Characterization of ß-amino- and γ-amino butyric acid-induced citrus seeds germination under salinity using nanoLC-MS/MS analysis.

KEY MESSAGE: BABA or GABA induces salinity acclimation during citrus seeds germination via alternation of specific proteins (e.g., citrin). The impact of four elicitors, namely hydrogen peroxide (H2O2), ß-amino butyric acid (BABA), γ-amino butyric acid (GABA) and hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS), in citrus seed germination under salinity (150 mM NaCl) was tested. The germination potential was adversely affected by NaCl-alone treatment. Pretreatment with H2O2 or the NaHS-H2S donor prior to salinity had no significant effect in germination process, however, BABA and GABA substantially improved seed acclimation to salinity, as evidenced by increased germination percentage and radicle length. Total soluble proteins of radicle and cotyledons were separated by 1DE SDS-PAGE and proteins zones were analyzed by mass spectrometry. In total, 27 and 3 proteins were identified in radicle and cotyledons, respectively. The identified proteins mainly include redox-regulated enzymes (i.e., glutathione S-transferase, dehydroascorbate reductase, Mn-superoxide dismutase, glutathione peroxidase), energy-related proteins (i.e., isocitrate lyase, malate synthase, pyruvate decarboxylase), stress proteins (i.e., stress-related protein, miraculin, thaumatin, disulfide isomerase), storage proteins (i.e., vicilin, Pis v 1 allergen 2S albumin) and transcriptional regulators (i.e., MarR family transcriptional regulator, MADS544 protein). Pretreatments with BABA or GABA altered the accumulation of protein zones exclusively corresponding to citrin, indicating that this protein may serve as a marker for salinity acclimation in citrus seeds.

Comparative Physiological and Proteomic Analysis Reveal Distinct Regulation of Peach Skin Quality Traits by Altitude.

The role of environment in fruit physiology has been established; however, knowledge regarding the effect of altitude in fruit quality traits is still lacking. Here, skin tissue quality characters were analyzed in peach fruit (cv. June Gold), harvested in 16 orchards located in low (71.5 m mean), or high (495 m mean) altitutes sites. Data indicated that soluble solids concentration and fruit firmness at commercial harvest stage were unaffected by alitute. Peach grown at high-altitude environment displayed higher levels of pigmentation and specific antioxidant-related activity in their skin at the commercial harvest stage. Skin extracts from distinct developmental stages and growing altitudes exhibited different antioxidant ability against DNA strand-scission. The effects of altitude on skin tissue were further studied using a proteomic approach. Protein expression analysis of the mature fruits depicted altered expression of 42 proteins that are mainly involved in the metabolic pathways of defense, primary metabolism, destination/storage and energy. The majority of these proteins were up-regulated at the low-altitude region. High-altitude environment increased the accumulation of several proteins, including chaperone ClpC, chaperone ClpB, pyruvate dehydrogenase E1, TCP domain class transcription factor, and lipoxygenase. We also discuss the altitude-affected protein variations, taking into account their potential role in peach ripening process. This study provides the first characterization of the peach skin proteome and helps to improve our understanding of peach's response to altitude.

Integrated analysis of metabolites and proteins reveal aspects of the tissue-specific function of synthetic cytokinin in kiwifruit development and ripening.

UNLABELLED: Fruit development and ripening depends on highly coordinated phyto-hormonal activities. Although the role of synthetic cytokinin N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) in promoting fruit growth has been established, knowledge regarding the underlying mechanism is still lacking. Here, we characterize the effect of CPPU application 20d after full bloom at pre- and post-harvest biology of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. 'Hayward'). Data revealed that CPPU stimulates kiwifruit growth through the enlargement of small cells. During fruit development, the abundance of 16 proteins that are mainly related to defence was increased by CPPU while CPPU altered the expression of 19 polar metabolites in outer pericarp. Sugar homeostasis, cell wall modifications, TCA cycle and myo-inositol pathway were mostly affected by CPPU in kiwifruit during development. Upon postharvest ripening at 20°C following 2months of cold storage (0°C), CPPU suppressed ethylene production and retained central placenta softening, indicating that CPPU induced tissue-dependent disturbances in climacteric ripening. Nineteen central placenta proteins and up to 15 metabolites of outer pericarp and central placenta tissues were affected by CPPU in ripened kiwifruits. These observations amplified our understanding in the regulation of fruit development and ripening by exogenously supplied cytokinins. BIOLOGICAL SIGNIFICANCE: This study demonstrates that CPPU application, apart from fruit development, influenced also the kiwifruit climacteric ripening behaviour. An insight on the action of CPPU during kiwifruit development is provided, showing that it is partially based on a general stimulation of TCA cycle and myo-inositol pathway along with alternation in sugar and cell wall metabolism. Data also revealed that CPPU regulates ethylene biosynthesis and influences central placenta softening, indicating that this tissue may play a prominent role in kiwifruit ripening. Also, this work provides a first characterization of the ripening-affected central placenta proteins that offers insights into kiwifruit ripening. The current study provides a baseline of information for understanding the metabolic processes that are regulated by exogenous cytokinin during fruit development and ripening.

