H2O2/ABA Signal Pathway Participates in the Regulation of Stomata Opening of Cucumber Leaves Under Salt Stress by Putrescine

bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272120; this version posted August 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 H2O2/ABA signal pathway participates in the regulation of

2 stomata opening of cucumber leaves under salt stress by

3 putrescine 4 Siguang Ma, Mohammad Shah Jahan, Shirong Guo, Mimi Tian, Ranran Zhou, Hongyuan Liu, Bingjie

5 Feng, Sheng Shu*

6 College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

7 E-mail address for each author: 8 Siguang Ma: [email protected] Shirong Guo: [email protected] 9 Mohammad Shah Jahan: [email protected] Mimi Tian: [email protected] 10 Ranran Zhou: [email protected] Hongyuan Liu: [email protected] 11 Bingjie Feng: [email protected] Sheng Shu*: [email protected] 12 13 Date of submission: August 28, 2020 14 Number of supplementary tables are 1; Number of figures are 9; Number of supplementary figures are 3. 15 Number of word count: 6363. 16 17 Highlight 18 Exogenous putrescine alleviates photosynthesis inhibition in salt-stressed cucumber seedlings by regulating 19 stomatal-aperture. 20 21 Abstract 22 The stomatal-aperture is imperative for plant physiological metabolism. The function of polyamines (PAs) in 23 stomatal regulation under stress environment largely remains elucidate. Herein, we investigated the regulatory 24 mechanism of exogenous putrescine (Put) on the stomatal opening of cucumber leaves under salt stress. The 25 results revealed that Put relieved the salt-induced photosynthetic inhibition of cucumber leaves by regulating

26 stomatal-apertures. Put application increased hydrogen peroxide (H2O2) and decreased abscisic acid (ABA) 27 content in leaves under salt stress. The inhibitors of diamine oxidase (DAO), polyamine oxidase (PAO), 28 nicotinamide adenine dinucleotide phosphate oxidase (NADPH) are AG, 1,8-DO and DPI, respectively and 29 pre-treatment with these inhibitors up-regulated key gene NCED of ABA synthase and down-regulated key gene

30 GSHS of reduced glutathione (GSH) synthase. The content of H2O2 and GSH were decreased and ABA content 31 was increased and its influenced trend is AG>1,8-DO>DPI. Moreover, the Put induced down-regulation of ABA

32 content under salt stress blocked by treatment with H2O2 scavenger (DMTU) and GSH scavenger (CNDB). 33 Additionally, the application of DMTU also blocked the increase of GSH content. Collectively, these results

34 suggest that Put can regulate GSH content by promoting H2O2 generation through polyamine metabolic pathway, 35 which inhibits ABA accumulation to achieve stomatal regulation under salt stress. 36 Keywords: Abscisic acid, Cucumber, Hydrogen peroxide, Putrescine, Glutathione, Salt stress, Stomata 37 38 Introduction 39 In order to meet the growing food demand, it's urgent to develop a stress resistance adaptive mechanism, which 40 helps to increase crop yield (Fahad et al., 2015). Carbon dioxide absorption capacity of the most plant leaves

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41 become lower due to the salt stress (Elbasan et al., 2020), and has a significant effect on net photosynthetic rate

42 (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular carbon dioxide concentration (Ci) (Moles

43 et al., 2016; Elbasan et al., 2020). If Ci decreases with the decrease of Gs, the decrease of Pn is caused by stomatal 44 limitation, otherwise, it is caused by non-stomatal limitation. Therefore, the effects of salt stress on plants can be 45 divided into stomatal limitation period and non-stomatal limitation period (Hejnak et al., 2016; Xia et al., 2017; 46 Yang et al., 2019). At the early stage of salt stress, stomatal closure is the main limiting factor (Sudhir et al., 2004; 47 Yildiztugay et al., 2020) and decrease the activity of ribose 1,5-diphosphate carboxylase/oxygenase (Rubisco), 48 destroy the photosynthetic system, and the chloroplast structure/function or thylakoid energy over excitation are 49 the main limiting factors, at the later stage of salt stress (Chen et al., 2020; Sudhir et al., 2004; Demetriou et al.,

50 2007; Shu et al., 2012). In turn, Pn function inhibited and carbohydrate metabolism becomes destroyed, thus 51 reducing dry matter accumulation (Jurczyk et al., 2016). Therefore, it is essential to increase stomatal pore size 52 during stomatal limitation period to resist salt stress injury. 53 In recent years, a large number of research reports on the role of PAs and ABA in resistance of higher plants to salt 54 stress has been published. Putrescine (Put), spermidine (Spd) and spermine (Spm) are the most abundant PAs in 55 plants, which are state in the free, conjugated and bound form in plants (Pál et al., 2015). One of the main 56 functions of PAs is to balance the ROS (reactive oxygen species) level that induced by NaCl stress and provide a 57 suitable physiological environment for cell survival and metabolism (Saha et al., 2015; Seo et al., 2019).

58 Exogenous Put could effectively improve Gs and Pn of cucumber leaves under salt stress (Zhang et al., 2009). In 59 addition, PAs may also act as stress messengers in plant response to different stress signals, as a regulatory signal 60 molecule, induce different transcriptional responses (Liu et al., 2007; DU et al., 2019; Marco et al., 2011). ABA, 61 as a plant hormone, plays an important role in the adaptive response to abiotic stresses that cause cell water loss

62 (Zheng et al., 2019). Since, Gs is largely controlled by ABA, the change of ABA concentration is often used to 63 explain the change of stomatal morphology (Verslues et al., 2006). Studies have shown that PAs participates in 64 signal transduction through the interaction with other hormones (Bitrián et al., 2012), such as ABA (Alert et al., 65 2011; Zhu et al., 2020). Espasandin et al. (2014) found that biochemical and morphological responses of cell water 66 loss were related to Put level and Put regulated ABA biosynthesis at the transcriptional level to control ABA 67 content in response to drought, indicating that these two plant growth regulators have complex cross-sectional 68 effects in stress response. However, it is still unclear how to Put interacts with ABA to regulate stomatal changes 69 during stomatal limitation period under salt stress and hence, we conducted the current experiment to extends our

70 knowledge about this regulation. H2O2 is produced by the catalysis of PAs catabolism enzyme DAO and PAO,

71 which are located in the apoplast and plays an important role in plant defense against abiotic stresses (Alcázar et

72 al., 2010; Tavladoraki et al., 2012; Guo et al., 2014; Fraudentali et al., 2019). The other two enzymes that catalyze

73 the production of H2O2 are NOX and CWPOD (Rejeb et al., 2015; Passardi et al., 2004). It is noteworthy that GSH

74 acts downstream of H2O2 in a number of processes, and GSH has been shown to negatively regulates 75 ABA-induced stomatal closure (Ogawa, 2005; Okuma et al., 2011; Akter et al., 2013; Asgher et al., 2020).

