High-throughput screening of rice for nitrosative-stress response and the identification of effective pH range for Nitric Oxide Donor S-Nitrocysteine

Bong Gyu Mun, Chan Ju Lee, Adil Hussain, Gang Sub Lee, Sang Uk Lee, Kyung Min Kim, Byung Wook Yun

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Nitric oxide (NO)-mediated signaling regulates growth, development, and responses to biotic and abiotic stresses in plants. It involves various NO-derivatives, such as NO radical, nitroxyl anion (NO -), peroxynitrite (ONOO -), and S-nitrosothiols (SNOs). Many of these SNOs including S-nitrosocysteine (CysNO), can modify intracellular protein thiols through trans-nitrosation reactions. Various NO donors have been used as tools to unravel the fundamental mechanisms of NO signaling. However, the effect of NO donors can be analyzed only under specific conditions. Here, we report a high-throughput screening system for identifying mutant lines exhibiting differential responses to the frequently used NO donor CysNO. T o determine the effect of CysNO on seed germination and growth at various pH levels, we grew Arabidopsis thaliana seeds on MS medium with various concentrations of CysNO at a pH range of 2-5.8. The pH was adjusted using different volumes of HCl. Seeds were found to be highly sensitive to pH below 4.5 at any given CysNO concentration; therefore, the effects of the CysNO treatments could not be determined. A similar effect of pH was observed in the case of rice plants. However, when the same concentrations of CysNO were prepared using EPPS (3-[4-(2-Hydroxyethyl)-1-piperazinyl] propanesulfonic acid) buffer to maintain pH at a suitable level, significantly different responses to CysNO were observed among wild-type and mutant rice lines, indicating the importance of optimum pH conditions. Using this technique, we identified several rice Ac/Ds transposon mutant lines that showed tolerance or sensitivity to exogenous NO stress. In conclusion, pH is a critical factor for plant germination and growth. However, information about changes of pH caused CysNO solution was lacking. In this study we demonstrated the effective pH range for maximum efficiency and absorbance of CysNO using EPPS buffer system in rice. Hence, the EPPS-based buffer system is more efficient for high-throughput screening in rice against nitrosative stress induced by the nitric oxide donor S-Nitrocysteine.

Original languageEnglish
Pages (from-to)41-47
Number of pages7
JournalInternational Journal of Agriculture and Biology
Issue number1
StatePublished - 2017


  • CysNO
  • EPPS buffer
  • High-throughput screening
  • Nitric oxide
  • PH


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