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.

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.

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.

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.

Proteomics reveals the overlapping roles of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity.

Hydrogen peroxide (H(2)O(2)) and nitric oxide (*NO) are key reactive species in signal transduction pathways leading to activation of plant defense against biotic or abiotic stress. Here, we investigated the effect of pre-treating citrus plants (Citrus aurantium L.) with either of these two molecules on plant acclimation to salinity and show that both pre-treatments strongly reduced the detrimental phenotypical and physiological effects accompanying this stress. A proteomic analysis disclosed 85 leaf proteins that underwent significant quantitative variations in plants directly exposed to salt stress. A large part of these changes was not observed with salt-stressed plants pre-treated with either H(2)O(2) or sodium nitroprusside (SNP; a *NO-releasing chemical). We also identified several proteins undergoing changes either in their oxidation (carbonylation; 40 proteins) and/or S-nitrosylation (49 proteins) status in response to salinity stress. Both H(2)O(2) and SNP pre-treatments before salinity stress alleviated salinity-induced protein carbonylation and shifted the accumulation levels of leaf S-nitrosylated proteins to those of unstressed control plants. Altogether, the results indicate an overlap between H(2)O(2)- and *NO-signaling pathways in acclimation to salinity and suggest that the oxidation and S-nitrosylation patterns of leaf proteins are specific molecular signatures of citrus plant vigour under stressful 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.

Effects of 4-month Fe deficiency exposure on Fe reduction mechanism, photosynthetic gas exchange, chlorophyll fluorescence and antioxidant defense in two peach rootstocks differing in Fe deficiency tolerance.

Fe deficiency was imposed by omission of Fe (-Fe), or by inclusion of bicarbonate (supplied as 20 mM NaHCO3) in the nutrient solution in two contrasting peach rootstocks (GF-677; tolerant to Fe deficiency and Cadaman; sensitive to Fe deficiency) for 4 months. In the Fe-deprived leaves and roots, and especially in those treated with bicarbonate, a decrease in Fe concentrations was recorded. Omission of Fe resulted in an increase of the activity of root Fe(III)-chelate reductase (FCR) in both rootstocks, whereas FCR activity decreased in the bicarbonate-treated roots of Cadaman. The results obtained from the FCR assay were confirmed by an agarose-based staining technique used to localize FCR activity. Also, an agar-pH-test revealed that the roots of GF-677 exposed to (-Fe) treatment induced a strong H+ extrusion. In addition, Fe deficiency resulted in reduction of the total chlorophyll (CHL) content. Apart from the (-Fe)-treated leaves of GF-677, Fe deficiency caused a decline in the photosynthetic rate (P(n)) and stomatal conductance (g(s)), without changes of the intercellular CO2 concentration (C(i)), as well as a reduction in the maximum quantum yield of PSII (F(v)/F(m)) and the ratio between variable to initial fluorescence F(v)/F0. The above changes were particularly evident for the bicarbonate-treated leaves of Cadaman. On the other hand, Fe deficiency resulted in an increase of leaf superoxide dismutase (SOD) activity and a depression of catalase (CAT) activity in the leaves and roots, irrespective of the rootstock. Although the non-enzymatic antioxidant activity (FRAP values) was increased in the roots of both rootstocks exposed to -Fe treatment, however, FRAP values were stimulated in the (-Fe)-treated leaves of GF-677 and decreased in the bicarbonate-treated leaves of Cadaman. The H2O2 content was increased in Fe-deprived tissues except for the (-Fe)-treated leaves and roots of GF-677. As a result of Fe deficiency, peroxidase (POD) activity and isoform expression were diminished in the tissues of Cadaman. However, in the tissues of GF-677 subjected to -Fe treatment POD activity was increased whereas an additional POD isoform was detected in the roots suggesting that expression of POD isoforms might be an important attribute linked to the tolerance to Fe deficiency.