76 Therefore, it is imperative to solve whether and how H2O2 and GSH play a role in PAs induced stomatal response 77 as well as find out the crosstalk between PAs and ABA. 78 We mainly explored the positive regulatory effect of exogenous Put on cucumber leaf stomata under salt stress and

79 speculated the interaction relationship among the Put, ABA, H2O2 and GSH, and finally concluded the internal 80 mechanism on how exogenous Put improving cucumber photosynthetic capacity under salt stress. 81 82 Materials and methods 83 Plant material and treatments

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84 Cucumber variety "Jinyou 4" was used in this experiment, which was provided by Tianjin Kerun Research 85 Institute, China. Uniform seeds were soaked in distilled water followed by placed into a constant temperature 86 shaker (28 ± 1°C, 200 rpm) for germination. After 24 h, germinated seeds were sown in a 32 hole seedling tray 87 filled with quartz substrates, and cultured in Solar Greenhouse at Nanjing Agricultural University, where the 88 diurnal temperature 28 ± 1°C/19 ± 1°C (day/night), and the relative humidity 60-75% were maintained. After 89 second true leaves were fully expanded, seedlings were transplanted into plastic containers for pre-culture with 90 half-strength nutrient solution (pH 6.5 ± 0.1, EC 2.0-2.2 mS·cm−1), and air pump was used to stabilize dissolved 91 oxygen (40 mh-1). When third true leaves were developed, the following treatments were applied: the seedlings 92 cultured in half-strength Hoagland nutrient solution with or without 75 mM NaCl. The leaves were sprayed with 8 93 mM Put, 0.1 mM glutathione ethyl ester (GSHmee), 2 mM D-arginine (D-Arg, an inhibitor of putrescine 94 synthesis), 10 mM aminoguanidine hydrochloride (AG, ketamine oxidase inhibitor), 2 mM,1,8-octanediamine 95 (1,8-DO, polyamine oxidase inhibitor), 1 mM diphenyl iodide chloride (DPI, NADPH oxidase inhibitor), 5 mM 96 salicylyl hydroxamic acid (SHAM), 0.1 mM 1-chloro-2,4-dinitrobenzene (CNDB, GSH scavenger). The 97 concentrations of the above treatments were determined according to the preliminary-experiments. Before each 98 treatment, the inhibitors were sprayed in leaves 12 and 6 h in advance and placed in incubator with light intensity 99 of 1000 μmol·m-2·S-1 for 6 h to ensure the completely opened the stomata. Leaves were sprayed with Put every day 100 at 5.00 p.m. The second functional leaf was collected and quickly put into liquid nitrogen and stored in -80°C for 101 subsequent analysis. 102 Determination of growth indexes, photosynthetic parameters and leaf surface temperature 103 The plant height, stem diameter and fresh weight of the above-ground of cucumber seedlings were measured with 104 a ruler, digital caliper and electronic balance, respectively. To determine the dry weight seedlings were dried at

105 100°C for 48 h. Net photosynthetic rate (Pn), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) 106 were measured using a portable photosynthesis system (Li-6400, USA) at 9:00 to 11:00 a.m. The leaf chamber -2 -1 -1 107 temperature (25 ± 1)°C, the light intensity 1000 μmol·m ·s , CO2 concentration (380 ± 10) μmol·L , and the 108 relative humidity 60%-70% were maintained during the data measurement. The stomatal limitation was

109 determined with this formula: Ls=(Ca-Ci)/Ca*100%, where, Ca is the atmospheric CO2 concentration. The second 110 functional leaf was selected and measured by infrared thermal imager (Testo 8751, Testo, Schwarzwald, Germany), 111 and the image was processed by Testo Comfort Software Basic 5.0 (Testo, Schwarzwald, Germany). 112 Determination of stomatal index 113 The area near the central vein of the second functional leaf of the seedling was finely cut. Gently scrape off the 114 mesophyll on the transparent tape with flat tweezers and water. The adhesive tape with lower skin is made into 115 pieces. The slides were observed with a fluorescence microscope (Leica DM2500, Solms, Germany) at 10×20 116 times, and photos were taken by Leica application suite V3 (Leica, Solms, Germany). The length and width of 117 pores were measured by ImageJ software (Rawak Software Inc., Stuttgart, Germany). At least 40 stomas were 118 randomly selected for each treatment.

119 Determination of H2O2 and reduced glutathione (GSH) content in guard cells

120 According to An et al. (2016), the content of H2O2 was determined. The changes of H2O2 content in guard cells

121 were observed by 2, 7- dichlo rodihydrofluorescein diacetate (H2DCF-DA; Sigma, USA). The leaves were cut and

122 placed in the buffer solution containing of 50 μM H2DCF-DA (50 mM KCl, 0.1 mM CaCl2, and 10 mM MES, pH 123 6.1) for 20 minutes (dark). After washing three times with non-dye buffer solution, immediately strong adhesive 124 tape was used to stick to the back of the blade. Scrape off the mesophyll from the front of the blade with flat end 125 tweezers, and observed the adhesive tape with lower epidermis. The content of GSH in stomata was determined by 126 monochlorobimane (MCB, Sigma, USA) with the treatment method of Akter et al. (2010). At room temperature, 127 the epidermis of the second functional leaf near the main vein of the seedling was removed and placed for staining

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128 in a buffer solution containing of 100 μm MCB for 2 h. In guard cells, MCB and GSH form fluorescent glutathione 129 S-bimane (GSB). The images were observed by laser confocal microscope with the excitation 488/405 nm, 130 481-549/400-495 nm excitation signals were collected to observe DCF and GSB; Zeiss LSM 800 META. 131 Chloroplast auto-fluorescence was used as control (561nm excitation, 562-700nm excitation signal). The image 132 was analyzed with ImageJ software (Rawak Software Inc ., Stuttgart, Germany), and each process was repeated 133 three times. The image obtained by CLSM with Carl Zeiss LSM software (ZEN 2018) using a 20x magnification 134 objective.

