Athanassios Molassiotis

Oxidative and nitrosative‐based signaling and associated post‐translational modifications orchestrate the acclimation of citrus plants to salinity stress

Reactive oxygen and nitrogen species are involved in a plethora of cellular responses in plants; however, our knowledge on the outcomes of oxidative and nitrosative signaling is still unclear. To better understand how oxidative and nitrosative signals are integrated to regulate cellular adjustments to external conditions, local and systemic responses were investigated in the roots and leaves of sour orange plants (Citrus aurantium L.) after root treatment with hydrogen peroxide (H(2) O(2) ) or sodium nitroprusside (a nitric oxide donor), followed by NaCl stress for 8 days. Phenotypic and physiological data showed that pre-exposure to these treatments induced an acclimation to subsequent salinity stress that was accompanied by both local and systemic H(2) O(2) and nitric oxide (NO) accumulation. Combined histochemical and fluorescent probe approaches showed the existence of a vascular-driven long-distance reactive oxygen species and NO signaling pathway. Transcriptional analysis of genes diagnostic for H(2) O(2) and NO signaling just after treatments or after 8 days of salt stress revealed tissue- and time-specific mechanisms controlling internal H(2) O(2) and NO homeostasis. Furthermore, evidence is presented showing that protein carbonylation, nitration and S-nitrosylation are involved in acclimation to salinity stress. In addition, this work enabled characterization of potential carbonylated, nitrated and nitrosylated proteins with distinct or overlapping signatures. This work provides a framework to better understand the oxidative and nitrosative priming network in citrus plants subjected to salinity conditions.

Hydrogen peroxide- and nitric oxide-induced systemic antioxidant prime-like activity under NaCl-stress and stress-free conditions in citrus plants.

We tested whether pre-treatments of roots with H(2)O(2) (10mM for 8h) or sodium nitroprusside (SNP; 100microM for 48h), a donor of ()NO, could induce prime antioxidant defense responses in the leaves of citrus plants grown in the absence or presence of 150mM NaCl for 16d. Both root pre-treatments increased leaf superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities, and induced related-isoform(s) expression under non-NaCl-stress conditions. When followed by salinity, certain enzymatic activities also exhibited an up-regulation in response to H(2)O(2) or SNP pre-exposure. An NaCl-stress-provoked decrease in the ascorbate redox state was partially prevented by both pre-treatments, whereas the glutathione redox state under normal and NaCl-stress conditions was increased by SNP. Real-time imaging of ()NO production was found in vascular tissues and epidermal cells. Furthermore, NaCl-induced inhibition in ()OH scavenging activity and promotion of ()OH-mediated DNA strand cleavage was partially prevented by SNP. Moreover, NaCl-dependent protein oxidation (carbonylation) was totally reversed by both pre-treatments as revealed by quantitative assay and protein blotting analysis. These results provide strong evidence that H(2)O(2) and ()NO elicit long-lasting systemic primer-like antioxidant activity in citrus plants under physiological and NaCl-stress conditions.

Polyamines reprogram oxidative and nitrosative status and the proteome of citrus plants exposed to salinity stress

The interplay among polyamines (PAs) and reactive oxygen and nitrogen species (RNS and ROS) is emerging as a key issue in plant responses to salinity. To address this question, we analysed the impact of exogenous PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)] on the oxidative and nitrosative status in citrus plants exposed to salinity. PAs partially reversed the NaCl-induced phenotypic and physiological disturbances. The expression of PA biosynthesis (ADC, SAMDC, SPDS and SPMS) and catabolism (DAO and PAO) genes was systematically up-regulated by PAs. In addition, PAs altered the oxidative status in salt-stressed plants as inferred by changes in ROS production and redox status accompanied by regulation of transcript expression and activities of various antioxidant enzymes. Furthermore, NaCl-induced up-regulation of NO-associated genes, such as NR, NADde, NOS-like and AOX, along with S-nitrosoglutathione reductase and nitrate reductase activities, was partially restored by PAs. Protein carbonylation and tyrosine nitration are depressed by specific PAs whereas protein S-nitrosylation was elicited by all PAs. Furthermore, we identified 271 S-nitrosylated proteins that were commonly or preferentially targeted by salinity and individual PAs. This work helps improve our knowledge on the plants response to environmental challenge.

