Influence of Citrus sunki and Poncirus trifoliata Root Extracts on Metabolome of Phytophthora parasitica.

Phytophthora parasitica is an oomycete pathogen that infects a broad range of crops of worldwide economic interest; among them are citrus species. In general, some Citrus and the rootstocks of related genera offer considerable resistance against P. parasitica; therefore, understanding the mechanisms involved in the virulence of this pathogen is crucial. In this work, P. parasitica secondary metabolite production was studied using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/ESI-Q-TOF-MS) combined with chemometric tools, and its metabolic profile was evaluated under the influence of Citrus sunki (a highly susceptible host) and Poncirus trifoliata (a resistant genotype) extracts. The root extracts of Citrus sunki had an influence on the growth and hyphae morphology, and the root extracts of P. trifoliata had an influence on the zoospore behavior. In parallel, the spatial distribution of several metabolites was revealed in P. parasitica colonies using MALDI-MSI, and the metabolite ion of m/z 246 was identified as the protonated molecule of Arg-Ala. The MALDI-MSI showed variations in the surface metabolite profile of P. parasitica under the influence of the P. trifoliata extract. The P. parasitica metabolome analysis using UHPLC-ESI-Q-TOF-MS resulted in the detection of Arg-Gln (m/z 303.1775), as well as L-arginine (m/z 175.1191) and other unidentified metabolites. Significant variations in this metabolome were detected under the influence of the plant extracts when evaluated using UHPLC-ESI-Q-TOF-MS. Both techniques proved to be complementary, offering valuable insights at the molecular level when used to assess the impact of the plant extracts on microbial physiology in vitro. The metabolites identified in this study may play significant roles in the interaction or virulence of P. parasitica, but their functional characterization remains to be analyzed. Overall, these data confirm our initial hypotheses, demonstrating that P. parasitica has the capabilities of (i) recognizing host signals and altering its reproductive programing and (ii) distinguishing between hosts with varying responses in terms of reproduction and the production of secondary metabolites.

Anti-Obesity Effects of Polymethoxyflavone-Rich Fraction from Jinkyool (Citrus sunki Hort. ex Tanaka) Leaf on Obese Mice Induced by High-Fat Diet.

Polymethoxyflavones (PMFs) are flavonoids exclusively found in certain citrus fruits and have been reported to be beneficial to human health. Most studies have been conducted with PMFs isolated from citrus peels, while there is no study on PMFs isolated from leaves. In this study, we prepared a PMF-rich fraction (PRF) from the leaves of Citrus sunki Hort ex. Tanaka (Jinkyool) and investigated whether the PRF could improve metabolic decline in obese mice induced by a high-fat diet (HFD) for 5 weeks. The HFD-induced obese mice were assigned into HFD, OR (HFD + orlistat at 15.6 mg/kg of body weight/day), and PRF (HFD + 50, 100, and 200 mg/kg of body weight/day) groups. Orlistat and PRF were orally administered for 5 weeks. At the end of the experiment, the serum biochemical parameters, histology, and gene expression profiles in the tissues of each group were analyzed. The body weight gain of the obese mice was significantly reduced after orlistat and PRF administration for 5 weeks. PRF effectively improved HFD-induced insulin resistance and dyslipidemia. Histological analysis in the liver demonstrated that PRF decreased adipocyte size and potentially improved the liver function, as it inhibited the incidence of fatty liver. PRF activated AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and hormone-sensitive lipase (HSL) in HFD-induced obese mice. Moreover, liver transcriptome analysis revealed that PRF administration enriched genes mainly related to fatty-acid metabolism and immune responses. Overall, these results suggest that the PRF exerted an anti-obesity effect via the modulation of lipid metabolism.

Complete chloroplast genome sequence of a medicinal landrace citrus Jinkyool (Citrus sunki Hort. ex Tanaka) in Jeju Island, Korea.

Citrus sunki (Jinkyool) is a medicinal landrace citrus belonging to the Rutaceae family. We determined the complete chloroplast genome (160,699 bp) of C. sunki CRS0085 in Jeju Island, Korea. The genome is composed of four distinct parts; a large single copy of 87,918 bp, a small single copy of 21,355 bp, and a pair of inverted repeat regions of 25,713 bp. A total of 134 genes including 89 protein-coding genes, 37 tRNA genes, and eight rRNA genes were identified. The phylogenetic tree showed that C. sunki CRS0085 has the closest relationship with C. reticulata within genus Citrus.