135 Determination of H2O2 and reduced glutathione content in leaves

136 The H2O2 content in leaves was estimated according to the method of Alexieva et al. (2001). For the calculation of 137 GSH, we followed the Rao et al. (1995) method. 138 Determination of polyamine content 139 The content of polyamine in leaves was determined according to Liu et al. (2002). 0.3g fresh leaves sample was 140 homogenized in 1.6 mL of precooled 5% perchloric acid (PCA) followed by kept in an ice bath for 1 h and then 141 centrifuged at 12000 rpm at 4°C for 20 min. The supernatant was collected to estimate the contents of free and 142 conjugated PAs, and the resulting precipitate was used to assay bound PAs. For estimation of free and conjugated 143 PAs, 0.7 mL of supernatant was transferred into a new tube, and 1.4 mL of 2 mol L-1 NaOH and 15 μL benzoyl 144 chloride were added with this supernatant, and the mixers were vortexed for 20 s followed by incubation at 37°C 145 for 30 min. After incubation, 2 mL saturated NaCl and 2 mL precooled ether were added in above mixers for 146 oscillation, and the whole solution was gently shaken followed by centrifugation at 3000 rpm for 5min. Later, 1mL 147 of ether phase solution was dried, and then re-dissolved it with the addition of 100 μL methanol (60% W/V) and 148 20 μL solution was injected to determine the free and conjugated PAs. For estimation of bound PAs, the remaining 149 precipitation was washed again with 5% PCA, followed by centrifugation at 1000 rpm for 5min to remove the 150 supernatant, and repeated it for 4 times. To suspension the precipitation, added 5 mL of 6 mol L-1 HCl and sealed 151 in an amperometric flask, hydrolyzed followed by incubation the solution at 110°C for 18 h. After incubation, the 152 solution was filtered, evaporated to dryness at 70°C, and the residue was re-suspended and dissolved with 1.6 mL 153 of 5% PCA, and centrifuged for 20 min at 12000 rpm at 4°C for 1h. The sample preparation of bound PAs 154 quantification, the above-mentioned procedure was used. The whole PAs determination was carried out by 155 ultra-performance liquid chromatography (Ultimate 3000, Thermo Scientific, San Jose, CA, USA). 156 Determination of abscisic acid content 157 For ABA extraction Dobrev and Kaminek (2002) protocol and for purification Albacete et al. (2008) method was 158 used. Briefly, 0.3 g of leaf was homogenized in 3 mL precooled 50% chromatographic methanol (v/v), and the 159 extracted was incubation at 4°C for 12h followed by centrifugation at 10000 rpm at 4°C for 10 min, and then, the 160 supernatant was stored at 4°C. Again, 2 mL of precooled 80% methanol was added to the residue, extracted at 4°C 161 for 12 h, and centrifuged at 10000 rpm at 4°C for 10 min. After that, 2mL of precooled 100% methanol was added 162 to the residue and extracted for 12 h, and centrifuged as the above condition. Finally, all the extracts were collected 163 and combined, and PVPP was added into the extract at the rate of 0.2 g-1·FW to adsorb phenols and pigments. 164 After shaking at 4°C for 60 min, centrifuged the mixed solution like the above condition. The supernatant was 165 passed slowly through the C18 column, collected in a centrifugal tube, and then kept into freeze-drying machine. 166 Thereafter, 2-5 mL of 50% methanol was added in supernatant for dissolution, passed through 0.22 μm organic 167 phase ultrafiltration membrane for determination of ABA. Chromatographic conditions: Hypersil ODS C18 168 column (250mm × 4.0mm, 5μm). The mobile phase: methanol and ultrapure water (0.5% glacial acetic acid added). 169 For determination of the quantitative value of ABA external standard calibration curve method was used, and each 170 sample repeated three times. 171 Determination of enzyme activity