Oxidative and nitrosative signaling in plants: Two branches in the same tree?

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) constitute key features underpinning the dynamic nature of cell signaling systems in plants. Despite their importance in many aspects of cell biology, our understanding of oxidative and especially of nitrosative signaling and their regulation remains poorly understood. Early reports have established that ROS and RNS coordinately regulate plant defense responses to biotic stress. In addition, evidence has accumulated demonstrating that there is a strong cross-talk between oxidative and nitrosative signaling upon abiotic stress conditions. The goal of this mini-review is to provide latest findings showing how both ROS and RNS comprise a coordinated oxidative and nitrosative signaling network that modulates cellular responses in response to environmental stimuli.

NO says more than 'YES' to salt tolerance Salt priming and systemic nitric oxide signaling in plants

Nitric oxide (NO) is now recognized as an important signaling molecule and there has been an increasing bulk of studies regarding the various functions of NO in plants exposed to environmental stimulus. There is also emerging evidence, although not extensive, that NO plays systemic signaling roles during the establishment of salt tolerance in many plant species. In this mini-review, we highlight several candidate mechanisms as being functional in this NO systemic signaling action. In addition, we outline data supporting that plants possess prime-like mechanisms that allow them to memorize previous NO exposure events and generate defense responses following salt stress.

Interplay between protein carbonylation and nitrosylation in plants

ROS and reactive nitrogen species (RNS) are key regulators of redox homeostasis in living organisms including plants. As control of redox homeostasis plays a central function in plant biology, redox proteomics could help in characterizing the potential roles played by ROS/RNS‐induced posttranslational modification in plant cells. In this review, we focus on two posttranslational modifications: protein carbonylation (a marker of protein oxidation) and protein S‐nitrosylation, both of which having recently emerged as important regulatory mechanisms during numerous fundamental biological processes. Here, we describe the recent progress in proteomic analysis of carbonylated and nitrosylated proteins and highlight the achievements made in understanding the physiological basis of these oxy/nitro modifications in plants. In addition, we document the existence of a relationship between ROS‐based carbonylation and RNS‐based nitrosylation thus supporting the finding that crosstalk between cellular signaling stress pathways induced by ROS and RNS could be mediated by specific protein modifications.

Proteomic signatures uncover hydrogen peroxide and nitric oxide cross-talk signaling network in citrus plants.

Hydrogen peroxide (H(2)O(2)) and nitric oxide ((•)NO) elicit numerous processes in plants. However, our knowledge of H(2)O(2) and (•)NO-responsive proteins is limited. The present study aimed to identify proteins whose accumulation levels were regulated by these signaling molecules in citrus leaves. To address this question, hydroponically grown citrus plants were treated by incubating their roots in the presence of H(2)O(2) or the (•)NO donor, sodium nitroprusside (SNP). Both treatments induced H(2)O(2) and (•)NO production in leaves, indicating occurrence of oxidative and nitrosative stress conditions. However, treated plants maintained their normal physiological status. The vascular system was shown to be involved in the H(2)O(2) and (•)NO systemic signaling as evidenced by real-time labeling of the two molecules. Comparative proteomic analysis identified a number of proteins whose accumulation levels were altered by treatments. They were mainly involved in photosynthesis, defense and energy. More than half of them were commonly modulated by both treatments, indicating a strong overlap between H(2)O(2) and (•)NO responses. Using a redox proteomic approach, several proteins were also identified as being carbonylation targets of H(2)O(2) and SNP. The analysis reveals an interlinked H(2)O(2) and (•)NO proteins network allowing a deeper understanding of oxidative and nitrosative signaling in plants.

