Exogenous myo-inositol alleviates salinity-induced stress in Malus hupehensis Rehd

Accepted Manuscript Exogenous myo-inositol alleviates salinity-induced stress in Malus hupehensis Rehd Lingyu Hu, Kun Zhou, Yangtiansu Li, Xiaofeng Chen, Bingbing Liu, Cuiying Li, Xiaoqing Gong, Fengwang Ma PII:

S0981-9428(18)30483-2

DOI:

https://doi.org/10.1016/j.plaphy.2018.10.037

Reference:

PLAPHY 5479

To appear in:

Plant Physiology and Biochemistry

Received Date: 3 September 2018 Revised Date:

14 October 2018

Accepted Date: 30 October 2018

Please cite this article as: L. Hu, K. Zhou, Y. Li, X. Chen, B. Liu, C. Li, X. Gong, F. Ma, Exogenous myoinositol alleviates salinity-induced stress in Malus hupehensis Rehd, Plant Physiology et Biochemistry (2018), doi: https://doi.org/10.1016/j.plaphy.2018.10.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Exogenous myo-inositol alleviates salinity-induced stress in Malus hupehensis Rehd.

2

Lingyu Hu1, Kun Zhou1, Yangtiansu Li, Xiaofeng Chen, Bingbing Liu, Cuiying Li, Xiaoqing

3

Gong*, Fengwang Ma*

4

State Key Laboratory of Crop Stress Biology for Arid Areas/Shaanxi Key Laboratory of Apple,

5

College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, PR China

6

*Corresponding author

7

E-mail

8

[email protected] (F. Ma)

9

1

(X.

Gong),

[email protected]

SC

[email protected]

&

M AN U

addresses:

RI PT

1

Co-first authors: These authors contributed equally to this work

Abstract

11

Myo-inositol mediates various physiological processes and stress responses. Here, we investigated

12

its role in Malus hupehensis Rehd. plants when grown hydroponically under saline conditions.

13

Salt-stressed plants showed reduced growth and marked declines in photosynthetic activity and

14

chlorophyll concentrations. However, pretreatment with 50 µM myo-inositol significantly

15

alleviated those inhibitions and enabled plants to maintain their photosynthetic capacity. In

16

addition to changing stomatal behavior, exogenous myo-inositol inhibited ROS accumulation and

17

Na+ uptake. In contrast, activities of antioxidant systems were enhanced, and expression was

18

elevated for genes involved in Na+ uptake (e.g., HKT1, NHX1, SOS1, and SOS2). This exogenous

19

application also provoked the accumulation of sugars or sugar alcohols, which partially

20

contributed to the maintenance of osmotic balance, and the scavenging of ROS, either directly or

21

indirectly. In summary, myo-inositol appears to alleviate the salt-induced inhibition of

22

physiological processes for M. hupehensis, not only by supporting the plant’s antioxidant defense

AC C

EP

TE D

10

ACCEPTED MANUSCRIPT system but also by mediating Na+ and K+ homeostasis and the osmotic balance.

24

Key words: Malus hupehensis Rehd.; myo-inositol; salt stress; ion homeostasis; antioxidant

25

system; osmotic balance.

26

Abbreviations:

27

APX, ascorbate peroxidase; AsA-GSH, ascorbate-glutathione; Chl, chlorophyll; Ci, intercellular

28

CO2 concentration; DAB, 3,3’-diaminobenzidine; DHAR, dehydroascorbate reductase; DO,

29

dissolved oxygen; GolS, galactinol synthase; GR, glutathione reductase; Gs, stomatal conductance;

30

H2O2, hydrogen peroxide; HKT1, High-affinity K+ transporter 1; IMP, inositol monophosphate

31

phosphatase; IMT, inositol O-methyltransferase; MDHAR, monodehydroascorbate reductase;

32

MIOX, myo-inositol oxygenase; MIPS1, myo-inositol 1-phosphate synthase 1; NBT, nitro blue

33

tetrazolium; NHX1, Na+/H+ antiporter 1; PBS, phosphate-buffered saline; Pn, net photosynthesis

34

rate; POD, peroxidase; qRT-PCR, quantitative real-time PCR; REL, relative electrolyte leakage;

35

RGR, relative growth rate; ROS, reactive oxygen species; RS, raffinose synthase; SEM, scanning

36

electron microscopy; SOD, superoxide dismutase; SOS, salt overly sensitive; TDW, total dry

37

weight; TFW, total fresh weight; Tr, transpiration rate

EP

TE D

M AN U

SC

RI PT

23

39 40

AC C

38 1. Introduction

Plant growth and productivity are constantly challenged by adverse environmental conditions.

41

Soil salinization is widespread in arid and semi-arid regions, particularly on irrigated lands in such

42

areas (Suzuki et al., 2016). One example, the Loess Plateau in China, is the largest and most ideal

43

region for growing apple trees (Malus × domestica Borkh.) in that country because of abundant

44

irradiance and a wide range in temperatures between day and night (Zhou et al., 2015). However,

ACCEPTED MANUSCRIPT 45

increased salinity in the soil has severely hampered the development of apple plantations and

46

resulted in economic losses for orchardists. Excessive salt accumulations trigger detrimental effects that are mainly attributed to two

48

problems: osmotic stress and ion toxicity. Increases in osmotic pressure, caused by excess salt in

49

the root zone, lead to significant reductions in water uptake that, in turn, slow cell division and

50

expansion, thereby diminishing cellular activity. Meanwhile, the external and internal

51

over-accumulation of Na+, a major toxic cation in salt-affected soil environments, disturbs K+

52

homeostasis and vital metabolic reactions, e.g., photosynthesis, resulting in the accumulation of

53

reactive oxygen species (ROS) such as superoxide radicals, hydroxyl radicals, and hydrogen

54

peroxide (H2O2), as well as the generation of toxic metabolites (Chinnusamy et al., 2006). To cope

55

with this, plants simultaneously invoke various defensive strategies, including biosynthesis of

56

osmoprotectants, alterations to the photosynthetic pathway, partitioning and exclusion of toxic Na+

57

ions, induction of antioxidant systems to scavenge ROS, stimulation of phytohormones, and

58

regulation of gene expression to improve salt tolerance (Hasegawa et al., 2000; Yang and Guo,

59

2018).

EP

TE D

M AN U

SC

RI PT

47

Myo-inositol is the most abundant isoform in plants, although chiro-, scyllo-, muco-, and

61

neo-inositols have also been reported in some species (Valluru and Ende, 2011). This versatile

62

compound is a critical factor in inositol metabolism, generating 37 distinct derivatives, including

63

inositol polyphosphates, D-glucuronic acid (a cell wall component), phosphatidylinositol

64

phosphate, members of the raffinose family, and methylated derivatives (ononitol and pinitol).

65

Under stress conditions, these protective compounds have dual functions as signals and key

66

metabolites. Myo-inositol and its derivatives participate in many stress-induced processes, such as

AC C

60

ACCEPTED MANUSCRIPT programmed cell death, immunity, and salt tolerance, which suggests that they have extensive

68

protective roles in plants (Loewus and Murthy, 2000; Kaur et al., 2013; Tan et al., 2013). Because

69

myo-inositol is also believed to be essential for plant growth, it is included in standard Murashige

70

and Skoog (MS) media (Murashige and Skoog, 1962; Ye et al., 2016). Its value in culturing

71

systems is supported by the fact that endogenous myo-inositol functions in multiple developmental

72

and physiological processes, such as the phosphatidylinositol signaling pathway, phosphate

73

storage, auxin storage and transport, and cell wall biosynthesis (Cui et al., 2013).

