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Calcium and Calmodulin Are Involved in Nitric Oxide-Induced Adventitious Rooting of Cucumber under Simulated Osmotic Stress.

ABSTRACT: Osmotic stress is a major form of abiotic stress that adversely affects growth and development of plants and subsequently reduces yield and quality of crops. In this study, the effect of nitric oxide (NO) and calcium (Ca²⁺) on the process of adventitious rooting in cucumber (Cucumis sativus L.) under simulated osmotic stress was investigated. The results revealed that the effect of exogenous NO and Ca²⁺ in promoting the development of adventitious roots in cucumber seedlings under simulated osmotic stress was dose-dependent, with a maximal biological response at 10 μM NO donor nitroprusside (SNP) or 200 μM Ca²⁺. The application of Ca²⁺ chelators or channel inhibitors and calmodulin (CaM) antagonists significantly reversed NO-induced adventitious rooting, implying that endogenous Ca²⁺/CaM might be involved in NO-induced adventitious rooting under osmotic stress. Moreover, intracellular Ca amount was also increased by NO in cucumber hypocotyls during the development of adventitious roots under osmotic stress. This increase of endogenous Ca²⁺ was inhibited by NO specific scavenger 2-(4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), nitrate reductase inhibitors tungstate (Na₂WO₄) and sodium azide (NaN₃)_. This gives an indication that Ca²⁺ might be a downstream signaling molecule in the adventitious root development by NO under osmotic condition. The results also show that NO or Ca²⁺ play a positive role in improving plant water status and photosynthetic system by increasing chlorophyll content and photochemical activity in leaves. Furthermore, NO and Ca²⁺ treatment might alleviate the negative effects of osmotic stress by decreasing membrane damage and reactive oxygen species (ROS) production by enhancing the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Therefore, Ca²⁺/CaM may act as a downstream signaling molecule in NO-induced development of adventitious root under simulated osmotic stress through improving the photosynthetic performance of leaves and activating antioxidative system in plants.

SUBMITTER: Niu L

PROVIDER: S-EPMC5623940 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nitric oxide (NO) acts an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. However, whether S-nitrosylation is a crucial molecular mechanism of NO is still largely unknown. In this study, 50 ?M S-nitrosoglutathione (GSNO) treatment was found to have a maximum biological effect on promoting adventitious rooting in cucumber. Meanwhile, removal of endogenous NO significantly inhibited the development of adventitious roots implying that NO is responsible for promoting the process of adventitious rooting. Moreover, application of GSNO resulted in an increase of intracellular S-nitrosothiol (SNO) levels and endogenous NO production, while decreasing the S-nitrosoglutathione reductase (GSNOR) activity during adventitious rooting, implicating that S-nitrosylation might be involved in NO-induced adventitious rooting in cucumber. Furthermore, the identification of S-nitrosylated proteins was performed utilizing the liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and biotin-switch technique during the development of adventitious rooting. Among these proteins, the activities and S-nitrosylated level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin alpha chain (TUA), and glutathione reductase (GR) were further analyzed as NO direct targets. Our results indicated that NO might enhance the S-nitrosylation level of GAPDH and GR, and was found to subsequently reduce these activities and transcriptional levels. Conversely, S-nitrosylation of TUA increased the expression level of TUA. The results implied that S-nitrosylation of key proteins seems to regulate various pathways through differential S-nitrosylation during adventitious rooting. Collectively, these results suggest that S-nitrosylation could be involved in NO-induced adventitious rooting, and they also provide fundamental evidence for the molecular mechanism of NO signaling during adventitious rooting in cucumber explants.

Project description:Previous studies have shown that both hydrogen gas (H2) and ethylene (ETH) play positive roles in plant adventitious rooting. However, the relationship between H2and ETH during this process has not been explored and remains insufficiently understood. In this study, cucumber (Cucumis sativus L.) was used to explore the proteomic changes in ETH-H2-induced rooting. Our results show that hydrogen-rich water (HRW) and ethylene-releasing compound (ethephon) at proper concentrations promote adventitious rooting, with maximal biological responses occurring at 50% HRW or 0.5 µM ethephon. ETH inhibitors aminoethoxyvinylglycine (AVG) and AgNO3 cause partial inhibition of adventitious rooting induced by H2, suggesting that ETH might be involved in H2-induced adventitious rooting. According to two-dimensional electrophoresis (2-DE) and mass spectrometric analyses, compared with the control, 9 proteins were up-regulated while 15 proteins were down-regulated in HRW treatment; four proteins were up-regulated while 10 proteins were down-regulated in ethephon treatment; and one protein was up-regulated while nine proteins were down-regulated in HRW+AVG treatment. Six of these differentially accumulated proteins were further analyzed, including photosynthesis -related proteins (ribulose-1,5-bisphosphate carall boxylase smsubunit (Rubisco), sedoheptulose-1,7-bisphosphatase (SBPase), oxygen-evolving enhancer protein (OEE1)), amino and metabolism-related protein (threonine dehydratase (TDH)), stress response-related protein (cytosolic ascorbate peroxidase (CAPX)), and folding, modification and degradation-related protein (protein disulfide-isomerase (PDI)). Moreover, the results of real-time PCR about the mRNA levels of these genes in various treatments were consistent with the 2-DE results. Therefore, ETH may be the downstream signaling molecule during H2- induced adventitious rooting and proteins Rubisco, SBPase, OEE1, TDH, CAPX and PDI may play important roles during the process.

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Calcium and Calmodulin Are Involved in Nitric Oxide-Induced Adventitious Rooting of Cucumber under Simulated Osmotic Stress.

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