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172 To determine the different enzyme activities, firstly, 0.5 g fresh sample was ground in 1.6 mL phosphoric acid 173 buffer (0.1mol L-1, pH 6.5) followed by centrifugation at 4°C for 20 min at 10000 rpm, and the supernatant was 174 collected for amine oxidase extraction. The 3 mL reaction system consisted of 0.1 M phosphate buffer, 2 mM N, 175 N-dimethylaniline, 0.5 mM 4-aminoantipyrine, 0.1 mL of 250 U mL peroxidase solution and 0.2 mL extraction 176 solution. The OD value was measured at 550 nm at 0 min and 30 min interval using an UV-visible 177 spectrophotometer (UV-2600, Unico, Shanghai, China). The activity of DAO was measured when Put was used to 178 start the reaction (Rea et al., 2004), and the PAO activity was measured by using Spm to start the reaction (Mhaske 179 et al., 2013). 180 The NOX activity was determined using plant NADPH oxidase ELISA Kit (MEIMIAN, China) with the 181 manufacturer's instructions. The CWPOD activity was measured according to Lin and Kao, (2001) using protocol. 182 Total RNA extraction and real-time fluorescent quantitative PCR (qRT-PCR) analysis 183 The total RNA content in leaves was extracted using the Total RNAsimple Kit (Tiangen Biotech Co., Ltd, Beijing, 184 China) in accordance with manufacturer’s guidelines. 1μg RNA was reverse transcribed into single-stranded 185 cDNA using the SuperScript First-strand Synthesis Kit (Takara Bio Inc., Tokyo, Japan). Gene-specific primers 186 were designed by Beacon Designer 7.9 software (Premier Biosoft International, CA, USA) based on nucleotide 187 sequences retrieved from a search of the National Center for Biotechnology Information databases (NCBI) and the 188 Cucumber Genome Database (http://cucumber.genomics.org.cn). StepOnePlus™ fluorescence quantitative PCR 189 (Applied Biosystems) and SYBR ® Premix Ex Taq™ II kit (Takara) were used for qRT-PCR target fragment. The 190 quantification of qRT-PCR was repeated three times. The thermal cycling conditions were as follows: 95°C for 5 191 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min, and finally extended at 95°C for 15 s. The relative 192 expression of genes was analyzed according to Jahan et al. (2019). The relative mRNA expression level was 193 calibrated with actin (internal standard gene) and compared with the control group. 194 195 Statistical analysis 196 At least five (5) seedlings per treatment were used to collect samples, and all measurements were repeated at least 197 3 times. Each experiment contains at least three biological replicates. All data were statistically analyzed by SPSS 198 17.0 software (SPSS Inc., Chicago, IL, USA), Comparisons of means were performed using Duncan’s multiple 199 range test at P ≤ 0.05 level of significance. The stomatal aperture data were collected from three independent 200 experiments. 201 202 Results 203 1. Effect of exogenous Put on growth of cucumber seedlings under salt stress 204 As shown in Fig. 1, after 7 days of treatment, there was no significant differences in the growth of cucumber 205 seedlings treated with Put alone. Exogenous Put significantly increased plant height, shoot fresh and dry weight 206 under salt stress. Conversely, compared with salt stress, these growth parameters increased by 36.58%, 8.12% and 207 17.75%, respectively. However, the stem diameter did not change significantly (P<0.05). These results indicated 208 that exogenous Put could alleviate salt-induced growth inhibition of cucumber seedling by increasing biomass 209 accumulation under salt stress. 210 2. Effects of exogenous Put on photosynthesis and stomatal characteristics of cucumber seedlings under salt 211 stress

212 One day (1) day after salt treatment, photosynthesis parameter namely: Pn and Gs were significantly reduced by 213 63.7% and 65%, respectively as compared with the control group (Fig. 2). On the other hand, foliar sprayed with 214 Put in salt-treated seedlings increased these components by 90.89% and 71.43%, respectively than those the salt

215 treatment alone. Interestingly, with the increases of treatment time, the improvement effect of exogenous Put on Pn

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216 and Gs was gradually obvious. After 7 days of salt treatment, Ci value was higher than that of the corresponding 217 control group for the first time, and Ls was lower than that of the control group, indicating that the salt stress was 218 mainly caused stomatal limitation and changed into non-stomatal limitation. Exogenous Put could prolong the 219 transformation time under salt stress environment (P<0.05). As shown in Figure S1, one day after Put treatment, 220 stomatal morphology and leaf surface temperature were significantly improved under salt stress. Since the 221 photosynthetic parameters changed significantly one day after treatment, we focused our eyes on this period. The

222 results showed that the decreased of Pn was mainly due to stomatal limitation. Exogenous Put could relieve

223 stomatal limitation by improving the cooperation among Gs, Ci and Pn under salt stress. 224 3. Effects of different treatments on the content and distribution of endogenous polyamines 225 The PAs content of Put treated in salt-stressed seedlings showed a trend of increasing first and then decreasing, 226 reaching the highest level after 6 h (Fig. 3A). To determine the response of Put, Spd and Spm, we analyzed their 227 distribution in total PAs. As shown in Fig. 3A, the contents of these three PAs in the control group were changed 228 smoothly and kept at the same order of magnitude. The content of total polyamines increased rapidly to the peak 229 and maintained this level only in the Put treatment seedlings and it was reached in the highest position and 230 recorded as 998.48 nmol·g-1 FW (P<0.05). After salt treatment, only Put content showed a trend of increasing first 231 and then decreasing, reaching the peak at 9 h, accounting for 64.40% of the total polyamine content. After the 232 seedlings were treated with Put under salt stress, the content of Put, Spd and Spm showed a trend of total 233 polyamines, reaching the peak after 6 h, accounting for 65.75%, 22.30% and 11.95% of total polyamines 234 respectively (P<0.05). The results showed that the content of PAs was changed by Put. To get more insights about 235 this, we quantified the expression pattern of Put synthetase genes ADC 1, 2, 3, 4. In salt-stressed with or without 236 Put treatment seedlings, ADC2, 3 showed a trend of increasing first and then decreasing. In respect to the control 237 group, ADC2 increased 1.76, 1.98 folds and ADC3 increased 2.52, 2.78times, respectively. It should be noted that 238 the expression levels of ADC2 and ADC3 genes were significantly increased by exogenous Put under salt stress 239 (P<0.05). As shown in Figure S3, under each treatment, the PAs content and ADC2, 3 changed significantly. The 240 results showed that exogenous Put could increase the content of polyamines in leaves by induced and non-induced 241 ways. Since ADC1 and ADC4 changed significantly after 1 day, which was beyond the time period of our study, 242 no further analysis was needed.

243 4. Regulatory function of Put on stomatal-aperture is related to H2O2 under salt stress 244 There was no significant change in stomatal aperture in solely Put treatment seedling leaves (Fig. 4A). Conversely, 245 in salt-stressed plants, the stomatal aperture decreased continuously, and reached a significant level after 3 h, and 246 decreased the lowest level after 24 h. However, exogenous Put could effectively alleviate the decrease of 247 stomatal-aperture under salt stress, and the effect was the best after 6 h, and remained stable. As shown in Fig. 4C, 248 the peak appeared only 1 h after Put treatment, which increased by 44.04% compared with the corresponding

249 control group and decreased to the control level after 3 h. The content of H2O2 was increased first and then 250 decreased both in salt with or without Put treatment, and these two peaks appeared after 1h and 6 h, respectively.