SC

RI PT

67

Although the physiological relevance of endogenous myo-inositol, especially its protective

75

roles under biotic and abiotic stresses, has been finely characterized through a transgenic approach

76

with many plants, including Arabidopsis thaliana (hereafter, Arabidopsis; Kaur et al., 2013),

77

Medicago falcata (Tan et al., 2013), Brassica juncea (Goswami et al., 2014), Ipomoea batatas

78

(Zhai et al., 2016), and Oryza sativa (Kusuda et al., 2015), the potential role of exogenous

79

myo-inositol applications in plant development has been examined in only a few studies. For

80

example, pretreatment with myo-inositol can alleviate salt-induced ion disequilibrium for

81

chromosomes and can retard DNA degradation in cells of Allium cepa (Chatterjee and Majumder,

82

2010). Such exogenous applications can also rescue the lesion phenotype associated with the

83

mips1 mutant in Arabidopsis (Donahue et al., 2010). Under normal conditions, exogenous

84

myo-inositol can restore the expression of Arabidopsis genes involved in stress responses, cell

85

wall biosynthesis, regulation of phytohormones, redox reactions, and chromosome modifications

86

(Ye et al., 2016).

AC C

EP

TE D

M AN U

74

87

Apple is one of the most important fruit crops world-wide. Although breeding for increased

88

salt tolerance is important, the long juvenile period of those plants and their high degree of genetic

ACCEPTED MANUSCRIPT heterozygosity mean that only limited progress has been made to develop salt-hardy cultivars

90

through traditional approaches. Nevertheless, we have previously reported that exogenous

91

application of melatonin (Li et al., 2012) or dopamine (Li et al., 2015), can significantly ease the

92

negative effects of salinity-induced stress in apple plants. Those successes then present new

93

strategies for improving the growth of plants under abiotic stresses. In the research described here,

94

we employed a hydroponics system and found that pretreatment with 50 µM myo-inositol

95

significantly alleviated salt-induced inhibition and enabled plants of M. hupehensis Rehd. to

96

maintain their normal growth. We also investigated the potential protective mechanism for

97

myo-inositol in antioxidative activity and ion and osmosis homeostasis. These findings will be

98

beneficial to further applications and examinations of the physiological role of myo-inositol under

99

different stress conditions.

100

2. Materials and methods

101

2.1. Plant materials and growing conditions

TE D

M AN U

SC

RI PT

89

All experiments were conducted at the Northwest A&F University, Yangling (N34°20,

103

E108°24), China, from March to July of 2016, 2017, and 2018 . Seeds of Malus hupehensis Rehd.

104

were stratified for 50 d at 4°C. Following germination, four seedlings each were planted in plastic

105

pots (12 cm×12 cm) filled with sand and placed in a greenhouse under natural conditions. After 40

106

d of growth, plants of similar size were transferred to a hydroponics system and cultured as

107

described by Bai et al. (2013). Briefly, the plants were grown in plastic tubs (52 cm×37 cm×15 cm)

108

containing 20 L of ½-strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950). All of

109

the tubs were wrapped with black plastic to restrict light exposure to the roots and were placed in a

110

growth chamber (23-25°C/15-18°C day/night). Light was provided by sodium lamps during a 14-h

AC C

EP

102

ACCEPTED MANUSCRIPT photoperiod (light intensity of 160 µmol m-2 s-1). The nutrient solution was continuously aerated

112

with an air pump, and dissolved oxygen (DO) concentrations were maintained at 8.0 to 8.5 mg L-1

113

by a DO controller (FC-680; Corporation of Super, Shanghai, China). The pH was adjusted to 6.5

114

± 0.1 with H3PO4 and the solution was changed every 3 d. After 3 weeks of culturing, plants with

115

uniform size were selected for our experiments.

116

2.2. Screening for optimum concentration of myo-inositol

RI PT

111

We applied 0, 1, 10, 50, 100, or 150 µM myo-inositol to the roots for 10 d before introducing

118

our salt-stress treatment (200 mM NaCl) for 11 d. The myo-inositol was refreshed every 5 d along

119

with the nutrient solution. On Days 6 and 11, we calculated the salt injury index (SI) as follows:

M AN U

SC

117

SI= (0 × n0 + 1 × n1 + 2 × n2 + 3 × n3 + 4 × n4) / (4 × n)

121

Where n0, n1, n2, n3, and n4 were the numbers of plants coded on an injury scale of 0

122

through 4 (0 = no symptoms, no lesions on leaves; 1 = a few leaf tips or edges yellowing, with

123

<10 spots that were not coalesced on a leaf; 2 = approximately 50% of the tips or edges

124

browning, >10 spots per leaf, and coalescence; 3 = most tips or edges browning or dead, and

125

leaves turned downward; 4 = whole plants dead or dying) (Li et al., 2015).

126

2.3. Induction of salt stress after myo-inositol pretreatment

EP

AC C

127

TE D

120

Based on the results from our screening of myo-inositol concentrations, we selected a

128

pretreatment level of 50 µM to apply to half of the plants in the hydroponics system 10 d before

129

the beginning of the stress period. The plants were then subdivided into four groups: 1) CK,

130

½-strength Hoagland’s nutrient solution only; 2) ST group, ½-strength Hoagland’s nutrient

131

solution supplemented with 200 mM NaCl; 3) MI group, ½-strength Hoagland’s nutrient solution

132

plus 50 µM myo-inositol; and 4) ST+MI group, ½-strength Hoagland’s nutrient solution to which

ACCEPTED MANUSCRIPT 133

was added 200 mM NaCl and 50 µM myo-inositol. The stress period spanned 12 d and included

134

three replicates.

135

2.4. Evaluation of photosynthetic characteristics We determined the net photosynthesis rate (Pn), intercellular CO2 concentration (Ci),

137

stomatal conductance (Gs), and transpiration rate (Tr) between 9:00 and 11:00 am with a portable

138

photosynthesis system (Li-6400; LICOR, Huntington Beach, CA, USA). All measurements were

139

performed at 1000 µmol photons m-2 s-1 and a constant airflow rate of 500 µmol s-1. The cuvette

140

CO2 concentration was set at 400 µmol CO2 mol-1 air. Data were collected from fully expanded,

141

fully light-exposed leaves at the same position from eight plants.

142

2.5. Observations of leaf stomata

M AN U

SC

RI PT

136

The fourth leaf was sampled from each selected plant in a treatment group and immediately

144

fixed with 4% glutaraldehyde [0.1 M phosphate-buffered saline (PBS) and 4% glutaraldehyde; pH

145

6.8]. After being rinsed with PBS four times (10 min each), the samples were dehydrated in a

146

graded ethanol series, vacuum-dried, and gold-coated. Scanning electron microscopy (SEM) was

147

performed on a JSM-6360LV microscope (JEOL Ltd., Tokyo, Japan). Stomata were randomly

148

counted in 20 different visual sections on the abaxial epidermis and the final tallies were used to

149

determine stomatal density. The stomatal apertures were measured randomly from 20 stomata on

150

those same specimens, using IMAGE J software.

151

2.6. Growth measurements

AC C

EP

TE D

143

152

We collected 12 plants per treatment on Days 0 (initial) and 12 (final) of the experiment and

153

recorded their heights from the stem base to the terminal bud. After the total fresh weight (TFW)

154

of each sample plant was determined, the total dry weights (TDWs) were obtained after the plants

ACCEPTED MANUSCRIPT 155

were oven-dried at 75°C for at least 72 h to a constant weight. The relative growth rate (RGR) was

156

calculated by the equation of Radford (Liang et al. 2017): RGR = (Ln DW2 - Ln DW1)/(T2 - T1),

158

Where DW2 was the plant dry weight at the final harvest time (T2) and DW1 was the plant

RI PT

157

159

dry weight at the initial time (T1).

160

2.7. Assessments of total chlorophyll, relative electrolyte leakage, and root architecture

Total chlorophyll (Chl) concentrations were measured along the same position for each

162

selected plant with a SPAD chlorophyll meter (SPAD-502 Plus; Konica Minolta). Data were

163

collected from four designated portions of each leaf. Three biological replicates were performed

164

per treatment, using at least six plants each.

M AN U

SC

161

Relative electrolyte leakage (REL) of the leaves was measured according to the method

166

described by Dionisio-Sese and Tobita (1998). Root images were collected with a scanner (Epson

167

Perfection V700 Photo; Seiko Epson Corporation) and the root architecture was analyzed with the

168

WinRHIZO® image analysis system (V4.1 c; Régent Instruments, Quebec City, QC, Canada).

