Genetically Modified Heat Shock Protein90s and Polyamine Oxidases in Arabidopsis Reveal Their Interaction under Heat Stress Affecting Polyamine Acetylation, Oxidation and Homeostasis of Reactive Oxygen Species

One Sentence Summary Heat shock proteins90 (HSP90s) induce acetylation of polyamines (PAs) and interact with polyamine oxidases (PAOs) affecting oxidation of PAs and hydrogen peroxide (H2O2) homeostasis in Arabidopsis thaliana. The chaperones, heat shock proteins (HSPs), stabilize proteins to minimize proteotoxic stress, especially during heat stress (HS) and polyamine (PA) oxidases (PAOs) participate in the modulation of the cellular homeostasis of PAs and reactive oxygen species (ROS). An interesting interaction of HSP90s and PAOs was revealed in Arabidopsis thaliana by using the pLFY:HSP90RNAi line against the four AtHSP90 genes encoding cytosolic proteins, the T-DNA Athsp90-1 and Athsp90-4 insertional mutants, the Atpao3 mutant and pharmacological inhibitors of HSP90s and PAOs. Silencing of all cytosolic HSP90 genes resulted in several-fold higher levels of soluble spermidine (S-Spd), acetylated Spd (N-8-acetyl-Spd) and acetylated spermine (N-1-acetyl-Spm) in the transgenic Arabidopsis thaliana leaves. Heat shock induced increase of soluble-PAs (S-PAs) and soluble hydrolyzed-PAs (SH-PAs), especially of SH-Spm, and more importantly of acetylated Spd and Spm. The silencing of HSP90 genes or pharmacological inhibition of the HSP90 proteins by the specific inhibitor radicicol, under HS stimulatory conditions, resulted in a further increase of PA titers, N-8-acetyl-Spd and N-1-acetyl-Spm, and also stimulated the expression of PAO genes. The increased PA titers and PAO enzymatic activity resulted in a profound increase of PAO-derived hydrogen peroxide (H2O2) levels, which was terminated by the addition of the PAO-specific inhibitor guazatine. Interestingly, the loss-of-function Atpao3 mutant exhibited increased mRNA levels of selected AtHSP90 genes. Taken together, the results herein reveal a novel function of HSP90 and suggest that HSP90s and PAOs cross-talk to orchestrate PA acetylation, oxidation, and PA/H2O2 homeostasis.