251 As compared with only salt treatment, the content of H2O2 was increased by 19.52% and 28.14% , respectively in

252 the salt with Put treated plants. Compared with the control, the H2O2 accumulation in the salt treatment group 253 increased by 71.56% and 234.78%, respectively. As shown in Fig. 4B, the changing trend of DCF fluorescence

254 intensity in stomata is similar to that of H2O2 content in leaves. These results suggest that H2O2 may participate in 255 the regulation process in the form of signal molecules. 256 5. The alleviating effect of Put on stomata under salt stress is based on the regulation of ABA content by

257 H2O2

258 Although the content of endogenous PAs and H2O2 reached the peak after 6 h, the stomatal-aperture changed 259 significantly after 3 h. The results showed that the signal of stomatal changed was completed at/or before 3 h.

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260 Therefore, we have chosen 3 h time point for further study. As shown in Fig. 5A, C, D, the stomatal-aperture of 261 salt with Put treatment decreased to the level of salt treatment compared with that before application of D-Arg and 262 DMTU and the fluorescence intensity of DCF in stomata decreased by 0.2, 0.77 times, respectively. The results 263 employed that Put had a certain specificity on stomatal-aperture regulation under salt stress, and endogenous PAs

264 played a regulatory role in the upstream of H2O2. The ABA as a key signal substance in stomatal regulation under 265 salt stress has been deeply studied. In order to determine whether ABA is involved in the regulation process, we 266 determined the endogenous content of ABA in each treatment condition. As shown in Fig. 5B, Put alone had no 267 significant effect on ABA content. Exogenous application of Put markedly inhibited the increase of ABA content 268 under salt treatment, and it was 0.79 folds lower than salt-stressed leaf. The ABA content of salt with Put treatment 269 decreased by 6.36 times compared with that before application of DMTU, but the stomatal aperture increased to a

270 significant level. Supplemental application of H2O2, the ABA content decreased by 4.77 times, but the stomatal- 271 aperture is reduced to a very significant level. These results indicate that ABA is involved in the regulation of Put

272 on stomata, and PAs and H2O2 play a role in the upstream of ABA. In order to further elucidation of the underlying

273 molecular mechanism on how H2O2 affects ABA, we quantified transcript abundance of the four genes that induce 274 ABA synthesis gene RD29B, ABA signal transduction key gene Snrk, and ABA signal pathway negative 275 regulation gene ABI under each treatment condition (Fig. 5E). Interestingly, only ABA synthesis gene NCED 276 showed significant effect on the different treatments and it was consistent with the changes in endogenous ABA 277 content.

278 6. H2O2 effect on the ABA content through the catalysis of DAO, PAO, and NOX

279 Based on the main pathways of H2O2 production in plants, we hypothesized that H2O2 might be catalyzed by DAO, 280 PAO, CWPOD and NOX. As displayed in Fig. 6A, C, D, the stomatal-aperture and DCF fluorescence intensity of 281 salt with Put treatment were not affected by SHAM pre-treatment. The stomatal-aperture of salt with Put treatment 282 decreased to a very significant level than without DPI treatment. At the same time, the fluorescence intensity of 283 DCF was reduced by 13.25%. Compared with SHAM treatment, the stomatal diameter of DPI treatment decreased 284 to a significant level, and DCF fluorescence intensity decreased by 12.54%.In response to pre-treatment with DPI, 285 the stomatal-aperture of AG treatment was significantly reduced, and the fluorescence intensity of DCF decreased 286 by 38.12%. The stomatal-aperture of 1,8-DO treatment was lower than that of DPI treatment, but higher than that 287 of AG treatment, whereas, the DCF fluorescence intensity decreased by 15.9% and increased by 35.90% 288 respectively. Further, we verified the inhibitory effects of these four kinds of inhibitors and as shown in Figure S3, 289 these inhibitors have a significant inhibitory effect on their corresponding enzyme activity as well as their

290 encoding gene expression. These results indicate that H2O2 plays a regulatory role mainly through the maintaining 291 of DAO, PAO and NOX catalysis. However, there is no significant involved was found of CWPOD for the 292 regulation of ABA content. As indicated in Fig. 6B, E, the endogenous ABA content and the transcript abundance 293 of NCED in salt-stressed with Put treatment increased by 25.96% and 38.86%, respectively than that of the prior 294 treatment of SHAM. In respect to the pre-treatment of SHAM, the ABA content and mRNA level of NCED in DPI 295 treatment increased by 29.68% and 16.09%, respectively. Similarly, the ABA content and NCED expression of 296 AG treatment increased by 125.16% and 80.19% respectively than that of SHAM treated plants and compared 297 with the prior application of DPI, it increased by 73.65% and 56.1 2%, respectively. In addition, in contrast with 298 the pre-treatment of SHAM/DPI/AG, the endogenous ABA level and NCED gene expression of 1,8-DO treatment 299 increased by 148.37/73.65/10.28% and 64.22/56.12/9.4%, respectively. The gene expression of RD29B, Snrk and 300 ABI did not show regular changes under various treatment combinations, so no further analysis was conducted.

301 These results suggest that H2O2 is mainly catalyzed by DAO, PAO and NOX, and H2O2 plays a key regulatory role 302 in the upstream of ABA.

303 7. GSH is downstream of H2O2, which is involved in the alleviation mechanism of exogenous Put on stomata

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304 of cucumber leaves under salt stress

305 The above results have shown that GSH induced by H2O2 and has a negative effect on ABA-mediating stomatal 306 closure. So we assumed that GSH might play a role in this process. The GSH content did not change significantly 307 in solely Put treated plants. However, compared with the control group, the fluorescence intensity of GSB 308 increased by 99.35% (Fig. 7A, B, C). The GSB fluorescence intensity and GSH content of salt-stressed plants were 309 increased by 186.49% and 84.84%, respectively than that of its corresponding control group. In respect to salt 310 treatment, GSB fluorescence intensity and GSH content of salt with Put treatments were increased by 46.7% and 311 68.85%, respectively. However, the GSB fluorescence intensity and GSH content of salt with Put treatment 312 decreased by 20.09% and 40.78%, respectively than that of the D-Arg treated plants. Similarly, it decreased by 313 77.40% and 72.82%, respectively than those of the DMTU pre-treatment plants. Interestingly, after supplementing