169

2.8. Assays of antioxidant enzyme activities and determination of ROS accumulations

EP

TE D

165

Activities of superoxide dismutase (SOD), peroxidase (POD), monodehydroascorbate

171

reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), and

172

ascorbate peroxidase (APX), as well as H2O2 levels, were determined by using detection kits

173

according to the manufacturer’s instructions (Suzhou Comin Biotechnology Co., Ltd, Suzhou,

174

China). At the end of the experimental period, we used leaf samples to detect the presence of H2O2

175

and the superoxide radical (O2−)by staining with DAB (3,3’-diaminobenzidine) and NBT (nitro

176

blue tetrazolium), respectively (Dai et al., 2018).

AC C

170

ACCEPTED MANUSCRIPT 177

2.9. Measurements of soluble sugars and sugar alcohols Soluble sugars were extracted and derivatized according to the protocol of Wang et al. (2010),

179

with a minor modification. Briefly, 100 mg of leaf sample was extracted in 1.4 mL of 75%

180

methanol, and ribitol was added as the internal standard. After the non-polar metabolites were

181

fractionated into chloroform, we transferred 2 or 50 µL of the polar phase into individual 1.5-mL

182

centrifuge tubes. The samples were then vacuum-dried and derivatized with methoxamine

183

hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide. All of those derivatives were

184

transferred into 2.0-mL Eppendorf vials to analyze the metabolites with a Shimadzu

185

GC/MS-2010SE (Shimadzu Corporation, Tokyo, Japan). The metabolites were distinguished and

186

quantified by comparing their fragmentation patterns with those from a mass spectral library

187

generated on our GC/MS system as well as from an annotated quadrupole GC/MS spectral library

188

downloaded

189

mpg.de/csbdb/gmd/msri/gmd msri.html). Values were calculated based on their corresponding

190

standard curves and internal standards.

191

2.10. Determinations of Na+ and K+

SC

Golm

M AN U

the

Metabolome

Database

mpimp-golm.

EP

Concentrations of Na+ and K+ were measured by flame spectrometry (M410; Sherwood

193

Scientific, Cambridge, UK), as described by Liang et al. (2017).

194

2.11.Quantitative real-time PCR analysis

195

(http://csbdb.

TE D

from

AC C

192

RI PT

178

Total RNA was extracted with a Wolact® plant RNA isolation kit (Wolact, Hong Kong,

196

China), and the first-strand cDNA was synthesized using a PrimeScriptTM RT reagent Kit with the

197

gDNA Eraser (Perfect Real Time) (Takara, Tokyo, Japan). Quantitative real-time PCR (qRT-PCR)

198

was performed on a CFX96 real-time PCR system (BIO-RAD, USA), using SYBR Premix Ex

ACCEPTED MANUSCRIPT TaqII (Takara). The reference gene was Malus elongation factor 1 alpha (EF-1α; DQ341381)

200

(Shao et al., 2014). The qRT-PCR primers (Table 1) were designed with Primer Premier 5 software

201

(Biosoft International, Palo Alto, CA, USA). Each experiment involved three biological replicates,

202

and the relative expression level for each gene was calculated according to the 2-∆∆CT method

203

(Livak and Schmittgen, 2001).

204

2.12. Statistical analysis

We used SPSS software (version 16.0) for the statistical analysis. All data were subjected to

SC

205

RI PT

199

one-way ANOVA, and values were presented as means ± SD (standard deviation).

207

3. Results

208

3.1. Screening of myo-inositol concentrations

M AN U

206

To examine whether myo-inositol can affect the tolerance of Malus hupehensis seedlings

210

under salt stress, we used a hydroponics system to investigate the performance of plants after

211

pretreatment with different concentrations of that compound. As shown in Table 2, such

212

applications alleviated the salt-induced damage in leaves and SI values differed among treatment

213

groups. When compared with groups that were not pre-treated, plants in the 50 µM myo-inositol

214

group showed the greatest decline in SI (by 21.6% on Day 6 and 34.4% on Day 11), followed by

215

the plants in the 100 µM myo-inositol group, which showed decreases of 13.4% on Day 6 and

216

32.8% on Day 11. Application of 150 µM myo-inositol benefited plants before Day 6, but harmed

217

them after that point. Therefore, we selected 50 µM myo-inositol as the concentration to use in

218

further experiments.

219

3.2. Effects of exogenous myo-inositol on photosynthetic capacity and plant growth

220

AC C

EP

TE D

209

To further our understanding of its role, we applied 50 µM myo-inositol to the roots of

ACCEPTED MANUSCRIPT designated plants before inducing salt stress. After 8 d of observation, the leaves of stressed plants

222

were severely withered while plants that had received the pretreatment showed only slight

223

symptoms of stress (Fig. 1A). Although the net photosynthesis rate was significantly lower in the

224

ST group than in the CK group, myo-inositol supplementation narrowed that gap, with plants in

225

the ST+MI group showing higher Pn levels than those in the ST group (Fig. 2A). Similar trends

226

were noted for Ci, Gs, and Tr. While the stomatal density was decreased in salt-stressed leaves

227

when compared with the control, exogenous myo-inositol alleviated that decline. The size of the

228

stomatal aperture, an important determinant of the photosynthetic process, was obviously

229

up-regulated by myo-inositol (Fig. 2B, C).

M AN U

SC

RI PT

221

Although stress conditions significantly inhibited plant growth, the application of 50 µM

231

myo-inositol markedly eased that inhibition (Table 3 and Fig. 1B). For example, on Day 12,

232

growth in the ST group was severely inhibited, as indicated by their declines in height (17.27 cm),

233

TDW (1.27 g), TFW (2.49 g), and RGR (24.16 g kg-1 d-1), all of which were somewhat lower than

234

the values recorded for plants in the CK group (21.88 cm, 1.85 g, 3.56 g, and 42.08 g kg-1 d-1,

235

respectively) and in the ST+MI group (19.62 cm, 1.48 g, 3.08 g, and 32.69 g kg-1 d-1, respectively).

236

By comparison, the height, TDW, TFW, and RGR of plants in the MI group were 24.82 cm, 2.14 g,

237

4.09 g, and 49.87 g kg-1 d-1, respectively (Table 3). Our examination of root architecture on Day

238

12 revealed damage due to salt stress (Fig. 1B). Root growth was suppressed in the ST group, with

239

values being somewhat lower for average length (219.76 cm), volume (0.20 mm3), surface area

240

(22.60 cm2), and numbers of tips (503) and forks (945) than their corresponding samples from the

241

CK group (374.94 cm, 0.32 mm3, 37.46 cm2, 861, and 1692, respectively) and the ST+MI group

242

(303.00 cm, 0.27 mm3, 29.01 cm2, 786, and 1485, respectively). When compared with the CK

AC C

EP

TE D

230

ACCEPTED MANUSCRIPT tissues, root lengths, root volumes, surface area, and numbers of tips and forks were decreased by

244

41.4%, 37.5%, 39.7%, 41.6%, and 44.1% respectively, for the ST plants. However, for the ST+MI

245

plants, those corresponding values were significantly higher than for the ST plants, with respective

246

increases of 22.2%, 22.3%, 17.1%, 32.8%, and 31.9%. Furthermore, after 12 d of myo-inositol

247

treatment, root growth was improved under normal conditions, producing increases in the MI

248

plants of 24.0%, 39.3%, 30.8%, 25.1%, and 21.9% for root length, root volume, surface area and

249

number of root tips and root forks, respectively, when compared with the CK group. Nevertheless,

250

average root diameters did not differ among treatment groups. These results suggested that

251

myo-inositol has a possible role in modulating the growth of the apple root system.

252

3.3. Effects of myo-inositol pretreatment on total chlorophyll concentrations and relative

253

electrolyte leakage

M AN U

SC

RI PT

243

Total chlorophyll concentrations were monitored to determine how they might be influenced

255

by myo-inositol. As shown in Figure 3B, this compound had no apparent effect on Chl levels

256

under normal conditions, and SPAD levels were almost the same between the CK and MI groups.

