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

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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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 1 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. 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 2 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. 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.
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