Effects of polyamines on the expression of antioxidant genes and proteins in citrus plants exposed to salt stress

Although there are accumulating reports that polyamines are involved in abiotic/oxidative stress responses, their role is not yet fully understood. Salt stress is one of the most devastating abiotic stresses which seriously interrupt plant growth and productivity. The present study attempts to examine the effects of root treatments with putrescine (Put, I mM), spermidine (Spd, ImM) and spermine (Spm, ImM) on polyamine homeostasis, as well as on several antioxidant-related genes and proteins in the leaves of citrus plants (Citrus aurantium L.) exposed to 150 mM NaCI for 15 d. Analysis of endogenous levels of free polyarnines in NaCl-stressed plant tissues reveals the existence of a polyamine transport system from roots to leaves. Real-time analysis of reactive oxygen species (ROS) by confocal laser scanning microscopy (CLSM) showed an over-accumulation of superoxide anion (02) and hydrogen peroxide (H202) in the stomata of citrus plants exposed to salt stress. Exogenously applied polyamines to salinized nutrient solution induced the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and ascrobic oxidase (AO) whereas it caused the opposite effect on peroxidase (POD), guaiacol peroxidase (GPO D) and ascorbate peroxidase (APX). The effect of polyamines was further examined by determining the plant's antioxidant gene expression profile following a quantitative real-time RT-PCR approach. The overall results indicate that the interaction between different polyamines can be dispersed throughout the citrus plant, and provide additional information suggesting that polyamines may act as a biological mediator allowing citrus plants to activate specific antioxidant responses against salinit

Pre- and Post-harvest Melatonin Application Boosted Phenolic Compounds Accumulation and Altered Respiratory Characters in Sweet Cherry Fruit

The aim of the present study was to investigate the impact of exogenous melatonin (0. 5 mM) application through pre-harvest foliar spray and postharvest immersion, alone or in combination, on ripening parameters of sweet cherry (cv. Ferrovia) fruit and their relationship with bioactive compounds and gene expression at harvest as well after cold storage (0°C) for 12 days and subsequent room temperature (20°C) exposure for 8 h. Although several ripening traits were not influenced by melatonin, the combining pre- and post-harvest treatments delayed fruit softening at post-cold period. Preharvest spray with melatonin depressed fruit respiration at time of harvest while all applied treatments induced respiratory activity following cold, indicating that this anti-ripening action of melatonin is reversed by cold. Several genes related to the tricarboxylic acid cycle, such as PaFUM, PaOGDH, PaIDH, and PaPDHA1 were upregulated in fruit exposed to melatonin, particularly following combined pre- and post-harvest application. The accumulation of phenolic compounds, such as neochlorogenic acid, chlorogenic acid, epicatechin, procyanidin B1, procyanidin B2+B4, cyanidin-3-O-galactoside, and cyanidin-3-O-rutinoside along with the expression of several genes involved in phenols biosynthesis, such as PaSK, PaPAL, Pa4CL, PaC4H, and PaFNR were at higher levels in melatonin-treated cherries at harvest and after cold exposure, the highest effects being observed in fruits subjected to both pre- and post-harvest treatments. This study provides a comprehensive understanding of melatonin-responsive ripening framework at different melatonin application conditions and sweet cherry stages, thereby helps to understand the action of this molecule in fruit physiology

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

The effect of H2O2 and NO on the antioxidant mechanism of sour orange seedlings (Citrus aurantium L.) grown under salinity conditions