257

However, salt stress significantly diminished Chl concentrations in the ST group beginning on

258

Day 8 of treatment, and their detected SPAD levels were significantly reduced to 29. By contrast,

259

a similar reduction began on Day 12 in the ST+MI group, with a detected SPAD level of 30 that

260

was also much higher than that measured from the ST group. Under stress conditions, values for

261

REL increased in the leaf samples. However, that rise was significantly slowed in response to

262

myo-inositol (Fig. 3B).

263

3.4. ROS accumulations and activities of antioxidant enzymes

264

AC C

EP

TE D

254

At the end of the experimental period, results from DAB- and NBT-staining revealed more

ACCEPTED MANUSCRIPT intense brown coloration or blue patches on the ST leaves than on those from the ST+MI group.

266

This indicated that the myo-inositol-mediated alleviation was closely coupled with the ROS level

267

(Fig. 3A). By Days 4, 8, and 12, H2O2 levels in the ST group had increased by 101.2%, 170.4%,

268

and 267.0%, respectively, over those measured in the CK group. By contrast, exogenous

269

application of 50 µM myo-inositol was linked with a significant decrease in H2O2 production, with

270

levels being 60.5% (Day 4), 66.6% (Day 8), and 105% (Day 12) lower in the ST+MI group than

271

in the ST plants (Fig. 3B).

SC

RI PT

265

Our investigation of the antioxidant system demonstrated that salt stress significantly

273

increased SOD and POD activities, thereby suggesting that these enzymes have protective roles.

274

For example, on Days 4 and 8, SOD activity was significantly higher in ST+MI plants than in ST

275

plants (Fig. 4A). For POD activity, levels were not statistically different between the ST and

276

ST+MI plants on Day 4, but they were significantly higher in the ST+MI group than in the ST

277

group between Days 8 and 12 d (Fig. 4A). Similar trends were noted for the capacity of the

278

ascorbate -glutathione (AsA-GSH) cycle, while MDHAR and APX activities in the ST+MI group

279

peaked on Day 8 and were higher than those in the CK group. On Days 4 and 12, DHAR activity

280

was lower in the ST group than in the ST+MI group, while exogenous myo-inositol had no effect

281

on GR activity on either Day 8 or 12 (Fig. 4B).

282

3.5. Concentrations of soluble sugars and sugar alcohols

TE D

EP

AC C

283

M AN U

272

Soluble sugars and sugar alcohols are important metabolites and signals, and most of them

284

can be induced by stress conditions. As shown in Figure 5, the concentrations of sucrose, fructose,

285

sorbitol, glucose, myo-inositol, and galactose were significantly elevated by salt treatment. When

286

compared with plants in the ST group, levels of sucrose and fructose were relatively lower in the

ACCEPTED MANUSCRIPT ST+MI group on Days 4, 8, and 12. However, plants in the ST+MI group accumulated much more

288

glucose, myo-inositol, and galactose on Days 4 and 8, but less on Day 12. No significant

289

difference in sorbitol concentrations was observed between the ST and ST+MI groups. These

290

findings indicated that exogenous myo-inositol could alter the levels of endogenous soluble sugars

291

and sugar alcohols under saline conditions.

292

3.6. Accumulations of Na+ and K+

RI PT

287

Because salinity is known to disturb Na+ and K+ homeostasis and negatively affect plant

294

growth, we measured their concentrations in the leaves at the end of the experiment. Compared

295

with the CK group, levels were either dramatically increased (Na+) or decreased (K+) by salt stress.

296

Although the Na+ concentrations did not differ between the control and pretreated plants under

297

normal conditions, those levels were significantly reduced under stress for plants that had received

298

the pretreatment. By contrast, the K+ concentration was slightly elevated by myo-inositol under

299

normal conditions, but this supplementation had no effect on the Na:K ratio. Consequently,

300

upregulation of that ratio was obviously alleviated by myo-inositol when salinity was introduced

301

(Fig. 6A).

302

3.7. qRT-PCR analysis of related genes

M AN U

TE D

EP

AC C

303

SC

293

To identify the possible regulatory mechanism for myo-inositol, we analyzed the expression

304

of genes for its synthesis, i.e., myo-inositol 1-phosphate synthase 1 (MIPS1) and inositol

305

monophosphate phosphatase (IMP), as well as those involved in its metabolic pathway, i.e.,

306

myo-inositol oxygenase (MIOX), galactinol synthase (GolS), and raffinose synthase (RS).

307

Although all detected genes were significantly induced by stress treatment, supplementation with

308

myo-inositol delayed this type of induction such that those genes showed the same or relatively

ACCEPTED MANUSCRIPT higher expression (Fig. 7). For example, MIPS1 was induced by salt stress and expression peaked

310

on Day 4. However, that trend was slowed in pretreated plants, with expression peaking much

311

later, i.e., on Day 8 in the ST+MI group and at a much higher level than that detected in the ST

312

group. Expression patterns were similar among IMP, MIOX, GolS, and RS, with peaks appearing

313

on Day 4 in the ST group but being delayed until Day 8 in the ST+MI group. We also noted that

314

myo-inositol up-regulated the expression of RS under normal conditions.

RI PT

309

We also used qRT-PCT to monitor the expression of genes that function in the salt overly

316

sensitive (SOS) pathway. Those genes are closely related to genes that control the balance between

317

Na+ and K+. Here, HKT1 (High-affinity K+ transporter 1) and NHX1 (Na+/H+ antiporter 1) were

318

strongly induced by salinity treatment and their expression was much higher in the ST+MI group

319

than in the ST group (Fig. 6B). This indicated that pretreatment with myo-inositol improved their

320

expression in stressed plants. While both SOS1 and SOS2 were up-regulated by salinity, with

321

expression peaking on Day 4, the myo-inositol supplement delayed that trend. Finally, transcript

322

levels of SOS1 and SOS2 peaked on Day 8 in the ST+MI group and were higher than those

323

detected in the ST group.

EP

TE D

M AN U

SC

315

325 326

AC C

324 4. Discussion

Soil salinization severely reduces crop productivity and is an escalating problem in cultivated

327

lands, especially in arid and semi-arid regions (Zhu, 2001). Endogenous supplementation with

328

myo-inositol can confer multiple tolerances to abiotic and biotic stresses by inducing

329

overexpression of the key myo-inositol synthesis gene, MIPS (Kaur et al., 2013; Tan et al., 2013;

330

Goswami et al., 2014; Kusuda et al., 2015; Zhai et al., 2016). Such applications, although not as

ACCEPTED MANUSCRIPT 331

widely studied, can also alleviate salt-induced damage in plants (Chatterjee and Majumder, 2010;

332

Donahue et al., 2010). In this study, we found that pretreatment with exogenous myo-inositol can

333

significantly enhance salt tolerance in Malus hupehensis. Our experiments demonstrated that salinity stress was associated with significant decreases in

335

numerous growth parameters, including plant heights, total fresh and dry weights, and relative

336

growth rates (Table 3). However, pre-treating the plants with myo-inositol significantly relieved

337

those inhibitory effects, while also improving performance under normal conditions. This

338

compound closely interacts with phytohormones and regulates growth (Loewus and Murthy, 2000).

339

For example, myo-inositol can be conjugated to IAA to form IAA-MI, facilitating the

340

long-distance transport of auxin within plants (Loewus and Murthy, 2000; Chen and Xiong, 2010).

341

The rate of photosynthesis is also closely related to overall growth and is constantly regulated by

342

external environmental factors, such as salt stress (Parida and Das, 2005). In particular, saline

343

conditions inhibit growth by diminishing photosynthetic capacity and interrupting cell division

344

and expansion (Zhu, 2001). We observed declines in the net photosynthesis rate, intercellular CO2

345

concentration, stomatal conductance, and transpiration rate when salt stress was introduced (Fig.