314 treatment with H2O2, it was increased by 199.28% and 56.92%, respectively. To further gain more evidence, we 315 quantified the GSH synthase gene expression, as shown in Fig. 7C, the changes of GSHS1, 2 could fit the changing

316 trend of GSH under each treatment. These results deduce that H2O2 regulates GSH content at the transcript level. 317 8. GSH plays a regulatory role on ABA content 318 In order to further illustrated the relationship between GSH and ABA, we used GSHee and CNDB. As shown in 319 Fig. 6A, the effect of exogenous GSHee on stomatal aperture under salt stress was similar to that of exogenous Put. 320 The stomatal aperture of salt with Put treatment decreased to the level of salt treatment compared with the CNDB 321 pre-treatment plants. Surprisingly, the DCF fluorescence intensity of salt with Put treatment had no significant 322 changes as compared to the salt with GSHee treatment (Fig. 6C, D, 8A, B). However, in respect to salt with or 323 without Put treatments, the GSB fluorescence intensity of salt with GSHee treatment increased by 28.38% and 324 decreased by 5.86%, respectively, and in contrast, the content of GSH increased by 96.77% and decreased by 325 18.45%, respectively. The DCF fluorescence intensity of salt with Put treatment had no significant effect as 326 compared to prior CNDB treatment. But the GSB fluorescence intensity and GSH content were reduced by

327 77.97% and 87.38%, respectively compared with the salt with Put treatment. It is suggested that H2O2 can affect 328 stomatal aperture by affecting GSH content. As shown in Fig. 6B, E, the ABA content and NCED expression of 329 salt with Put treatment increased by 118.70% and 154.89% respectively, as compared to prior CNDB treatment, 330 inferring that the regulation of exogenous Put on stomata depends on the change of GSH content under salt stress. 331 As shown in Fig. 8A, B, C, the GSB fluorescence intensity and GSH content of salt with Put treatment reduced by 332 37.39% and 24.27% respectively, as compared to prior SHAM treatment. Compared with the pre-treatment with 333 SHAM, the GSB fluorescence intensity of DPI treatment increased by 13.38%, GSH content had no significant 334 change. The GSB fluorescence intensity and GSH content of salt with Put treatment reduced by 62.31% and 335 84.47% respectively, as compared to prior AG treatment. Compared with the pre-treatment of SHAM, the GSB 336 fluorescence intensity and GSH content of AG treatment reduced by 39.80% and 79.49%, respectively. The GSB 337 fluorescence intensity and GSH content of salt with Put treatment reduced by 56.14% and 72.82%, respectively, as 338 compared to prior 1,8-DO treatment. Compared with the pre-treatment of AG, the GSB fluorescence intensity and 339 GSH content of AG treatment reduced by 16.35% and 75.00%, respectively. However, these were decreased by 340 29.92% and 64.10%, respectively, compared with pre-treatment of SHAM treatment. As shown in Fig. 8C, the 341 changing trend of GSH1, 2 gene expression was consistent with the above GSH content. These results indicating

342 that H2O2 produced by DAO, PAO and NOX is involved in the regulation of ABA by GSH. 343 344 Discussion 345 Recently, most of the photosynthesis based research under salt stress has mainly focused on improving the stability 346 of photosynthetic organs and antioxidant capacity (Ioannidis et al., 2012; Saha et al., 2015). However, very few 347 studies have been emphasized on the mechanism of PAs to enhance photosynthesis by improving

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348 stomatal-aperture in the form of the signal during the stomatal limitation period. H2O2 plays an important role in 349 the signal transduction process of resisting salt stress (Li et al., 2015; Niu and Liao, 2016). It is noteworthy that

350 H2O2 is one of the direct products of oxidative metabolism of PAs, which lays a foundation for the study of 351 polyamine signaling in the stomatal limitation period under salt stress (Pottosin et al., 2014). In the present study, 352 the results showed that exogenous Put could effectively regulate the stomatal-aperture closure of cucumber leaves 353 under salt stress through signal transduction pathway.

354 Exogenous Put alleviated the growth inhibition of cucumber seedlings by improving Gs during stomatal 355 limitation period under salt stress 356 Exogenous application of PAs have been an effective tool to improve crop stress resistance and crop productivity 357 in response to a variety of abiotic stresses (Lee et al., 2012; Hao et al., 2012). In this study, exogenous Put 358 significantly alleviated the salt-induced growth inhibition effect of cucumber seedlings during the stomatal 359 limitation period under salt stress (Fig. 1), which is consistent with the previous results (Zhang et al., 2009). After 360 salt treatment, as shown in Fig. 2A, the changing from stomatal limitation to non-stomatal limitation was observed 361 in this study and this kind of similar changing trend also found by Munns et al. (1993). Exogenous Put not only

362 increased Pn by improving Gs (Sun et al., 2016), but also delayed the transition of two periods, which is mainly due 363 to the protective effects of Put on photosynthetic organ stability (Ioannidis et al., 2012), the effectiveness of loop 364 electron transport chain (Wu et al., 2019) and gas exchange persistence (Zhang et al., 2009) under salt stress. In 365 contrast, exogenous Put alone does not seem to cause significant changes, which is consistent with the results of 366 Wu et al. (2019), suggesting that exogenous Put may be stimulated by other substances.

