Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings

Abstract

Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins’ structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24–72 h with CAN (10 or 50 μM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant’s secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 μM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the formation of carbonyl groups than to nitration, and are detected particularly after 24 h. Thus, oxidative stress and oxidative modifications of proteins seems to be of significant importance in the rapid response of plants to CAN.

Canavanine-Induced Decrease in Nitric Oxide Synthesis Alters Activity of Antioxidant System but Does Not Impact S-Nitrosoglutathione Catabolism in Tomato Roots

“…This PTM, mediated by ONOO - , could be considered as a marker of nitro-oxidative stress. As demonstrated earlier, short term (24 h) CAN supplementation of tomato seedlings resulted in an increase in 3-NT concentration in root proteins as compared with plants growing in water and corresponds well to ONOO - formation (Krasuska et al, 2016a). For that reason, a next step of this work was to identify 3-NT-immunopositive proteins that were abundant in roots of CAN-treated plants.…”

“…Although the possibility that plants have NOS protein or adequate protein complex of a structure unrelated to a mammalian type is under consideration, functionality of the reaction was demonstrated in various plant tissue (Corpas and Barroso, 2017). In our previous research, it was shown that CAN, a commonly used inhibitor of mammalian isoform of NO synthase, led to restriction in NO formation in tomato roots (Krasuska et al, 2016a). This observation was the impulse to check CAN impact on one of the putative pathways of NO synthesis in plants.…”

“…In our previous experiments, tomato ( Solanum lycopersicum L.) seedlings were cultured for 24 or 72 h in 10-µM or 50-µM CAN resulting in inhibition of root growth in 50 or 100%, respectively ( Figure 1 ). Malformations in root morphology (shorter and thicker roots, limited number of lateral roots) were accompanied by modification in NO and peroxynitrite (ONOO - ) production and localization (Krasuska et al, 2016a; Krasuska et al, 2016b). CAN restricted NO emission and transiently (after 24 h) enhanced ONOO - production suggesting direct impact of this NPAA on NO biosynthesis (Krasuska et al, 2016a; Krasuska et al, 2016b).…”

“…Malformations in root morphology (shorter and thicker roots, limited number of lateral roots) were accompanied by modification in NO and peroxynitrite (ONOO - ) production and localization (Krasuska et al, 2016a; Krasuska et al, 2016b). CAN restricted NO emission and transiently (after 24 h) enhanced ONOO - production suggesting direct impact of this NPAA on NO biosynthesis (Krasuska et al, 2016a; Krasuska et al, 2016b). In the current work, we have measured Arg-dependent NOS-like activity in tomato roots to prove that CAN is an inhibitor of Arg-dependent NO formation in plants.…”

“…Supplementation of tomato seedlings with CAN led to increased superoxide radical (O 2 •- ) generation and elevated hydrogen peroxide (H 2 O 2 ) concentration in the roots, accompanied by stimulation of protein carbonyl groups formation (Krasuska et al, 2016a). Based on these data, we have proposed that CAN-induced oxidative burst could be potentially due to the disruption of the cellular antioxidant system, as NO regulates many enzymes involved in modulation of ROS level (Begara-Morales et al, 2016).…”