346

2). Decreases in photosynthesis are usually connected with imbalances in the levels of leaf

347

photosynthetic pigments (Netondo et al., 2004). Likewise, we found that total chlorophyll

348

concentrations in the leaves were lower under stress conditions. However, those reductions were

349

significantly alleviated in plants that had been pre-treated with myo-inositol. This exogenous

350

application improved photosynthetic activity and plant growth in stressed plants, as reflected in

351

changes to stomatal apertures. Stomatal behavior enabled those plants to modulate transpirational

352

water losses and the uptake of CO2, both of which closely affect photosynthesis.

AC C

EP

TE D

M AN U

SC

RI PT

334

ACCEPTED MANUSCRIPT Salinity presents two main challenges to plants -- osmotic stress and ion toxicity. This

354

osmotic stress usually leads to the production of ROS such as H2O2, superoxides, and

355

hydroxy/peroxy radicals. These ROS can destroy normal metabolism through oxidative damage to

356

DNA, proteins, and lipids, thereby causing membrane dysfunction and cell death (Miller et al.,

357

2010). We found here that levels of salt-induced H2O2 and O2− were obviously decreased in

358

response to exogenous myo-inositol, as evidenced by the lower REL values in pretreated plants

359

that did not incur serious damage to their cellular membranes (Fig. 3). The antioxidant system in

360

plants is usually employed to eliminate excess ROS and maintain a dynamic balance of ROS in

361

vivo. After SOD converts superoxide to H2O2, POD catalyzes its breakdown (Das and

362

Roychoudhury, 2014; Ismail et al., 2014). When compared with our CK group of plants, the

363

induction of salt stress was associated with greater concentrations of O2− and H2O2, as well as

364

higher SOD and POD activities, and more severe damage to cellular membranes. However, for

365

stressed plants that had been pre-treated with myo-inositol, the increments in levels of O2− and

366

H2O2 and membrane damage were eased while SOD and POD activities were also improved. A

367

previous study has shown that an increase in myo-inositol can enhance SOD activity and decrease

368

concentrations of H2O2 and malondialdehyde in stressed plants (Zhai et al., 2016). Moreover, in

369

pathogen-induced responses, myo-inositol is thought to act downstream of ROS, and the specific

370

level of myo-inositol is a critical factor that determines whether oxidative stress induces or blocks

371

defense responses (Chaouch and Noctor, 2010). The AsA-GSH cycle also has a crucial role in the

372

response to salt-induced stress. Ascorbate peroxidase works in conjunction with that cycle and has

373

a critical function in scavenging ROS in plant tissues (Shigeoka et al., 2002; Wang et al., 2012). In

374

our study, the activities of APX, MDHAR, DHAR, and GR were higher under salt stress, but

AC C

EP

TE D

M AN U

SC

RI PT

353

ACCEPTED MANUSCRIPT 375

myo-inositol pretreatment further enhanced the activities of APX and MDHAR (Fig. 4B). All of

376

these results suggest that exogenous myo-inositol can improve activity by the antioxidant system

377

and then stabilize ROS levels under saline conditions. One important strategy for achieving salt tolerance under stress conditions is the

379

re-establishment of ionic and osmotic homeostasis (Zhu, 2001). Ionic toxicity is primarily caused

380

by excess accumulations of sodium in the cytoplasm or organelles, which leads to an ion

381

imbalance and disruption of normal plant growth (Dai et al., 2018). Nelson et al. (1999) have

382

shown that exogenous applications of myo-inositol enhance the uptake and transport of sodium

383

through the xylem, which demonstrates the importance of this compound in regulating ion toxicity

384

and osmotic homeostasis. We also found that the myo-inositol pretreatment enhanced salt

385

tolerance, as evidenced by lower Na+ accumulations and a reduced Na:K ratio. The SOS pathway,

386

which exports Na+ out of the cells, is activated by salt stress and is clearly important for

387

modulating plant Na+/K+ homeostasis (Himabindu et al., 2016; Yang and Guo, 2018). Our results

388

indicated that myo-inositol could mediate other potential pathways to improve salt tolerance at

389

earlier stages, based on our findings that upregulation of SOS1 and SOS2 was delayed.

390

Furthermore, the ion transporter HKT1 helps exclude Na+ from the leaves (Munns and Tester,

391

2008), and the Na+/H+ antiporter NHX1 is related to Na+ compartmentation (Brini and Masmoudi,

392

2012). Overexpression of AtHKT1 and MdNHX1 significantly enhances salt tolerance (Møller et

393

al., 2009; Li et al., 2013). Our qRT-PCR results showed that HKT1 and NHX1 were greatly

394

up-regulated by salt stress, and this tendency was further enhanced when plants were treated with

395

myo-inositol before being exposed to saline conditions (Fig. 6B). We believe that this direct

396

communication of exogenous myo-inositol was also beneficial to the ionic and osmotic

AC C

EP

TE D

M AN U

SC

RI PT

378

ACCEPTED MANUSCRIPT 397

homeostasis in the roots (see also Chatterjee and Majumder, 2010). All of our results imply that

398

myo-inositol helps maintain the ion balance by reducing Na+ concentrations and the Na:K ratio,

399

thereby conferring salt tolerance by the plant. Under salinity stress, plants accumulate various compatible solutes in the cytoplasm,

401

including sugars, complex sugars, polyols, proline, and glycine betaine, by which they sustain

402

their osmotic equilibrium and support the absorption of water (Gil et al., 2011; Redillas et al.,

403

2012; Bertrand et al., 2015; Hu et al., 2015). In addition to carbon storage, soluble sugars such as

404

sucrose, glucose, fructose, and starch have valuable protective roles in osmotic adjustments,

405

detoxification of ROS, and stabilization of membrane integrity, enzymes, and proteins (Rosa et al.,

406

2009; Zhu et al., 2016). Our experiments revealed that salt stress increased the concentrations of

407

sucrose, fructose, glucose, and galactose (Fig. 5). While pretreatment with myo-inositol enhanced

408

the levels of glucose and galactose in stressed plants, it did not influence the amounts of sucrose or

409

fructose. This indicated that myo-inositol could only partially regulate the accumulation of soluble

410

sugars to withstand the effects of salt stress. Sugar alcohols such as sorbitol (Gao et al., 2001),

411

mannitol (Abebe et al., 2003), myo-inositol (Kumari and Parida, 2018), and its methylated

412

derivative pinitol (Krasensky and Jonak, 2012) are also largely accumulated and help protect

413

plants under salt stress. In fact, myo-inositol can be catalyzed to ononitol by inositol

414

O-methyltransferase (IMT) before the ononitol is then epimerized to pinitol (Bohnert and Jensen,

415

1996). In Nicotiana tabacum, the overexpression of MIPS and IMT from halo-tolerant plants

416

increases the concentrations of myo-inositol, pinitol, and ononitol, and also enhances salt tolerance

417

(Majee et al., 2004; Patra et al., 2010). Here, exogenous supplementation with myo-inositol

418

induced the expression of two myo-inositol synthesis genes -- MIPS1 and IMP -- and boosted the

AC C

EP

TE D

M AN U

SC

RI PT

400

ACCEPTED MANUSCRIPT 419

accumulation of myo-inositol under saline conditions. We also determined that myo-inositol regulated metabolic changes further downstream (Fig.

421

7). Myo-inositol can be transferred to raffinose by sequential catalysis of GolS and RS (Nishizawa

422

et al., 2008). The raffinoses also have an important role in osmoprotection and ROS-scavenging

423

(ElSayed et al., 2014). Results from our qRT-PCR analysis showed that transcripts of GolS and RS

424

were induced by salinity and were even more abundant in the ST+MI group (Fig. 7). This is

425

consistent with previous reports that both GolS and RS are up-regulated by multiple types of

426

stresses to increase the accumulation of galactinol and raffinose (ElSayed et al., 2014; Lahuta et

427

al., 2014). Taken together, we have determined that myo-inositol alleviates salt-induced inhibition

428

of growth by Malus hupehensis, not only acting in antioxidant defenses but also mediating Na+

429

and K+ homeostasis and the osmotic balance.