Salinity is one of the most serious environmental stresses reducing plant production. Many studies have been conducted lately aiming at stimulating the salinity defence and adaptation metabolic pathways. Hydrogen peroxide and NO• are considered among the signaling molecules mediating the plant response metabolic pathways. In the present PhD thesis, the hypothesis of triggering the sour orange (Citrus aurantium) salinity adaptation mechanism via previous exogenous pre-treatment with Η2Ο2 or SNP (NO• donor) was studied. For this purpose 5-month old sour orange seedlings were pre-treated with Η2Ο2 (10 mΜ, 8 h) or SNP (100 μΜ, 48 h) and then hydroponically cultured for 16 days with or without 150 mM NaCl. After the end of the pre-treatment period (day 0), leaf and root samples were collected as well as at the days 2, 4, 8 and 16. In the leaves of NaCl-treated seedlings, more severe salinity symptoms were observed than in the leaves treated with Η2Ο2+NaCl or SNP+NaCl. During the experimental period, the roots in salinity treatments (NaCl, Η2Ο2+NaCl, SNP+NaCl) had high Na+ and Cl- concentrations. Comparing to the ionic concentration in the leaves of the NaCl treatment, the Na+ and Cl- were lower until the 16th day in the leaves of Η2Ο2+NaCl treated seedlings and until the 4th (Na+) and 8th (Cl-) day, in the leaves of the SNP+NaCl treatment. NO• was detected in the leaf vascular bundles and the epidermal cells. Hydrogen peroxide or SNP pretreatment increased leaf and root Η2Ο2 content from day 0 until day 16th. Salinity treatments induced Η2Ο2 and MDA production in the plant tissues. However, in comparison to NaCl treatment, Η2Ο2 concentration was higher in the leaves of Η2Ο2+NaCl treatment and in the SNP+NaCl roots. Moreover MDA concentration was lower in the leaves of Η2Ο2+NaCl and SNP+NaCl treatments compared to NaCl treatment. Root and leaf deoxyribose and plasmid pBR322 DNA, both protecting against HO• were in general decreased due to Η2Ο2, SNP, Η2Ο2+NaCl, SNP+NaCl application and predominantle to NaCl. The latter caused furthermore the detection of prooxidant compounds in the root extracts the 8th and 16th day. Hydrogen peroxide pretreatment increased immediately (day 0) leaf Put content and decreased leaf Spd and root Put. At the same time (day 0), SNP pretreatment increased root Spd and decreased leaf Put and Spd. Salinity treatments caused time and tissue-dependent changes in PAs content. The most notable changes include the leaf Put and Spd decrease due to all salinity treatments, the Put decrease the NaCl and SNP+NaCl roots and the transient increase of Spm in the leaves (the 2nd day in all salinity treatments; the 4th day in Η2Ο2+NaCl treatment) and Spd in the roots (2nd and 4th day in Η2Ο2+NaCl and SNP+NaCl treatments; 8th day in Η2Ο2+NaCl treatment). The NaCl και SNP+NaCl treatments caused a gradient decrease of the root total antioxidant power (FRAP values). FRAP values remained unchanged in the leaves of NaCl treatment but increased in the leaves of Η2Ο2, SNP, Η2Ο2+NaCl, SNP+NaCl treatments. The Η2Ο2 pretreatment decreased the ratio ΑA/(ΑA+DHA) in the roots (during the whole experimental period) and in the leaves (only at day 0) whereas the SNP pretreatment in the roots (until the 4th day). During the course of the experiment, apart from the leaves of Η2Ο2+NaCl treated plants, the ratio ΑA/(ΑA+DHA) decreased in all remaining salinity treatments. The glutathione redox state (GSH/GSH+GSSG) was reduced in the roots of the NaCl treated plants, but generally increased in the leaves and the roots of the seedlings treated with SNP or SNP+NaCl and remained unchanged in those treated with Η2Ο2 or Η2Ο2+NaCl.Η αλατότητα αποτελεί σημαντικό πρόβλημα για τα καλλιεργουμενα φυτά. Τα τελευταία έτη πραγματοποιήθηκαν ερευνητικές προσπάθειες προκειμένου να ενεργοποιηθούν οι μεταβολικές οδοί άμυνας και προσαρμογής των φυτών στην αλατότητα. Το Η2Ο2 και το NO• θεωρούνται μόρια επαγωγής μοριακών ερεθισμάτων για την ενεργοποίηση των μεταβολικών οδών που συνδέονται με τους αμυντικούς μηχανισμούς των φυτών. Έτσι, στην παρούσα διδακτορική διατριβή διερευνήθηκε εάν η εξωγενής προμεταχείριση σποροφύτων νεραντζιάς (Citrus aurantium) με Η2Ο2 ή με νιτροπρωσσικό νάτριο (SNP- δότης NO•) μπορεί μετέπειτα να προκαλέσει αντοχή στην αλατότητα. Για το σκοπό αυτό, σπορόφυτα νεραντζιάς ηλικίας 5 μηνών προμεταχειρίσθηκαν με Η2Ο2 (10 mΜ, 8 h) ή με SNP (100 μΜ, 48 h) και στη συνέχεια αναπτύχθηκαν υδροπονικά χωρίς ή με 150 mM NaCl για 16 ημέρες. Δείγματα φύλλων και ριζών παραλήφθηκαν αμέσως μετά τις προμεταχειρίσεις (ημέρα 0) καθώς και τη 2, 4, 8 και 16η ημέρα. Έντονα συμπτώματα αλατότητας παρατηρήθηκαν στα φύλλα της μεταχείρισης με NaCl και ηπιότερα στα φύλλα με Η2Ο2+NaCl ή με SNP+NaCl. Oι ρίζες των μεταχειρίσεων αλατότητας (NaCl, Η2Ο2+NaCl, SNP+NaCl) διατηρούσαν τις συγκεντρώσεις Na+ και Cl- σε υψηλά επίπεδα σε όλη τη διάρκεια του πειράματος. Οι συγκεντρώσεις Na+ και Cl- παρέμειναν σε χαμηλότερα επίπεδα στα φύλλα της μεταχείρισης Η2Ο2+NaCl μέχρι τη 16η ημέρα, ενώ της μεταχείρισης SNP+NaCl μέχρι την 4η (Νa+)και την 8η (Cl-) ημέρα, συγκριτικά με τη μεταχείριση NaCl. Το NO• ανιχνεύθηκε κυρίως στις ηθμαγγειώδεις δεσμίδες και στα επιδερμικά κύτταρα των φύλλων. Η προμεταχείριση με Η2Ο2 ή με SNP είχε ως αποτέλεσμα η συγκέντρωση του Η2Ο2 να αυξηθεί στις ρίζες και στα φύλλα των σποροφύτων από την έναρξη (ημέρα 0) ως το τέλος του πειράματος. Οι μεταχειρίσεις αλατότητας συντέλεσαν στην αυξημένη παραγωγή Η2Ο2 και μαλονδιαλδεΰδης (MDA) στους φυτικούς ιστούς. Όμως, η συγκέντρωση του Η2Ο2 ήταν υψηλότερη, συγκριτικά με τη μεταχείριση NaCl, στα φύλλα της μεταχείρισης Η2Ο2+NaCl και στις ρίζες της μεταχείρισης SNP+NaCl. Επίσης, η συγκέντρωση της MDA στα φύλλα των μεταχειρίσεων Η2Ο2+NaCl και SNP+NaCl ήταν χαμηλότερη από αυτή της μεταχείρισης NaCl. Σε γενικές γραμμές, η ικανότητα των ριζών και των φύλλων να προστατεύουν το μόριο της δεσοξυριβόζης και το πλασμιδιακό pBR322 DNA από τις HO• μειώθηκε εξαιτίας της προσθήκης Η2Ο2, SNP, Η2Ο2+NaCl, SNP+NaCl, ενώ ιδιαίτερη μείωση διαπιστώθηκε στη μεταχείριση NaCl, στις ρίζες των σποροφύτων της οποίας την 8η και τη 16η ημέρα ανιχνεύθηκε ποσότητα ουσιών με προοξειδωτική δράση. Η προμεταχείριση με Η2Ο2 είχε ως άμεσο αποτέλεσμα (ημέρα 0) την αύξηση της πουτρεσκίνης (Put) στα φύλλα, τη μείωση της (σπερμιδίνης) Spd στα φύλλα και τη μείωση της Put στις ρίζες. Επίσης, την ίδια χρονική στιγμή (ημέρα 0) η προμεταχείριση με SNP συντέλεσε στην αύξηση της Spd στις ρίζες και την μείωση της Put και Spd στα φύλλα. Μεταβολές στη συγκέντρωση των πολυαμινών (PΑs)- εξαρτώμενες από χρόνο και τον ιστό διαπιστώθηκαν στις μεταχειρίσεις αλατότητας με αξιοσημείωτες τη μείωση της Put και της Spd στα φύλλα όλων των μεταχειρίσεων αλατότητας, τη μείωση της Put στις ρίζες των μεταχειρίσεων NaCl και SNP+NaCl, καθώς και την παροδική αύξηση της σπερμίνης (Spm) στα φύλλα (2η ημέρα, σε όλες της μεταχειρίσεις αλατότητας; 4η ημέρα, μεταχείριση Η2Ο2+NaCl) και της Spd στις ρίζες (2η και 4η ημέρα, Η2Ο2+NaCl και SNP+NaCl; 8η ημέρα, Η2Ο2+NaCl)