Physiological and proteomic approaches to address the active role of ozone in kiwifruit post-harvest ripening

Post-harvest ozone application has recently been shown to inhibit the onset of senescence symptoms on fleshy fruit and vegetables; however, the exact mechanism of action is yet unknown. To characterize the impact of ozone on the post-harvest performance of kiwifruit (Actinidia deliciosa cv. ‘Hayward’), fruits were cold stored (0 °C, 95% relative humidity) in a commercial ethylene-free room for 1, 3, or 5 months in the absence (control) or presence of ozone (0.3 μl l−1) and subsequently were allowed to ripen at a higher temperature (20 °C), herein defined as the shelf-life period, for up to 12 days. Ozone blocked ethylene production, delayed ripening, and stimulated antioxidant and anti-radical activities of fruits. Proteomic analysis using 1D-SDS-PAGE and mass spectrometry identified 102 kiwifruit proteins during ripening, which are mainly involved in energy, protein metabolism, defence, and cell structure. Ripening induced protein carbonylation in kiwifruit but this effect was depressed by ozone. A set of candidate kiwifruit proteins that are sensitive to carbonylation was also discovered. Overall, the present data indicate that ozone improved kiwifruit post-harvest behaviour, thus providing a first step towards understanding the active role of this molecule in fruit ripening.

ANTIOXIDANT AND ANATOMICAL RESPONSES IN SHOOT CULTURE OF THE APPLE ROOTSTOCK MM 106 TREATED WITH NACL, KCL, MANNITOL OR SORBITOL

To determine whether the major influence of high salinity is caused by the osmotic component or by salinity-induced specific ion toxicity, we compared the effects of mannitol, sorbitol, NaCl and KCl (all in concentratuions corresponded to osmotic potential −1.0 MPa) on the antioxidant and anatomical responses of the apple rootstock MM 106 explants grown in the Murashige and Skoog (MS) medium. All the compounds had a significant influence on explants mineral composition and reduced the leaf water content, whereas mannitol and salts decreased chlorophyll (Chl) content and increased proline content. Superoxide dismutase (SOD), peroxidase (POD) and non-enzymatic antioxidant activities as well as H2O2 content were increased in the leaves and stems. In addition, in the leaves of explants exposed to NaCl an additional Mn-SOD isoform was revealed, while specific POD isoforms were detected in the leaves and stems treated with NaCl or KCl. However, catalase activity was depressed in the salt-treated leaves. At the ultrastructural level, the NaCl-treated leaves had the thickest lamina, due to an extensive increase of the size of epidermal and mesophyll cells. Also, an increase of the relative volume of the intercellular spaces in response to NaCl was observed. The results suggest that Na accumulation is the first candidate for the distinct antioxidant and anatomical responses between saline and osmotically generated stress in the MM 106 explants.

Nitrosative responses in citrus plants exposed to six abiotic stress conditions

Nitrosative status has emerged as a key component in plant response to abiotic stress; however, knowledge on its regulation by different environmental conditions remains unclear. The current study focused on nitrosative responses in citrus plants exposed to various abiotic stresses, including continuous light, continuous dark, heat, cold, drought and salinity. Morphological observations and physiological analysis showed that abiotic stress treatments were sensed by citrus plants. Furthermore, it was revealed that nitrosative networks are activated by environmental stress factors in citrus leaves as evidenced by increased nitrite (NO) content along with the release of NO and superoxide anion (O₂⁻) in the vascular tissues. The expression of genes potentially involved in NO production, such as NR, AOX, NADHox, NADHde, PAO and DAO, was affected by the abiotic stress treatments demonstrating that NO-derived nitrosative responses could be regulated by various pathways. In addition, S-nitrosoglutathione reductase (GSNOR) and nitrate reductase (NR) gene expression and enzymatic activity displayed significant changes in response to adverse environmental conditions, particularly cold stress. Peroxynitrite (ONOO⁻) scavenging ability of citrus plants was elicited by continuous light, dark or drought but was suppressed by salinity. In contrast, nitration levels were elevated by salinity and suppressed by continuous light or dark. Finally, S-nitrosylation patterns were enhanced by heat, cold or drought but were suppressed by dark or salinity. These results suggest that the nitrosative response of citrus plants is differentially regulated depending on the stress type and underscore the importance of nitrosative status in plant stress physiology.