M AN U

SC

RI PT

420

431

5. Conclusions

TE D

430

We discovered here that pre-treating apple roots with 50 µM myo-inositol prior to the

433

imposition of salt stress partially alleviates the inhibitory effect that salinity would normally have

434

on whole-plant growth. Such pretreatment also significantly slowed the declines in photosynthetic

435

parameters and total chlorophyll concentrations that are generally associated with saline

436

conditions. This supplementation reduced the extent of salt-related oxidative damage by

437

improving the plant’s antioxidant system. Levels of Na+ and Na:K ratio were also lower in

438

pretreated plants under saline conditions because of the positive effect this compound has on

439

osmotic stress and ion toxicity. Concentrations of soluble sugars and sugar alcohols in those

440

stressed plants, as well as the expression of genes for myo-inositol metabolism, were modified by

AC C

EP

432

ACCEPTED MANUSCRIPT this exogenous supplement. These responses suggest that, as an important metabolite (or signal),

442

myo-inositol can regulate ionic and osmotic homeostasis and ROS-scavenging. With this scientific

443

foundation, we can conduct future explorations of the mechanism by which myo-inositol improves

444

salt tolerance in apple plants.

RI PT

441

445 446

Author contributions

L. Hu and F. Ma designed the experiments; L. Hu performed the experiments with assistance

448

from K. Zhou, Y. Li, X. Chen, B. Liu, and C. Li; L. Hu and K. Zhou performed the data analyses

449

and wrote the manuscript; and F. Ma and X. Gong critically revised the article.

450

Conflict of interest

M AN U

451

SC

447

The authors declare that there are no conflicts of interest. Acknowledgements

453

This work was supported by the earmarked fund for the China Agriculture Research System

454

(CARS-27). The authors are grateful to Priscilla Licht for help in revising our English

455

composition.

EP

456

TE D

452

References

458

Abebe, T., Guenzi, A.C., Martin, B., Cushman, J.C., 2003. Tolerance of mannitol-accumulating

459

AC C

457

transgenic wheat to water stress and salinity. Plant Physiol. 131, 1748-1755.

460

Bai, T.H., Li, C.Y., Li, C., Liang, D., Ma, F.W., 2013. Contrasting hypoxia tolerance and

461

adaptation in Malus species is linked to differences in stomatal behavior and photosynthesis.

462

Physiol. Plant. 147, 514-523.

463

Bertrand, A., Dhont, C., Bipfubusa, M., Chalifour, F.P., Drouin, P., Beauchamp, C.J., 2015.

ACCEPTED MANUSCRIPT 464

Improving salt stress responses of the symbiosis in alfalfa using salt-tolerant cultivar and

465

rhizobial strain. Appl. Soil Ecol. 87, 108-117.

467 468 469

Bohnert, H.J., Jensen, R.G., 1996. Strategies for engineering water stress tolerance in plants. Trends Biotechnol. 14, 89-97.

RI PT

466

Brini, F., Masmoudi, K., 2012. Ion transporters and abiotic stress tolerance in plants. ISRN Mol. Biol. 3, 1-13.

Chaouch, S., Noctor, G., 2010. Myo-inositol abolishes salicylic acid-dependent cell death and

471

pathogen defence responses triggered by peroxisomal hydrogen peroxide. New Phytol. 188,

472

711-718.

476 477 478 479 480 481

M AN U

Chen, H., Xiong, L., 2010. Myo-inositol-1-phosphate synthase is required for polar auxin transport

TE D

475

cepa: prevention by inositol pretreatment. Protoplasma 245, 165-172.

and organ development. J. Biol. Chem. 285, 24238-24247. Chinnusamy, V., Zhu, J.H., Zhu, J.K., 2006. Salt stress signaling and mechanisms of plant salt tolerance. Genet. Eng. 27, 141-177.

EP

474

Chatterjee, J., Majumder, A.L., 2010. Salt-induced abnormalities on root tip mitotic cells of Allium

Cui, M., Liang, D., Wu, S., Ma, F.W., Lei, Y.S., 2013. Isolation and developmental expression

AC C

473

SC

470

analysis of L-myo-inositol-1-phosphate synthase in four Actinidia species. Plant Physiol. Biochem. 73, 351-358.

482

Dai, W.S., Wang, M., Gong X.Q., Liu, J.H., 2018. The transcription factor FcWRKY40 of

483

Fortunella crassifolia functions positively in salt tolerance through modulation of ion

484

homeostasis and proline biosynthesis by directly regulating SOS2 and P5CS1 homologs.

485

New Phytol. doi: 10.1111/nph.15240.

ACCEPTED MANUSCRIPT

487 488 489

Das, K., Roychoudhury, A., 2014. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Front. Environ. Sci. 2, 53. Dionisio-Sese, M.L., Tobita, S., 1998. Antioxidant response of rice seedlings to salinity stress. Plant Sci. 135, 1-9.

RI PT

486

Donahue, J.L., Alford, S.R., Torabinejad, J., Kerwin, R.E., Nourbakhsh, A., Ray, W.K., Hernick,

491

M., Huang, X., Lyons, B.M., Hein, P.P., Gillaspy, G.E., 2010. The Arabidopsis thaliana

492

myo-inositol 1-phosphate synthase1 gene is required for myo-inositol synthesis and

493

suppression of cell death. Plant Cell 22, 888-903.

495

M AN U

494

SC

490

ElSayed, A.I., Rafudeen, M.S., Golldack, D., 2014. Physiological aspects of raffinose family oligosaccharides in plants: protection against abiotic stress. Plant Biol. 16, 1-8. Gao, M., Tao, R., Miura, K., Dandekar, A.M., Sugiura, A., 2001. Transformation of Japanese

497

persimmon (Diospyros kaki Thunb.) with apple cDNA encoding NADP-dependent

498

sorbitol-6-phosphate dehydrogenase. Plant Sci. 160, 837-845.

TE D

496

Gil, R., Lull, C., Boscaiu, M., Bautista, I., Lidón, A., Vicente, O., 2011. Soluble carbohydrates as

500

osmolytes in several halophytes from a Mediterranean salt marsh. Not. Bot. Hortic. Agrobot.

501

39, 9-17.

AC C

EP

499

502

Goswami, L., Sengupta, S., Mukherjee, S., Ray, S., Mukherjee, R., Majumder, A.L., 2014.

503

Targeted expression of L-myo-inositol 1-phosphate synthase from Porteresia coarctata

504 505 506 507

(Roxb.) Tateoka confers multiple stress tolerance in transgenic crop plants. J. Plant Biochem. Biotechnol. 23, 316-330.

Hasegawa, P.M., Bresson, R.A., Zhu, J.K., Bohnert, H.J., 2000. Plant cellular and molecular responses to high salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 463-499.

ACCEPTED MANUSCRIPT 508

Himabindu, Y., Chakradhar, T., Reddy, M.C., Kanygin, A., Redding, K.E., Chandrasekhar, T.,

509

2016. Salt-tolerant genes from halophytes are potential key players of salt tolerance in

510

glycophytes. Environ. Exp. Bot. 124, 39-63. Hoagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plants without soil.

512

California Agriculture Experiment Station Circular 347. College of Agriculture, University of

513

California, Berkeley, CA, USA.

RI PT

511

Hu, L.X., Zhang, P.P., Jiang, Y., Fu, J.M., 2015. Metabolomic analysis revealed differential

515

adaptation to salinity and alkalinity stress in Kentucky bluegrass (Poa pratensis). Plant Mol.

516

Biol. Rep. 33, 56-68.

518

M AN U

517

SC

514

Ismail, A., Takeda, S., Nick, P., 2014. Life and death under salt stress: same players, different timing? J. Exp. Bot. 65, 2963-2979.

Kaur, H., Verma, P., Petla, B.P., Rao, V., Saxena, S.C., Majee, M., 2013. Ectopic expression of the

520

ABA-inducible dehydration-responsive chickpea L-myo-inositol 1-phosphate synthase 2

521

(CaMIPS2) in Arabidopsis enhances tolerance to salinity and dehydration stress. Planta 237,

522

321-335.

