Biochemical and proteomic approaches to investigating effects of IAA-aspartate in pea (Pisum sativum L.) seedlings during osmotic shock.

Osmotic shock is the first step of high salt or drought action that involves biochemical and molecular changes during plant response to these unfavorable conditions. Indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp) is the main amide conjugate of auxin in pea (Pisum sativum L.) tissues. Although the exact molecular mechanism of the IAA-Asp action is unknown, this conjugate's indole-3-acetic acid (IAA)-independent biological activity has been observed during physiological and stress conditions. In this work, we investigated the effect of IAA-Asp alone, as well as in combination with NaCl or polyethylene glycol (PEG) (osmotic shock) on reduced/oxidized glutathione (GSH/GSSG) ratio, activities of enzymes modulating glutathione concentration, protein S-glutathionylation, and IAA homeostasis. We did not observe the hydrolysis of IAA-Asp to IAA in pea seedlings, which, together with other results, suggests that IAA-Asp modulates plant response to abiotic stimuli independently of IAA. Moreover, despite the effect of IAA-Asp on the enzymes responsible for IAA conjugation, no changes in this phytohormone level were visible. Furthermore, 3h plant treatment with IAA-Asp increased the activity of glutathione reductase (GR), which correlates with an elevated GSH/GSSG ratio. On the contrary, more extended (48h) incubation with IAA-Asp diminished the GSH/GSSG ratio and increased the activity of glutathione peroxidase (GPX). IAA-Asp reduced GR activity during salt treatment but did not affect the GSH/GSSG ratio. Similarly, under plant incubation with PEG, IAA-Asp did not change the GSH/GSSG ratio but increased glutathione S-transferase (GST) activity. We also analyzed the effect of IAA-Asp on pea protein S-glutathionylation. Increased S-glutathionylation of heat shock 70 kDa protein (HSP70) was observed after plant treatment with IAA-Asp, PEG, or IAA-Asp combined with PEG. The proteomic analysis also revealed that IAA-Asp diminished S-glutathionylation of lipoxygenase during plant incubation with PEG. Thus, we suggest that IAA-Asp modulates redox status in pea during oxidative stress and under normal physiological conditions.