Comparative Evaluation of Secondary Metabolite Chemodiversity of Citrus Genebank Collection in Greece: Can the Peel be More than Waste?

Citrus fruits are among the most economically important crops in the world. In the global market, the Citrus peel is often considered a byproduct but substitutes an important phenotypic characteristic of the fruit and a valuable source of essential oils, flavonoids, carotenoids, and phenolic acids with variable concentrations. The Mediterranean basin is a particularly dense area of autochthonous genotypes of Citrus that are known for being a source of healthy foods, which can be repertoires of valuable genes for molecular breeding with the focus on plant resistance and quality improvement. The scope of this study was to characterize and compare the main phenotypic parameters (i.e., peel thickness, fruit volume, and area) and levels of bioactive compounds in the peel of fruits from the local germplasm of Citrus in Greece, to assess their chemodiversity regarding their polyphenolic, volatile, and carotenoid profiles. A targeted liquid chromatographic approach revealed hesperidin, tangeretin, narirutin, eriocitrin, and quercetin glycosides as the major polyphenolic compounds identified in orange, lemon, and mandarin peels. The content of tangeretin and narirutin followed the tendency mandarin > orange > lemon. Eriocitrin was a predominant metabolite of lemon peel, following its identification in lower amounts in mandarin and at least in the orange peel. For these citrus-specific metabolites, high intra- but also interspecies chemodiversity was monitored. Significant diversity was found in the essential oil content, which varied between 1.2 and 3% in orange, 0.2 and 1.4% in mandarin, and 0.9 and 1.9% in lemon peel. Limonene was the predominant compound in all Citrus species peel essential oils, ranging between 88 and 93% among the orange, 64 and 93% in mandarin, and 55 and 63% in lemon cultivars. Carotenoid analysis revealed different compositions among the Citrus species and accessions studied, with ß-cryptoxanthin being the most predominant metabolite. This large-scale metabolic investigation will enhance the knowledge of Citrus peel secondary metabolite chemodiversity supported by the ample availability of Citrus genetic resources to further expand their exploitation in future breeding programs and potential applications in the global functional food and pharmaceutical industries.

Hydrogen Sulfide: A Potent Tool in Postharvest Fruit Biology and Possible Mechanism of Action.

Hydrogen sulfide (H2S), an endogenous gaseous molecule, is considered as a signaling agent, in parallel with other low molecular weight reactive substances, mainly hydrogen peroxide (H2O2) and nitric oxide (NO), in various plant systems. New studies are now revealing that the postharvest application of H2S, through H2S donors such as sodium hydrosulfide (NaSH) or sodium sulfide (Na2S), can inhibit fruit ripening and senescence programs in numerous fruits. We discuss here current knowledge on the impact of H2S in postharvest physiology of several climacteric and non-climacteric fruits such as banana, apple, pear, kiwifruit, strawberry, mulberry fruit, and grape. Although there is still a considerable lack of studies establishing the mechanisms by which H2S signaling is linked to fruit metabolism, we highlight several candidate mechanisms, including a putative cross-talk between H2S and ethylene, reactive oxygen and nitrogen species, oxidative/nitrosative stress signaling, sulfate metabolism, and post-translational modification of protein cysteine residues (S-sulfhydration) as being functional in this H2S postharvest action. Understanding H2S metabolism and signaling during postharvest storage and the interplay with other key player molecules would therefore provide new, improved strategies for better fruit postharvest storage. To achieve this understanding, postharvest fruit physiology research will need to focus increasingly on the spatial interaction between H2S and ethylene perception as well as on the interplay between S-sulfhydration/desulfhydration and S-nitrosylation/denitrosylation under several postharvest conditions.

Foliar Nutrition, Biostimulants and Prime-Like Dynamics in Fruit Tree Physiology: New Insights on an Old Topic.

Despite the fact that the usage of foliar nutrients has long history, many aspects of fertilization through leaves are still unknown. Herein, we review the current knowledge regarding the canopy fertilization putting special emphasis on Fe nutrition and briefly provide insights into the nanofertilizer technology of the foliar feeding of fruit crops. In addition, this paper discusses the main aspects of the foliar application of biostimulants regarding crucial factors of fruit cropping systems, such as fruit yield/size, tolerance to environmental stresses, and nutrient availability. Also, we specifically discuss the role of hydrogen peroxide (H2O2) and nitric oxide (NO) as priming molecules and their possible cross-talk with biostimulants in fruit tree physiology. Finally, a view of the key issues for future fundamental and applied research in the topic is put forward.

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.

Citrus Plants: A Model System for Unlocking the Secrets of NO and ROS-Inspired Priming Against Salinity and Drought.

Plants treated with chemical compounds can develop an enhanced capacity to resist long after being subjected to (a)biotic stress, a phenomenon known as priming. Evidence suggests that reactive oxygen species (ROS) and reactive nitrogen species (RNS) coordinately regulate plant stress responses to adverse environmental conditions; however, the mechanisms underlying this function remain unknown. Based on the observation that pre-exposure of citrus (Citrus aurantium L.) roots to the NO donor sodium nitroprusside (SNP) or to H2O2 prior to NaCl application can induce acclimation against subsequent stress we characterized the changes occurring in primed citrus tissues using several approaches. Herein, using this experimental model system, we provide an overview of our current knowledge of the possible mechanisms associated with NO and H2O2 priming to abiotic stresses, particularly concerning salinity and drought. The data and ideas presented here introduce six aspects of priming behavior in citrus under abiotic stress that provide knowledge necessary to exploit priming syndrome in the context of sustainable agriculture.

Roles of sodium hydrosulfide and sodium nitroprusside as priming molecules during drought acclimation in citrus plants.

Emerging evidence suggests that the gaseous molecules hydrogen sulfide (H2S) and nitric oxide (NO) enhances plant acclimation to stress; however, the underlying mechanism remains unclear. In this work, we explored if pretreatment of citrus roots with NaHS (a H2S donor) or sodium nitroprusside (SNP, a NO donor) for 2 days (d) could elicit long-lasting priming effects to subsequent exposure to PEG-associated drought stress for 21 d following a 5 d acclimation period. Detailed physiological study documented that both pretreatments primed plants against drought stress. Analysis of the level of nitrite, NOx, S-nitrosoglutahione reductase, Tyr-nitration and S-nitrosylation along with the expression of genes involved in NO-generation suggested that the nitrosative status of leaves and roots was altered by NaHS and SNP. Using a proteomic approach we characterized S-nitrosylated proteins in citrus leaves exposed to chemical treatments, including well known and novel S-nitrosylated targets. Mass spectrometry analysis also enabled the identification of 42 differentially expressed proteins in PEG alone-treated plants. Several PEG-responsive proteins were down-regulated, especially photosynthetic proteins. Finally, the identification of specific proteins that were regulated by NaHS and SNP under PEG conditions provides novel insight into long-term drought priming in plants and in a fruit crop such as citrus in particular.

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 plant's response to environmental challenge.

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.