367 Endogenous PAs and H2O2 inhibited the increase of ABA content by signal transduction 368 After salt treatment, as shown in Fig. 4A, stomatal aperture continued to decrease (Zhang et al., 2020) and reached 369 the lowest level at 24 h. Exogenous Put could effectively alleviate this trend. However, some studies have found 370 that exogenous Put promotes stomatal closure (An et al., 2008). This may be occurred due to the difference of 371 species and treatment conditions, so this is the first time, we investigated the positive regulatory effect of

372 exogenous Put on cucumber stomata under salt stress. The PAs and H2O2 content showed a typical signal change 373 trend with the supplementation of Put under salt stress, and Freitas et al. (2018) studied found the similar effect on 374 ethylene signaling. It should be noted that exogenous Put alone increased the content of endogenous PAs, even 375 exceeded than the salt treatment, but had no significant effect on stomatal-aperture, which further indicated that 376 exogenous Put might be needed for modulation of salt effect to play a positive role. As shown in Fig. 2A, Put had 377 the most significant changes compared to Spd and Spm (Yang et al., 2007), which may be happened due to the fact

378 that Put is the precursor of Spd and Spm synthesis (Pál et al., 2015). By analyzing the key Put synthase genes, it

379 was found that exogenous Put increased ADC expression after salt treatment, but exogenous Put alone did not 380 affect the expression of ADC, which indicated that the increasing mode of PAs was different under different 381 treatment conditions, and this part still needs to further study. 382 As mentioned above, even if complete signal changes are observed, the change of stomatal-aperture may occur 383 before the signal change due to the delay of the measurement method and operation process. This kind of similar 384 problem also appears in the study of Xia et al. (2014). It has been widely recognized that ABA content increases 385 and stomatal movement is modulated by ABA under salt stress (Hedrich et al., 2018). In the current study, 386 exogenous Put decreased ABA transcript abundance under salt stress. In addition, pre-treatment with D-Arg could 387 eliminate the stomatal alleviation through the exogenous Put treatment under salt stress, and ABA content returned 388 to the same level as of salt treatment. Similarly, pre-treatment with DMTU, the stomatal-aperture returned to the

389 identical control level, but ABA content increased rapidly. However, replenishing with H2O2, the stomatal-aperture 390 decreased to the salt treatment level, and the ABA content was lower than that of the salt treatment level. These

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391 findings suggested that H2O2 plays an important role in regulating ABA content downstream of Put, but it is not

392 unique. There may be other pathways regulating ABA content. It also indicates that H2O2 may directly regulate 393 stomatal-aperture changes (Roubelakisangelakis et al., 2008).

394 The sources of H2O2 in plants are diverse (Foyer and Noctor, 2005). As mentioned above, NOX, CWPOD, DAO

395 and PAO are considered as the most important sources of H2O2 in plants (Simonovicová et al., 2004; Rejeb et al.,

396 2015). As shown in Fig. 6, except for the CWPOD, the other three enzymes seem to be involved in the regulation 397 of this process. When DAO and PAO activities were inhibited, endogenous ABA content, DCF fluorescence 398 intensity and stomatal aperture were significantly affected. This may be due to the increase of endogenous PAs 399 content after exogenous Put supplementation, which induces the balance mechanism of PAs, and a large number of

400 PAs are decomposed to produce H2O2. Although some studies have found that H2O2 after PAs decomposition can 401 promote stomatal closure (An et al., 2008), this may differ from species to species and treatment conditions. In

402 conclusion, the role of H2O2 produced by PAs metabolism is still controversial due to its dual functions, and to 403 clarify these dual effect needs to be further study.

404 GSH downstream of H2O2 regulates ABA content at the transcription level 405 The GSH is an important regulator of redox homeostasis in plant cells. GSH affects downstream components of 406 ABA signaling pathway and inhibits stomatal closure by regulating redox status in guard cells (Jahan et al., 2008; 407 Okuma et al., 2011; Akter et al., 2013). However, the effect of GSHee pre-treatment as identical as to that of 408 exogenous Put (Fig. 6, 7). Pre-treatment with CNDB eliminated the alleviating effect of Put on salt stress. 409 Different from D-Arg, removing GSH did not affect the fluorescence intensity of DCF, implying that GSH

410 controls stomatal aperture by regulating ABA transcription level. The H2O2 can increase GSH and GSH/GSSG

411 ratio (An et al., 2012; Wimalasekera et al., 2011), and GSH is considered to play critical/pivotal role in H2O2 412 signaling pathway (Mhandi et al., 2010). Pre-treatment with D-Arg reduced the fluorescence intensity of GSB

413 under salt stress combined with Put treatment plants. After removing H2O2, the fluorescence intensity of GSB

414 decreased and supplementation with H2O2, it returned to the same level as salt treatment. These results confer that

415 H2O2 can regulate GSH in a certain concentration range. As shown in Fig. 8, the effect of pre-application of 4

416 types of H2O2 inhibitors on the fluorescence intensity of GSH is similar to that of DCF in Fig. 6, further indicating

417 that the H2O2 regulates GSH is mainly derived from the catalysis of DAO and PAO. 418 In summary, our research results show that exogenous Put enhanced the photosynthesis activities of cucumber 419 seedlings by delaying stomata closure under salt stress, thereby improving plants tolerance to salt stress. This

420 process mainly mediated by H2O2 signaling pathway. Under salt stress conditions, the exogenous Put increased 421 endogenous PAs content and enhanced the activities of DAO, PAO, NOX as well as the expression pattern of their

422 encoding genes further up-regulated, and thus t promoting the formation of H2O2 signal molecules. When the

423 concentration of H2O2 reached a certain level, the increase of ABA content in cucumber leaves inhibited by 424 inducing the change of GSH content. Finally, the whole mechanism alleviated salt-stress induced stomatal closure

425 in cucumber leaves (Fig. 9). To insights more understanding about stomatal closure under salt stress via H2O2/ 426 ABA-mediated signaling pathway, our current knowledge may be useful, and we suggest to apply an in-depth 427 molecular approach to elucidate the better mechanism. 428 429 430 Data availability statement 431 The data supporting the findings of this study are available from the corresponding author, (Sheng Shu), upon 432 request. 433

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434 Acknowledgements 435 This work was funded by The National Key Research and Development Program of China (2018YFD1000800), 436 and was sponsored by the China Agriculture Research System (CARS-23-B12).

