Hydrogen peroxide acts upstream of nitric oxide in heat shock pathway in Arabidopsis seedings
Lei Wang¹, Yunjing Guo¹, Shuo Zhou, Lixiu Jia, Liqun Zhao* 

College of Life Sciences, Hebei Normal University, Shijiazhuang 050024。
¹These authors contributed equally to the work.
*Corresponding Author: Tel:(+86 311)8078 7530; Fax:(+86 311)8078 7549; e-mail: zhaoliqun@hebtu.edu.cn

Abstract
We even reported that nitric oxide (NO) functioned as a signal in thermotolerence. To clarified its relationship with hydrogen peroxide (H₂O₂), we investigated the effects of heat on three types of Arabidopsis thaliana seedlings: wide type, AtrbohB, AtrbohD and AtrbohB/D (for NADPH oxidase-defective mutants), which all exhibited reduced endogenous H₂O₂ levels, and noa1(rif1) (for nitric oxide associated1/resistant to inhibition by fosmidomycin1). After heat shock (HS), the NO levels of AtrbohB, AtrbohD and AtrbohB/D seedlings were lower than that of wide type, the lowest one for AtrbohB/D seedlings; however, the H₂O₂ level of noa1(rif1) seedlings was not clear different from that of wide type. Treatment of AtrbohB, AtrbohD and AtrbohB/D seedlings with H₂O₂, sodium nitroprusside or S-nitroso-N-acetylpenicillamine revealed that both of internal H₂O₂ and NO partially rescued their heat sensitivity; however, no obvious effect was observed for noa1(rif1) seedlings after treatment of H₂O₂, suggesting that NO is involved in H₂O₂ signal transduction as a downstream factor. This point was verified by phenotypic analysis and thermotolerance testing using seedlings of two nia2 (nitrate reductase 2-mediated NO generation) -overexpressing transgenic lines in an AtrbohB/D background. Electrophoretic mobility-shift and western-blot analyses demonstrated that after HS treatment, H₂O₂ through NO stimulated the DNA-binding activity of HS transcription factors and the accumulation of heat shock protein 21 (HSP21). These data indicate that H₂O₂ acts upstream of AtCaM3 in thermotolerance, which is dependent on increased HS transcription factor DNA-binding activity and HSP accumulation.

Key Words: Arabidopsis; heat shock; hydrogen peroxide; nitric oxide