524

EP

Krasensky, J., Jonak, C., 2012. Drought, salt, and temperature stress-induced metabolic

AC C

523

TE D

519

rearrangements and regulatory networks. J. Exp. Bot. 63, 1593-1608.

525

Kumari, A., Parida, A.K., 2018. Metabolomics and network analysis reveal the potential

526

metabolites and biological pathways involved in salinity tolerance of the halophyte

527

Salvadora persica. Environ. Exp. Bot. 148, 85-99.

528

Kusuda, H., Koga, W., Kusano, M., Oikawa, A., Saito, K., Hirai, M.Y., Yoshida, K.T., 2015.

529

Ectopic expression of myo-inositol 3-phosphate synthase induces a wide range of metabolic

ACCEPTED MANUSCRIPT 530

changes and confers salt tolerance in rice. Plant Sci. 232, 49-56. Lahuta, L.B., Pluskota, W.E., Stelmaszewska, J., Zablinska, J., 2014. Dehydration induces

532

expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of

533

pea (Pisum sativum L.). J. Plant Physiol. 171, 1306-1314.

RI PT

531

Li, C., Wang, P., Wei, Z.W., Liang, D., Liu, C.H., Yin, L.H., Jia, D.F., Fu, M.Y., Ma, F.W., 2012.

535

The mitigation effects of exogenous melatonin on salinity-induced stress in Malus

536

hupehensis. J. Pineal. Res. 53, 298-306.

SC

534

Li, C., Wei, Z.W., Liang, D., Zhou, S.S., Li, Y.H., Liu, C.H., Ma, F.W., 2013. Enhanced salt

538

resistance in apple plants overexpressing a Malus vacuolar Na+/H+ antiporter gene is

539

associated with differences in stomatal behavior and photosynthesis. Plant Physiol. Biochem.

540

70, 164-173.

542

Li, C., Sun, X.K., Chang, C., Jia, D.F., Wei, Z.W., Li, C.Y., Ma, F.W., 2015. Dopamine alleviates

TE D

541

M AN U

537

salt-induced stress in Malus hupehensis. Physiol. Plant. 153, 584-602. Liang, B.W., Li, C.Y., Ma, C.Q., Wei, Z.W., Wang, Q., Huang, D., Chen, Q., Li, C., Ma, F.W.,

544

2017. Dopamine alleviates nutrient deficiency-induced stress in Malus hupehensis. Plant

545

Physiol. Biochem. 119, 346-359.

547

AC C

546

EP

543

Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 25, 402-408.

548

Loewus, F.A., Murthy, P.P.N., 2000. Myo-inositol metabolism in plants. Plant Sci. 150, 1-19.

549

Majee, M., Maitra, S., Dastidar, K.G., Pattnaik, S., Chatterjee, A., Hait, N.C., Das, K.P., Majumder,

550

A.L., 2004. A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia

551

coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial

ACCEPTED MANUSCRIPT 552

overexpression, characterization, and functional introgression into tobacco-conferring salt

553

tolerance phenotype. J. Biol. Chem. 279, 28539-28552.

555

Miller, G., Suzuki, N., Ciftci-Yilmaz, S., Mittler, R., 2010. Reactive oxygen species homeostasis and signaling during drought and salinity stresses. Plant Cell Environ. 33, 453-467.

RI PT

554

Møller, I.S., Gilliham, M., Jha, D., Mayo, G.M., Roy, S.J., Coates, J.C., Haseloff, J., Tester, M.,

557

2009. Shoot Na+ exclusion and increased salinity tolerance engineered by cell type-specific

558

alteration of Na+ transport in Arabidopsis. Plant Cell 21, 2163-2178.

563 564 565 566 567 568 569 570

M AN U

562

Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-497.

Nelson, D.E., Koukoumanos, M., Bohnert, H.J., 1999. Myo-inositol-dependent sodium uptake in

TE D

561

651-681.

ice plant. Plant Physiol. 119, 165-172.

Netondo, G.W., Onyango, J.C., Beck, E., 2004. Sorghum and salinity. II. Gas exchange and chlorophyll fluorescence of sorghum under salt stress. Crop Sci. 44, 806-811.

EP

560

Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59,

Nishizawa, A., Yabuta, Y., Shigeoka, S., 2008. Galactinol and raffinose constitute a novel function

AC C

559

SC

556

to protect plants from oxidative damage. Plant Physiol. 147, 1251-1263.

Parida, A.K., Das, A.B., 2005. Salt tolerance and salinity effects on plants: a review. Ecotox. Environ. Safe. 60, 324-349.

571

Patra, B., Ray, S., Richter, A., Majumder, A.L., 2010. Enhanced salt tolerance of transgenic

572

tobacco plants by coexpression of PcINO1 and McIMT1 is accompanied by increased level of

573

myo-inositol and methylated inositol. Protoplasma 245, 143-152.

ACCEPTED MANUSCRIPT 574

Redillas, M.C.F.R., Park, S.H., Lee, J.W., Kim, J.W., Jeong, J.S., Jung, H., Bang, S.W., Hahn, T.,

575

Kim, J., 2012. Accumulation of trehalose increases soluble sugar contents in rice plants

576

conferring tolerance to drought and salt stress. Plant Biotechnol. Rep. 6, 89-96. Rosa, M., Prado, C., Podazza, G., Interdonato, R., Gonzalez, J.A., Hilal, M., Prado, F.E., 2009.

578

Soluble sugars - metabolism, sensing and abiotic stress. Plant Signal. Behav. 4, 388-393.

579

Shao, Y., Qin, Y., Zou, Y.J., Ma, F.W., 2014. Genome-wide identification and expression profiling of the SnRK2 gene family in Malus prunifolia. Gene 552, 87-97.

SC

580

RI PT

577

Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y., Yoshimura, K., 2002.

582

Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 53, 1305-1319.

583

Suzuki, K., Yamaji, N., Costa, A., Okuma, E., Kobayashi, N. I., Kashiwagi, T., Katsuhara, M.,

584

Wang, C., Tanoi, K., Murata, Y., Schroeder, J.I., Ma, J.F., Horie, T., 2016. OsHKT1;

585

4-mediated Na+ transport in stems contributes to Na+ exclusion from leaf blades of rice at the

586

reproductive growth stage upon salt stress. BMC Plant Biol. 16, 22.

TE D

M AN U

581

Tan, J.L., Wang, C.Y., Xiang, B., Han, R.H., Guo, Z.F., 2013. Hydrogen peroxide and nitric oxide

588

mediated cold- and dehydration-induced myo-inositol phosphate synthase that confers

589

multiple resistances to abiotic stresses. Plant Cell Environ. 36, 288-299.

591

AC C

590

EP

587

Valluru, R., Ende, W.V.D., 2011. Myo-inositol and beyond-emerging networks under stress. Plant Sci. 181, 387-400.

592

Wang, H.C., Ma, F.F., Cheng, L.L., 2010. Metabolism of organic acids, nitrogen and amino acids

593

in chlorotic leaves of ‘Honeycrisp’ apple (Malus domestica Borkh) with excessive

594

accumulation of carbohydrates. Planta 232, 511-522.

595

Wang, P., Yin, L.H., Liang, D., Li, C., Ma, F.W., Yue, Z.Y., 2012. Delayed senescence of apple

ACCEPTED MANUSCRIPT 596

leaves by exogenous melatonin treatment: toward regulating the ascorbate–glutathione cycle.

597

J. Pineal Res. 53, 11-20.

599 600 601

Yang, Y.Q., Guo, Y., 2018. Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytol. 217, 523-539.

RI PT

598

Ye, W.X., Ren, W.B., Kong, L.Q., Zhang, W.J., Wang, T., 2016. Transcriptomic profiling analysis of Arabidopsis thaliana treated with exogenous myo-inositol. PLoS One 11, e0161949.

Zhai, H., Wang, F.B., Si, Z.Z., Huo, J.X., Xing, L., An, Y.Y., He, S.Z., Liu, Q.C., 2016. A

603

myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and

604

stem nematode resistance in transgenic sweet potato. Plant Biotechnol. J. 14, 592-602.

