MGBG treatments; NaCl + Spd + MGBG, combination of 354

NaCl, Spd and MGBG treatments. Seedlings were pretreated with 0.2 mM MGBG for 12 h, after 355

which they were treated for 2 h as described above. RBOHF is one of the encoding genes of 356

NOX. The values are means ± SE of three independent experiments. The different letters above 357

the columns indicate significant differences at P < 0.05 according to Duncan’s multiple range 358

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test. 359

The sources of H2O2 in response to short-term salt stress were further investigated. 360

NaCl treatment resulted in the enhancement of both the activity of three enzymes and 361

the expression of their encoding genes; these increases were more marked in the 362

salt-stressed plants treated with Spd (Fig. 3A-C), which was in accordance with the 363

change in the H2O2 content. However, it is worth noting that the extent of increase 364

varied with enzyme type. PAO showed the largest increase under the NaCl alone 365

treatment; compared with those in the control treatment, the PAO activity and gene 366

expression levels in the NaCl alone treatment increased by 1.8-fold and 2.3-fold, 367

respectively (Fig. 3B, E). The results suggested that the three tested enzymes, 368

specifically PAO, quickly responded to salt stress. In addition, MGBG was used to 369

further study whether PA synthesis was relevant to the increase; as a result, the 370

enzyme activities in the salt-stressed plants decreased markedly compared with those 371

in the NaCl alone treatment. In addition, more importantly, the activities of the three 372

enzymes showed different decreasing amplitudes, with the largest decrease observed 373

for PAO (55.1%), followed by NOX (33.0%) and then CWPOD (8.1%). Moreover, 374

similar results were observed for the expression levels of the genes encoding these 375

enzymes (Figs. 3D-F, S4). However, the negative effects of MGBG under salt stress 376

conditions were alleviated by exogenously applied Spd, suggesting that endogenous 377

PA biosynthesis was associated with the responses of the three enzymes to salt stress. 378

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379

Figure 4 Effects of inhibitors of CWPOD (SHAM), PAO (1,8-DO) and NOX (DPI) activity on 380

the H2O2 content in the roots of salt-stressed cucumber. Seedlings were treated for 2 h in different 381

nutrient solutions: 1, 75 mM NaCl (NaCl); 2, 75 mM NaCl and 0.1 mM Spd (NaCl + Spd); 3, 75 382

mM NaCl and 5 mM SHAM (NaCl + SHAM); 4, 75 mM NaCl, 0.1 mM Spd and 5 mM SHAM 383

(NaCl + Spd + SHAM); 5, 75 mM NaCl and 0.2 mM 1,8-DO (NaCl +1,8-DO); 6, 75 mM NaCl, 384

0.1 mM Spd and 0.2 mM 1,8-DO (NaCl + Spd + 1,8-DO); 7, 75 mM NaCl and 20 µM DPI (NaCl 385

+ DPI); or 8, 75 mM NaCl, 0.1 mM Spd and 20 µM DPI (NaCl + Spd +DPI). All the inhibitors 386

were added 12 h earlier. The values are means ± SE of three independent experiments. The 387

different letters above the columns indicate significant differences at P < 0.05 according to 388

Duncan’s multiple range test. 389

Next, three specific inhibitors of these enzymes were added to study the 390

relationships 391

between the enzymes and H2O2 levels under salt stress. It was clear that the increase 392

in the H2O2 content in the plants exposed to salt stress was inhibited prominently by 393

these inhibitors compared with the plants in the NaCl alone treatment (Fig. 4). 394

However, there were differences in the amplitude of the decreases in the H2O2 levels. 395

The inhibitive effect of 1,8-DO was the most significant; 1,8-DO caused the H2O2 396

content to decrease by up to 67 %. The inhibitive effect of DPI ranked second (35 %), 397

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and that of SHAM was lowest (29 %). These results suggest that PAO plays a main 398

role in the increase in H2O2 in response to salt stress. It should be noted that DPI 399

inhibited the PAO activity; thus, we estimated the PAO activity at 1, 2, and 6 h in 400

response to the addition of DPI or 1,8-DO. After 2 h of treatment, the PAO activity 401

was slightly inhibited by DPI (14.9 %), while more a potent inhibitive effect was 402

observed in the presence of 1,8-DO (63.9 %); this finding indicates that the 403

inhibitory effect of DPI on the PAO activity was not significant (Fig. S5). 404

405

3.5. PAO-mediated H2O2 production is involved in Spd-induced antioxidant defense 406

407

Figure 5 Effects of Spd on the MDA content (A) and O2•− generation rate (B) in the roots of 408

salt-stressed cucumber. The values are means ± SE of three independent experiments. The bars 409

represent the standard errors. The different letters indicate significant differences at P < 0.05 410

according to Duncan’s multiple range test. Except for treatment time, the treatment methods were 411

the same as those in the Table 1 notes; dynamic changes were measured in this figure. 412