In vivo and in vitro safety evaluation of fermented Citrus sunki peel extract: acute and 90-day repeated oral toxicity studies with genotoxicity assessment.

BACKGROUND: Citrus sunki Hort. ex Tanaka peel has been traditionally used as an ingredient in folk medicine due to its therapeutic effects on promotion of splenic health and diuresis as well as relief of gastrointestinal symptoms. Although a growing interest in health-promoting natural products and the development of highly concentrated products have facilitated consumption of C. sunki peel, its safety assessment has not been explored, posing a potential health risk. In this study, we carried out a series of systemic and genetic toxicity tests on fermented C. sunki peel extract (FCPE) to provide the essential information required for safe use in human. METHODS: We conducted acute and 90-day repeated oral toxicity studies in Sprague-Dawley rats to evaluate systemic toxicity, and three genotoxicity assays to measure bacterial mutation reversion, cellular chromosome aberration and in vivo micronucleus formation. RESULTS: Single oral administration of FCPE did not cause any clinical signs and lethality in all animals, establishing LD50 to be over 2000 mg/kg BW. Repeated administration of up to 2000 mg/kg BW FCPE for 90 days revealed no test substance-related toxicity as demonstrated in analysis of body weight gain, food/water intake, blood, serum biochemistry, organ weight and histopathology, collectively determining that the no-observable-adverse-effect-level of FCPE is over 2000 mg/kg BW. In addition, we detected no mutagenicity and clastogenicity in FCPE at 5000 µg/plate for the in vitro assays and 2000 mg/kg BW for the in vivo micronucleus test. CONCLUSION: FCPE did not cause systemic and genetic toxicity in our model systems at the tested dose levels. These results suggest a guideline for safe consumption of C. sunki peel in human.

QTLs and eQTLs mapping related to citrandarins' resistance to citrus gummosis disease.

BACKGROUND: Phytophthora nicotianae Breda de Haan (Phytophthora parasitica Dastur) causes severe damage to citrus crops worldwide. A population of citrandarins was created from the cross between the susceptible parent Citrus sunki Hort. Ex Tan. and the resistant parent Poncirus trifoliata (L.) Raf. cv. Rubidoux, both parents and two reference rootstocks (Rangpur lime and Swingle citrumelo) were grafted in a greenhouse on Rangpur lime. Inoculations were performed at 10 cm and 15 cm above the grafting region and the resulting lesions were evaluated by measuring the lesion length 60 days after inoculation. As control, non-inoculated plants of each genotype were used. In addition, we evaluated the expression of 19 candidate genes involved in citrus defense response 48 h after pathogen infection by quantitative Real-Time PCR (qPCR). We mapped genomic regions of Quantitative Trait Loci (QTLs) and Expression Quantitative Trait Loci (eQTLs) associated with resistance to P. parasitica in the linkage groups (LGs) of the previously constructed maps of C. sunki and P. trifoliata. RESULTS: We found disease severity differences among the generated hybrids, with lesion lengths varying from 1.15 to 11.13 mm. The heritability of the character was 65%. These results indicate that there is a great possibility of success in the selection of resistant hybrids within this experiment. The analysis of gene expression profile demonstrated a great variation of responses regarding the activation of plant defense pathways, indicating that citrandarins have several defense strategies to control oomycete infection. The information of the phenotypic and gene expression data made possible to detect genomic regions associated with resistance. Three QTLs and 84 eQTLs were detected in the linkage map of P. trifoliata, while one QTL and 110 eQTLs were detected in C. sunki. CONCLUSIONS: This is the first study to use eQTLs mapping in the Phytophthora-citrus interaction. Our results from the QTLs and eQTLs mapping allow us to conclude that the resistance of some citrandarins to the infection by P. parasitica is due to a favorable combination of QTLs and eQTLs transmitted by both parents.