Improving Quality of Fruit

Fruits are necessary for a balanced diet, and they are consumed for their vitamins, fiber, and other beneficial compounds [...

Improving Quality of Fruit

Fruits are necessary for a balanced diet, and they are consumed for their vitamins, fiber, and other beneficial compounds [...

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 2O 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 2O 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 2O 2 and NO signaling just after treatments or after 8 days of salt stress revealed tissue- and time-specific mechanisms controlling internal H 2O 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 condition

Involvement of AsA/DHA and GSH/GSSG Ratios in Gene and Protein Expression and in the Activation of Defence Mechanisms Under Abiotic Stress Conditions

In a persistently changing environment there are many adverse abiotic stress conditions such as cold, heat, drought, salinity, heavy metal toxicity and oxygen deprivation, which remarkably influence plant growth and crop production. Plant cells produce oxygen radicals and their derivatives, so-called reactive oxygen species (ROS) during various processes associated with abiotic stress. Moreover, the generation of ROS is the main means for higher plants to transmit cellular signalling information concerning the changing environmental conditions. Therefore, plants have evolved inducible redox state-based sensing mechanisms that are activated or amplified in response to adverse environmental conditions. Ascorbate and glutathione, the key cellular redox buffers, are used for both detoxification of ROS and transmission of redox signals. In recent years, it has become clear that abiotic stress conditions induce changes in the reduction/oxidation (redox) state of signalling molecules, which in turn modulate gene and protein expression to increase plant acclimation to abiotic stress. This important redox state-related branch of science has given several clues in understanding the adaptive plant responses to different stressful regimes. In this chapter, an overview of the literature is briefly presented in terms of the main function of ascorbate and glutathione in plant cells. Further more, we describe how important forms of abiotic stress regulate the expression of genes and proteins involved in the ascorbate and glutathione redox sensing system