The MDA content and O2•− generation rate was measured to examine the effects of 413

Spd on oxidative damage resulting from salt stress; as shown in Fig. 5, the values of 414

the two metrics increased greatly after 48 h and 24 h of treatment with NaCl, 415

respectively. The lowest increasing range was found in the treatment with NaCl and 416

Spd, which was nearly half the value of that in the treatment with only NaCl, showing 417

that exogenous Spd could reduce oxidative damage. In addition, it can be seen from 418

Fig. 5 that plants treated with NaCl, especially when NaCl was combined with Spd, 419

showed more clear changes in enzyme activities and gene expression levels of the 420

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three measured antioxidant enzymes (SOD, POD and CAT). The activities of SOD 421

and POD started to increase markedly at 8 h after NaCl treatment (Fig. 6A, B), and 422

rapid increases in CAT activity occurred 4 h later (Fig. 6C). For gene expression 423

levels, differences between treatments at each time point became significant at 2 h, 424

and further increases in gene expression were observed in salt-stressed plants treated 425

with Spd, which led to an almost 1-fold increase at 24 h compared to the levels in 426

plants exposed to salt stress alone (Fig. 6D-F). However, it was apparent that the 427

marked changes in enzyme activities and gene expression levels of the three 428

antioxidant enzymes occurred later than did the changes in the H2O2 content (Fig. 429

2A). 430

431

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Figure 6 Effects of Spd on the enzyme activities (A-C) and gene expression levels (D-F) of three 432

antioxidant enzymes (SOD, POD and CAT) in the roots of salt-stressed cucumber. The values are 433

the means ± SE of three independent experiments. The bars represent the standard error. Except 434

for treatment time, the treatment methods were the same as those in the Table 1 notes; dynamic 435

changes were measured in this figure. 436

437

438

Figure 7 Effects of the inhibitor of PAO activity (1,8-DO) and the H2O2 scavenger (DMTU) on 439

the enzyme activities (A-C) and gene expression levels (D-F) of three antioxidant enzymes (SOD, 440

POD and CAT) in the roots of salt-stressed cucumber. Seedlings were treated for 24 h in different 441

nutrient solutions: 1, 75 mM NaCl (NaCl); 2, 75 mM NaCl and 0.1 mM Spd (NaCl + Spd); 3, 75 442

mM NaCl and 0.2 mM 1,8-DO (NaCl +1,8-DO); 4, 75 mM NaCl, 0.1 mM Spd and 0.2 mM 443

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1,8-DO (NaCl + Spd + 1,8-DO); 5, 75 mM NaCl and 5 mM DMTU (NaCl + DMTU); or 6, 75 444

mM NaCl, 0.1 mM Spd and 5 mM DMTU (NaCl + Spd + DMTU). All inhibitors were added 12 445

h earlier. The values are the means ± SE of three independent experiments. The different letters 446

above the columns indicate significant differences at P < 0.05 according to Duncan’s multiple 447

range test. 448

Whether H2O2, mainly from PAO-mediated PA oxidation, was associated with the 449

change in antioxidant enzyme activities under salt stress was then studied via 1,8-DO 450

and DMTU; the results are shown in Fig. 7. A similar change trend in enzyme 451

activities and gene expression levels were observed, although the changes in the 452

latter were not as significant as were those in the former. The activities of these 453

enzymes and the expression level of SOD in the salt-stressed plants treated with the 454

two inhibitors were all significantly lower than those in the plants under salt stress 455

alone; however, the inhibitive effect of DMTU was more effective than that of 456

1,8-DO, which indicated that the responses of the antioxidant enzymes under salt 457

stress were mediated by PAO-induced H2O2 production. No significant differences 458

were found in their inhibitive effect, which might be because PAO is the main source 459

of H2O2. Furthermore, the enzyme activities and gene expression levels of SOD, 460

POD and CAT in the plants pretreated with 1,8-DO increased to their respective 461

levels in the plants treated with NaCl alone, and a slighter increase was observed in 462

the plants pretreated with DMTU compared with the plants treated with 1,8-DO. 463

