Proteomic Investigation of S-Nitrosylated Proteins During NO-Induced Adventitious Rooting of Cucumber.

Yue Wu,Jianming Xie,Jian Lv,Linli Hu,Xuemei Xiao,Jihua Yu,Weibiao Liao,Jian Yu,Lijuan Niu

doi:10.3390/ijms20215363

Yue Wu, Jianming Xie + Show 7 more

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https://doi.org/10.3390/ijms20215363

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Abstract

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 μM 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.

Highlights

Free radical nitric oxide (NO) is generated via non-enzymatic [1] and enzymatic pathways [2,3] in plants
In order to access the effects of exogenous GSNO on adventitious root development of cucumber, explants were cultivated with different concentrations of GSNO (0, 0.1, 1, 10, 50 and 100 μM)
A higher dose of GSNO (100 μM) obviously decreased the number and length of adventitious roots, indicating exogenous GSNO could have a concentration-dependent effect on adventitious rooting

Summary

Introduction

Free radical nitric oxide (NO) is generated via non-enzymatic [1] and enzymatic pathways [2,3] in plants. As a multifunctional physiological regulator, NO has been shown to be involved in every aspect of plant growth and every developmental process in plants [4]. An increasing body of evidence has indicated that NO could play an essential role in response to various abiotic stresses [5,6,7]. Previous studies suggested that NO could exert its effects depending on the cyclic guanosine monophosphate (cGMP) signaling pathway [8,9]. NO could promote the adventitious rooting of marigold through the cGMP-dependent pathway [10]. At present, increasing evidence demonstrates that S-nitrosylation might be involved in processes for regulating the growth, development, and stress responses in plants [14,15,16]

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