M AN U

SC

602

Zhou, S.S., Li, M.J., Guan, Q.M., Liu, F.L., Zhang, S., Chen, W., Yin, L.H., Qin, Y., Ma, F.W.,

606

2015. Physiological and proteome analysis suggest critical roles for the photosynthetic

607

system for high water-use efficiency under drought stress in Malus. Plant Sci. 236, 44-60.

TE D

605

Zhu, J.K., 2001. Plant salt tolerance. Trends Plant Sci. 6, 66-71.

609

Zhu, Y.X., Guo, j., Feng, R., Jia, J.H., Han, W.H., Gong, H.J., 2016. The regulatory role of silicon

610

on carbohydrate metabolism in Cucumis sativus L. under salt stress. Plant Soil. 406, 231-249.

EP

608

612

AC C

611

Table 1 Sequences of primers for qRT-PCR. Gene

Forward (5ʹ-3ʹ)

Reverse (5ʹ-3ʹ)

EF-1α SOS1 SOS2 HKT1 NHX1 MIPS1 IMP MIOX GolS RS

ATTCAAGTATGCCTGGGTGC TCCGGTTAATCCATCACACACCGT ACACGGGGAGGTAGTGACAA TCGTTCGCTATTTCGTGTCCTGCT AAGCGACAGTCCTGGAACATCAGT AGGCCAAGTTCCACTCATTCCAC GCCCTATGTGTTGGAATACCCG ATCCTTTACATAGGGCAGGGGCATA GCCAAGGGGTTGAGAAAGGTAAAAA GGTGCTGATTGGAATGGAGAAACA

CAGTCAGCCTGTGATGTTCC TTTGCTGCCCTGGAGGATTTGTTG CCTCCAATGGATCCTCGTTA TGGGCCTGAAAGAAGTGTTTGTGC TATTATCACTTGCTGCCGGAGGCT CAAGCCCTCAGTATGTTCTCCAG CACCTCATCAGTTTGGCTCACG TGGCTTAACTTTTTCGACATCAACTCG GGGTAAACGGGTTCGATCTCACG CCTTGAGAGGACAGAAATGGAAAAC

ACCEPTED MANUSCRIPT

Table 2 Effects of myo-inositol pretreatment (1-150 µM) on salt injury index (SI) for stressed Malus hupehensis plants. Day 6 Treatment group

SI

Decline

SI

Decline

ST ST + 1 µM ST + 10 µM ST + 50 µM ST + 100 µM ST + 150 µM

42.5% 40.8% 40.8% 33.3% 36.8% 35%

0 4% 4% 21.6% 13.4% 17.6%

60.4% 52.8% 44.8% 39.6% 40.6% 62.7%

0 12.6% 25.8% 34.4% 32.8% -3.8%

M AN U

TE D

Notes: Decline indicates relative degree of alleviation from damage due to salt stress if plant roots were pre-treated with exogenous myo-inositol. Treatment group ST: ½-strength Hoagland’s nutrient solution supplemented with 200 mM NaCl.

Table 3 Effects of 50 µM myo-inositol pretreatment on plants grown hydroponically for 12 d in the presence of 200 mM NaCl. Plant height (cm) Mean ± SD (%)

TDW (g) Mean ± SD (%)

TFW (g) Mean ± SD (%)

RGR (g kg-1 d-1) Mean ± SD (%)

CK MI ST ST+MI

21.88±0.81 (100) a 24.82±0.76 (113.6) b 17.27±0.59 (78.9) c 19.62±0.57 (89.7) d

1.85±0.06 (100) a 2.14±0.04 (115.7) b 1.27±0.07 (68.6) c 1.48±0.06 (80) d

3.56±0.23 (100) a 4.09±0.12 (114.9) b 2.49±0.14 (69.9) c 3.08±0.10 (86.5) d

42.08±2.47 (100) a 49.87±2.41 (118.5) b 24.16±1.98 (57.4) c 32.69±3.28 (77.7) d

EP

Treatment group

AC C

622 623 624 625 626 627 628 629

Day 11

SC

614 615 616 617 618 619 620 621

RI PT

613

630 631 632 633 634 635 636 637 638 639

Note: TDW (total dry weight) and TFW (total fresh weight) were measured from two-plant replicates; all data are means of six replicates ± SD. Within a column, values not followed by the same letter are significantly different (P <0.05). CK, control group; MI, group with 50 µM myo-inositol treatment under normal condition. ST, salt stress group; ST+MI, group with 50 µM myo-inositol treatment under salt stress. For each growth parameter, data in brackets indicate the percentage of the mean relative to the mean for the CK group.

640

Fig. 1. (A) Performance of aerial parts of Malus hupehensis plants grown hydroponically for 8 d,

Figure legends

ACCEPTED MANUSCRIPT 641

and (B) Architecture of Malus hupehensis root system on Day 12. CK, control; ST, 200 mM NaCl

642

treatment; MI, 50 µM myo-inositol-pretreated control; ST+MI, 50 µM myo-inositol pretreatment

643

under 200 mM NaCl. Data are means ± SD. Values not followed by same letter are significantly

644

different (P <0.05).

645 Fig. 2. (A) Photosynthetic indices of Malus hupehensis plants on Day 8, and (B) stomatal density

647

and stomatal aperture on Day 12, and (C) SEM images of stomata on Day 12. Data are means ±

648

SD. Values not followed by same letter are significantly different (P <0.05).

RI PT

646

SC

649

Fig. 3. (A) Observations on Day 12 from DAB- and NBT- staining of leaves, and (B) Chlorophyll

651

concentrations, relative electrolyte leakage, H2O2 concentrations in leaves from plants exposed to

652

200 mM NaCl. Data are means ± SD. Values not followed by same letter are significantly different

653

(P <0.05).

M AN U

650

654

Fig. 4. (A) activity of SOD and POD in leaves, and (B) activity of MDHAR, DHAR, GR, and

656

APX in leaves from plants exposed to 200 mM NaCl. FW, leaf fresh weight. Data are means ± SD.

657

Values not followed by same letter are significantly different (P <0.05).

658

TE D

655

Fig. 5. Effects of 50 µM myo-inositol pretreatment on levels of soluble sugars and sugar alcohols

660

in leaves from plants exposed to 200 mM NaCl. FW, leaf fresh weight. Data are means ± SD.

661

Values not followed by same letter are significantly different (P <0.05).

AC C

662

EP

659

663

Fig. 6. Effects of 50 µM myo-inositol pretreatment on ionic balance in leaves from plants exposed

664

to 200 mM NaCl. (A) Na+ and K+ concentrations and Na:K ratio, and (B) expression of genes

665

involved in ion homeostasis. Data are means ± SD. Values not followed by same letter are

666

significantly different (P <0.05).

667 668

Fig. 7. Effects of 50 µM myo-inositol pretreatment on expression of genes for myo-inositol

669

synthesis and metabolism in leaves from plants exposed to 200 mM NaCl. Data are means ± SD.

670

Values not followed by same letter are significantly different (P <0.05).

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 1.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 2.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 3.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 4.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 5.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 6.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 7.

ACCEPTED MANUSCRIPT Highlights: 1. Myo-inositol pretreatment alleviates the salt-induced damages in Malus hupehensis Rehd. seedlings.

RI PT

2. Myo-inositol enhances the activities of antioxidant systems and inhibits ROS accumulation induced by salinity.

accumulation under saline conditions.

SC

3. Myo-inositol promotes the expression of genes involved in Na+ uptake and reduces Na+

4. Myo-inositol provokes the accumulation of sugars or sugar alcohols and contributes to the

AC C

EP

TE D

M AN U

maintenance of osmotic balance under salt stress.

ACCEPTED MANUSCRIPT Contribution: L. Hu and F. Ma designed the experiments; L. Hu performed the experiments with assistance from K. Zhou, Y. Li, X. Chen, B. Liu, and C. Li; L. Hu and K. Zhou performed the data analyses

AC C

EP

TE D

M AN U

SC

RI PT

and wrote the manuscript; and F. Ma and X. Gong critically revised the article.