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 (N⁸-acetyl-Spd) and acetylated spermine (N¹-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, N⁸-acetyl-Spd and N¹-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.

Polyamine oxidases (PAOs) have been correlated with numerous physiological and developmental processes, as well as responses to biotic and abiotic stress conditions. Their transcriptional regulation is driven by signals generated by various developmental and environmental cues, including phytohormones. However, the inductive mechanism(s) of the corresponding genes remains elusive. Out of the five previously characterized Arabidopsis PAO genes, none of their regulatory sequences have been analyzed to date. In this study, a GUS reporter-aided promoter deletion approach was used to investigate the transcriptional regulation of AtPAO3 during normal growth and development as well as under various inductive environments. AtPAO3 contains an upstream open reading frame (uORF) and a short inter-cistronic sequence, while the integrity of both appears to be crucial for the proper regulation of gene expression. The full-length promoter contains several cis-acting elements that regulate the tissue-specific expression of AtPAO3 during normal growth and development. Furthermore, a number of TFBS that are involved in gene induction under various abiotic stress conditions display an additive effect on gene expression. Taken together, our data indicate that the transcription of AtPAO3 is regulated by multiple environmental factors, which probably work alongside hormonal signals and shed light on the fine-tuning mechanisms of PAO regulation.