464

4. Discussion 465

The significance of PAs in plants in the protection against different environmental 466

stresses has been well documented [27], and exogenous PA application is an effective 467

approach for enhancing crop tolerance [28, 29]. In the present study, exogenous Spd 468

could alleviate the suppressive effects of salt stress and restore the affected plants to 469

control levels. Several studies have shown that endogenous polyamine levels increase 470

under stress conditions, and increases are promoted by exogenous Spd application 471

[30-33]. Here, the total content, whether that of Put, Spd, or Spm, changed less under 472

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control conditions with or without exogenous Spd than under stress conditions. 473

However, a more rapid and significant increase in endogenous Spd levels was 474

observed after salt treatment (Fig. 1B), while the other two polyamines increased 475

relatively slowly (Fig. 1A, C). These results indicated that a relatively stronger 476

response to salt stress can be elicited by endogenous Spd and especially by 477

exogenous Spd. These results are consistent with those of a study in tomato [32]. By 478

contrast, minor changes in each polyamine in the Spd-treated plants not subjected to 479

stress can be explained by the balanced state of endogenous polyamine levels under 480

non-stress conditions, in which plant perception is less sensitive than that in the 481

stress treatments. In addition, whether these levels will be altered at later stages 482

needs to be studied further. As mentioned above, Spd is synthesized from Put and 483

further produces Spm. Therefore, on the one hand, smaller changes in Put levels in 484

the early stage might be due to the massive accumulation of endogenous Spd; on the 485

other hand, the synthesis of Spm was further promoted by the increase in Spd, which 486

might partly explain the slower change of Put and Spm relative to that of Spd. Overall, 487

exogenous Spd can induce the production of endogenous polyamines, especially Spd, 488

under salt stress. 489

With more intensive studies on how PAs regulate plant stress tolerance, the idea 490

that PAs should be considered compounds that are involved in complex signaling 491

systems rather than simple protective molecules has been demonstrated by increasing 492

numbers of reports [5, 34, 35]. As a signaling molecule, H2O2 can respond to abiotic 493

stress, including salt stress [36], and H2O2 being a product of PA catabolism as well as 494

the relationship between the two has garnered much attention from researchers. In this 495

study, the relationship between the two was investigated to determine whether their 496

relation varies among species. The inducing effect of Spd on H2O2 production was 497

first verified. The analysis of the time course of the change in the H2O2 content 498

revealed that H2O2 responded rapidly to salt stress in cucumber roots; H2O2 levels 499

increased sharply within 2 h after NaCl treatment, and exogenous Spd presented an 500

additional increase (Fig. 2A). These findings are in agreement with the results of Wu 501

et al. [37]. However, the salt stress-induced increase in H2O2 levels was suppressed by 502

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MGBG, which is an inhibitor of PA biosynthesis, and the inhibiting effect was 503

partially rescued by exogenously applied Spd compared with plants treated with only 504

NaCl (Fig. 2B, C). These data indicate that endogenous PAs are involved in H2O2 505

accumulation under salt stress and that, via the induction of endogenous PA 506

production, exogenous Spd can cause H2O2 levels to further increase. 507

Many studies have shown that several kinds of sources of H2O2 exist in plants [38, 508

39]. PAO, as noted above, is associated with H2O2 accumulation via PA oxidation. In 509

addition to PAO, NOX and CWPOD have been proposed to be important sources of 510

H2O2; these enzymes were shown to contribute partly to H2O2 production in barley 511

plants exposed to aluminum stress [40] and in Arabidopsis plants under salt or 512

mannitol stress [17]. Therefore, the pathway of H2O2 generation depends on plant 513

species and stress conditions. However, the different kinds of H2O2 sources as well as 514

the main source of H2O2 in cucumber under salt stress have yet to be clarified. The 515

changes in enzyme activities and gene expression levels showed that the three 516

enzymes responded rapidly to salt stress and that PAO presented the strongest 517

response (Fig. 3). Similar to H2O2, changes in the three enzymes were related to 518

endogenous PA biosynthesis induced by salt stress in accordance with the inhibitive 519

effect of MGBG, and these changes could be further enhanced under the effects of 520

exogenous Spd. Most importantly, the H2O2 content decreased to different degrees by 521

the addition of SHAM, 1,8-DO and DPI; these results made it clear that CWPOD, 522