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Fig. 1 Effect of exogenous put on Cucumber Seedling Growth under salt stress. Cont: The seedlings were cultured in normal nutrient solution, and the leaves were evenly sprayed with distilled water. Put (8mmol L-1): The seedlings were cultured in normal nutrient solution and sprayed with put evenly on the leaves. NaCl: The seedlings were cultured in 75mmol L-1 NaCl nutrient solution, and the leaves were evenly sprayed with distilled water; NaCl+Put: The seedlings were cultured in 75mmol L-1 NaCl solution, and Put was sprayed evenly on the leaves; A: After 7 days, exogenous Put alleviated the growth inhibition of cucumber seedlings under salt stress. B: After 7 days, the effect of exogenous Put on the phenotype of cucumber seedlings under salt stress. Each data represents the mean±SE of three independent experiments, the vertical bar represents SE (n=3), and different letters indicate the significant difference when P<0.05. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272120; this version posted August 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig. 2 Effects of exogenous Put on Pn, Gs, Ci, Ls of cucumber under salt stress. A: After 1, 3, 5 and 7 days, the

effects of exogenous Put on Pn (net photosynthetic rate), Gs (stomatal conductance), Ci (intercellular carbon dioxide concentration) and Ls (stomatal limitation ) of cucumber leaves under salt stress were studied. Each data represents the mean ± SE of three independent experiments, the vertical bar represents SE (n=3), and different letters indicate the significant difference when P<0.05.

Fig. 3 Effects of different treatments on PAs content distribution, total PAs content and gene expression of Put synthesis key enzyme in cucumber leaves within 24hours. A: Effects of different treatments on the contents of three main PAs (Putrescine, Put, Spermidine, Spd, Spermine, Spm) and total PAs in cucumber leaves within 24hours. B: The changes of key genes of put synthetase in 24 hours under different treatment conditions. Each data represents the mean ± SE of three independent experiments, the vertical bar represents SE (n=3), and different letters indicate the significant difference when P<0.05. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272120; this version posted August 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig. 4 Effects of exogenous Put on stomatal aperture and H2O2 content under salt stress for 24hours. A: The effect of each treatment on the stomatal aperture. Data were obtained from three independent experiments, ***P<0.001 (n=120 for each treatment), as analyzed by one-way ANOVA followed by a Tukey Kramer post hoc

test. B, C: Effects of different treatments on H2O2 content in guard cells and leaves. The fluorescence of DCF was green with a scale of 20μm. Each data represents the mean ± SE of three independent experiments, the vertical bar represents SE (n=3), and different letters indicate the significant difference when P<0.05.

Fig. 5 H2O2 and ABA participate in the alleviation mechanism of exogenous Put on stomatal closure of cucumber leaves under salt stress. A: The effect of each treatment on the stomatal aperture, Among them, 0.2

mM H2O2 of NaCl + put + DMTU + H2O2 treatment was sprayed on the leaves one hour before sampling. Data bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272120; this version posted August 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

were obtained from three independent experiments, *P<0.05, ***P<0.001 (n=120 for each treatment), as analyzed by one-way ANOVA followed by a Tukey Kramer post hoc test. B: Effects of different treatments on ABA content

in cucumber leaves. C, D: The effects of different treatments on H2O2 content and DCF fluorescence intensity in guard cells were studied, the fluorescence of DCF was green and the spontaneous fluorescence of chloroplast was red with a scale of 20 μm. E: The effect of each treatment on ABA synthase gene expression. Each data represents the mean ± SE of three independent experiments, the vertical bar represents SE (n=3), and different letters indicate the significant difference when P<0.05.

Fig. 6 The effects of four enzyme inhibitors (DAO, PAO, NOX and CWPOD) on stomatal aperture, H2O2 accumulation, ABA content and NCED expression in guard cells. A: Effects of 10 mM SHAM, 0.1 mM DPI, 10 mM AG and 2 mM 1,8-DO pretreatment on stomatal aperture under different treatments were studied. Data were obtained from three independent experiments, *P<0.05,**P<0.01,***P<0.001 (n=120 for each treatment), as analyzed by one-way ANOVA followed by a Tukey Kramer post hoc test. B: Effects of pre application of inhibitors and scavengers on ABA content under different treatment conditions were studied. C, D: Effects of pre bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272120; this version posted August 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

application of inhibitors and scavengers on H2O2 content and DCF fluorescence intensity in guard cells under different treatment conditions were studied. The fluorescence of DCF was green and the spontaneous fluorescence of chloroplast was red with a scale of 20 μm. E: Effects of pre application of inhibitors and scavengers on expression level of NCED of ABA synthase gene under different treatment conditions were studied. Each data represents the mean ± SE of three independent experiments, the vertical bar represents SE (n=3), and different letters indicate the significant difference when P<0.05.

Fig. 7 GSH is involved in the mechanism of exogenous Put to alleviate stomatal closure under salt stress. A, C: Effects of different treatments on GSH content and GSB fluorescence intensity in guard cells were studied. The fluorescence of GSB was yellow and the spontaneous fluorescence of chloroplast was red with a scale of 20μm. B: The effects of different treatments on GSH content and expression of GSHS1, 2 genes encoding GSH synthase were studied. Each data represents the mean ± SE of three independent experiments, the vertical bar represents SE (n=3), and different letters indicate the significant difference when P<0.05.

Fig. 8 The effects of four enzyme inhibitors (DAO, PAO, NOX, CWPOD) on GSH accumulation in guard cells, GSH content in leaves and expression of key genes GSHS1, 2 in leaves. A, C: Effects of 0.1mmol L-1 CNDB, 0.1mmol L-1 GSHmee pretreatment on GSH content and GSB fluorescence intensity in guard cells under different treatments were studied. The fluorescence of GSB was yellow and the spontaneous fluorescence of chloroplast was red with a scale of 20 μm. B: The effect of each treatment on GSH content and expression of GSHS1, 2 in leaves were studied. Each data represents the mean ± SE of three independent experiments, the vertical bar represents SE (n=3), and different letters indicate the significant difference when P<0.05.

Fig. 9 The model diagram of exogenous Put regulating photosynthesis of cucumber leaves by alleviating stomatal closure under salt stress. Put- putrescine, PAs-polyamines, DAO-diamine oxidase,PAO-polyamine

oxidase, NOX-nicotinamide adenine dinucleotide phosphate oxidase, H2O2-hydrogen peroxide, GSH-reduced glutathione, ABA-abscisic acid.