Caracterização física e química dos frutos nos diferentes quadrantes da planta e germinação de sementes do portaenxerto cítrico tangerineira 'Sunki' / Physical and chemical characterization of fruit in different plant quadrants and germination rootstock of citrus tangerine 'Sunki'

A tangerineira 'Sunki' apresenta grande relevância para a citricultura brasileira por apresentar importantes características para o melhoramento genético e por ser considerada um ótimo portaenxento. Neste contexto, objetivou-se com o presente trabalho estudar a caracterização dos frutos do portaenxerto cítrico 'Sunki', em função da posição dos mesmos na planta e também, avaliar a germinação de suas sementes. As características físicas avaliadas foram massa do fruto, diâmetros longitudinal e equatorial dos frutos, relação diâmetro longitudinal/diâmetro equatorial, número de gomos por frutos, massa de gomos e de casca e número de sementes viáveis por fruto. Quanto à caracterização química, foi avaliado acidez titulável, sólidos solúveis, "Ratio", pH e vitamina C. Para a avaliação dessas variáveis, os frutos foram obtidos dos quadrantes nordeste, sudoeste, noroeste e sudeste. Com relação à avaliação da germinação das sementes, foram utilizadas diferentes doses de ácido giberélico e Stimulate. As sementes, extraídas de frutos maduros, foram lavadas, secadas à sombra e armazenadas em BOD. Logo após, estas foram tratadas com biorreguladores por 24 horas. Depois da embebição, as sementes foram colocadas no germinador em papel germitest. Após o inicio do processo germinativo, as avaliações foram realizadas diariamente até o trigésimo quarto dia. Frutos da tangerineira 'Sunki' apresentam boa uniformidade quanto à posição dos frutos na copa; o uso de ácido giberélico e de bioestimulante é promissor para uso comercial na germinação de sementes desse portaenxerto.

The tangerine ' Sunki' are highly relevant for the Brazilian citrus industry by presenting important traits for genetic improvement and to be considered a great portaenxento. In this context, the objective of the present work to study the characterization of the fruits of citrus rootstock ' Sunki', depending on the position on the plan and also the germination of their seeds . The physical characteristics evaluated were fruit weight, longitudinal and equatorial diameters of the fruit longitudinal diameter / equatorial diameter, number of buds per fruit , weight of buds and bark and number of viable seeds per fruit. As for the chemical characterization was evaluated titratable, acidity, soluble solid, "Ratio ", pH and vitamin C. For the evaluation of these variables, the fruits were obtained from the northeast, southwest, northwest and southeast quadrants. Regarding the evaluation of seed germination, different doses of gibberellic acid and Stimulate were used. The seeds extracted from ripe fruit, washed, dried in the shade and stored in BOD. Immediately after, they were treated for 24 hours with plant growth regulators. After soaking, the seeds were placed in a germinator in germitest paper. After initiation of the germination process, the evaluations were performed daily until the thirty- fourth day. Fruits of 'Sunki' mandarin with uneven as the fruit position within the canopy, the use of gibberellic acid and growth promoter is promising for commercial use on seed germination of this rootstock.

Anti-apoptotic effect of fermented Citrus sunki peel extract on chemical hypoxia-induced neuronal injury / 한국영양학회지

PURPOSE: Neuronal apoptotic events induced by aging and hypoxic/ischemic conditions is an important risk factor in neurodegenerative diseases such as ischemia stroke and Alzheimer's disease. The peel of Citrus sunki Hort. ex Tanaka has long been used as a traditional medicine, based on multiple biological activities including anti-oxidant, anti-inflammation, and anti-obesity. In the current study, we examined the actions of fermented C. sunki peel extract against cobalt chloride (CoCl2)-mediated hypoxic death in human neuroblastoma SH-SY5Y cells. METHODS: Cell viability was measured by trypan blue exclusion. Expression of apoptosis related proteins and release of cytochrome c were detected by western blot. Production of intracellular reactive oxygen species (ROS) and apoptotic morphology were examined using 2',7'-dichlorofluorescin diacetate (DCF-DA) and 4',6-diamidino-2-phenylindole (DAPI) staining. RESULTS: Exposure to CoCl2, a well-known mimetic agent of hypoxic/ischemic condition, resulted in neuronal cell death via caspase-3 dependent pathway. Extract of fermented C. sunki peel significantly rescued the CoCl2-induced neuronal toxicity with the cell viability and appearance of apoptotic morphology. Cytoprotection with fermented C. sunki peel extract was associated with a decrease in activities of caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). In addition, increase in the intracellular ROS and release of cytochrome c from mitochondria to the cytosol were inhibited by treatment with extract of fermented C. sunki peel. CONCLUSION: Based on these data, fermented C. sunki peel extract might have a protective effect against CoCl2-induced neuronal injury partly through generation of ROS and effectors involved in mitochondrial mediated apoptosis.