PAO and NOX were all H2O2 sources in the roots of salt-stressed cucumber, and 523

among the three enzymes, the largest contribution was made by PAO. 524

There is a general consensus that salt stress leads to oxidative stress; O2•− is a 525

molecule that causes lipid peroxidation, one of whose products is MDA. In this study, 526

exogenous Spd applied to plants exposed to salt stress caused MDA levels and the 527

O2•− generation rate to decrease markedly compared with those in plants exposed to 528

NaCl alone (Fig. 5), which is in agreement with previous studies [41]. Antioxidant 529

defense is an important strategy in response to abiotic stress, and by enhancing the 530

antioxidant system, exogenously applied Spd can improve the stress tolerance of 531

plants [28, 42, 43]. Moreover, H2O2 is a key element in signal transduction pathways 532

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and leads to various stress responses. H2O2 derived from PA oxidation has been 533

shown to participate in leaf growth in maize plants under saline conditions [44] and 534

in PA-induced cell death [45]. In addition, Li et al. [12] reported that H2O2 signaling 535

plays an important role in PA-regulated tolerance to water stress in white clover. Spd 536

is oxidized by PAO in the apoplast, generating H2O2, which then induces tolerance 537

responses in tobacco [46]. Therefore, we speculated that H2O2 might participate in 538

mitigative effects of exogenous Spd. However, the complex relationship between 539

materials may vary with plant species, even within different plant organs, and the 540

signaling regulatory mechanisms underlying the protective effects of Spd in 541

cucumber subjected to salt stress have yet to be fully defined. Therefore, we 542

investigated the relationship between H2O2 and the alleviative effects of Spd on salt 543

stress-induced injuries based on the antioxidant system. First, the inducing effects of 544

exogenous Spd on the antioxidant enzyme were confirmed according to the higher 545

levels of enzyme activity and gene expression (Fig. 6). Considering that 546

PAO-mediated PA oxidation is the main source of H2O2 in salt-stressed cucumber 547

roots, 1,8-DO, which is a specific inhibitor of PAO activity, was then used to 548

examine their relationship. DMTU, an H2O2 scavenger, caused a marked decrease in 549

both antioxidant enzyme activity and gene expression levels under salt stress, and 550

there was no significant difference between this treatment and the NaCl alone 551

treatment. 1,8-DO also caused the same effect. It is suggested that PAO-mediated 552

H2O2 production is associated with the salt stress-induced antioxidant system in 553

cucumber, and exogenous Spd can further enhance this antioxidant defense. 554

However, the role of PA-derived H2O2 is still controversial due to its dual functions, 555

and the distinction between the two functions needs to be studied further. 556

557

5. Conclusion 558

In summary, our results show that, by strengthening the antioxidant capacity of 559

roots, exogenous Spd can improve salt tolerance in cucumber seedlings, and this 560

process is mediated by H2O2 that is mainly derived from PAO. Under salt stress 561

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conditions, with increased levels of endogenous PAs induced by exogenously applied 562

Spd, PAO activity as well as the expression of its encoding gene is further enhanced, 563

thus contributing to H2O2 accumulation. As a signaling molecule, H2O2 subsequently 564

induces enhanced antioxidant defense, and oxidative damage is reduced, which is 565

helpful for root growth under salt stress conditions. However, antioxidant defense is 566

just a response of the protective effect of Spd against salt stress, and more research is 567

needed. 568

569

Figure 8 A model explaining the role of Spd-mediated H2O2 signaling in the regulation of 570

antioxidant defense that is associated with salt stress tolerance in cucumber roots. 571

572

Acknowledgements 573

This work was supported by the National Natural Science Foundation of China 574

(No.31471869, No.31401919 and No. 31272209), the China Earmarked Fund for 575

Modern Agro-industry Technology Research System (CARS-25-C-03), and the 576

Priority Academic Program Development of Jiangsu Higher Education Institutions 577

(PAPD). 578

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1. The increase of endogenous PAs synthesis could induce H2O2 accumulation in roots of salt-stressed cucumber. 2. The main source of H2O2 in cucumber roots under salt stress was polyamine oxidation. 3. The enhancement of antioxidant capacity by Spd was mediated by PAO-induced H2O2.

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Shirong Guo was responsible for design and guidance of the research. Jianqiang Wu wrote the main manuscript text and performed the experiments. Chengcheng Li performed some of the experiments. Sheng Shu and Jin Sun modified this manuscript.