Targeted/untargeted metabolomics and antioxidant properties distinguish Citrus reticulata 'Chachi' from Citrus reticulata Blanco.

Dried citrus peel (DCP), also called "Chen Pi", has edible and medicinal value. However, the specific differences among various sources remain unknown. Herein, we collected six DCP species, namely, one Citrus reticulata 'Chachi' (CZG) and five Citrus reticulata Blanco (CRB). Targeted high-performance liquid chromatography and untargeted ultra-high-performance liquid chromatography-tandem mass spectrometry were employed to comprehensively compare the phenolic compounds and metabolites in DCP. Interestingly, 13 different phenolic compounds were noted in DCP. The total phenolic compound content in all CRB samples (58.86-127.65 mg/g) was higher than that of CZG (39.47 mg/g). Untargeted metabolomic revealed 1495 compounds, with 115 differentially expressed metabolites for CRBs and CZG, particularly flavonoids (38), terpenoids (15), and phenolic acids and derivatives (9). Lastly, antioxidant assays revealed that all CRB samples exhibited higher antioxidant activities compared with CZG. Therefore, our study results provide a theoretical basis for the high-value utilization of citrus peels and their metabolites.

Genome-wide analysis of UDP-glycosyltransferase family in Citrus sinensis and characterization of a UGT gene encoding flavonoid 1-2 rhamnosyltransferase.

UDP-glycosyltransferases (UGTs) play a crucial role in the glycosylation of secondary metabolites in plants, which is of significant importance for growth and response to biotic or abiotic stress. Despite the wide identification of UGT family members in various species, limited information is available regarding this family in citrus. In this study, we identified 87 UGT genes from the Citrus sinensis genome and classified them into 14 groups. We characterized their gene structures and motif compositions, providing insights into the molecular basis underlying discrepant functions of UGT genes within each evolutionary branch. Tandem duplication events were found to be the main driving force behind UGT gene expansion. Additionally, we identified numerous cis-acting elements in the promoter region of UGT genes, including those responsive to light, growth factors, phytohormones, and stress conditions. Notably, light-responsive elements were found with a frequency of 100 %. We elucidated the expression pattern of UGTs during fruit development in Citrus aurantium using RNA-seq and quantitative real-time PCR (qRT-PCR), revealing that 10 key UGT genes are closely associated with biosynthesis of bitter flavanone neohesperidosides (FNHs). Furthermore, we identified Ca1,2RhaT as a flavonoid 1-2 rhamnosyltransferase (1,2RhaT) involved in FNHs biosynthesis for the first time. Isolation and functional characterization of the gene Ca1,2RhaT from Citrus aurantium in vitro and in vivo indicated that Ca1,2RhaT encoded a citrus 1,2RhaT and possessed rhamnosyl transfer activities. This work provides comprehensive information on the UGT family while offering new insights into understanding molecular mechanisms regulating specific accumulation patterns of FNHs or non-bitter flavanone rutinosides (FRTs) in citrus.

Citrus peel powder alters the rheological properties and 3D printing performance of potato starch gel.

Owing to the growing interest in sustainable resource utilization, the current study explores the potential replacement of pectin with citrus peel powder (CP) in starch-based 3D food printing ink formulations. The effect of different concentrations of pectin (1 %, 2 %, 3 %) and CP (1 %, 2 %, 3 %) on the printing fidelity, microstructure, rheological and textural properties of potato starch gel were investigated. The results showed that the 3D printing performance of CP-added inks was higher than that of pectin-added inks at all tested concentrations. The storage modulus of CP-added ink was higher than that of pectin-added ink proving higher printing fidelity of CP-added inks. Additionally, hardness, gumminess, springiness and chewiness of food ink increased with an increase in the concentration of CP while decreased with an increase in concentration of pectin. Interestingly, pectin and CP-added inks displayed similar in vitro digestibility, suggesting an insignificant effect of replacing pectin with CP on in vitro glucose release. Moreover, the antioxidant activity of CP-added ink was higher than pectin-added ink demonstrating the potential applications of CP-added ink in functional ink development. Therefore, this study claims for effective replacement of pectin with CP in starch-based 3D food printing ink formulations as a promising sustainable additive.

Valorization of hydro-distillate of fruit peels of Citrus paradisi macfad. Cultivar. Foster: Chemical profiling, antioxidant evaluation and in vitro and in silico enzyme inhibition studies.

The major commercial crops in Pakistan are citrus fruit trees, which are farmed extensively and serve as the country's principal source of foreign exchange. A local citrus plant, Citrus paradisi, variety Foster is famous for its valuable fruit and fruit juice, however, tons of peels of this fruit are thrown as waste, which otherwise can be utilized in formulating nutraceutical and cosmetics. In the present study, essential oil of fruit peels was obtained through hydro-distillation, which was then analyzed through GC-MS analysis and studied for its antioxidant and enzyme inhibition potential. GCMS analysis revealed the presence of several components; major were found to be limonene, α-terpineol, caryophyllene, δ-amorphene, elemol, γ-eudesoml, nootkatone and di-isooctyl phthalate. Although, the oil showed weak free radical inhibition, it was potentially active in CUPRAC, FRAP, phosphomolybdenum and metal chelating antioxidant assays. The oil also exhibited anti-glucosidase, anti-amylase activities and also exhibited potent inhibition of the enzyme tyrosinase, which makes it strong candidate for nutraceuticals and skin care products. The docking studies also substantiate our results and caryophyllene, γ-eudesoml and nootkatone showed good binding affinity α-glucosidase and α-amylase and all tested compounds showed the higher binding affinity towards the enzyme tyrosinase.

Synthesis and characterization of Lanthanum Oxide nanoparticles using Citrus aurantium and their effects on Citrus limon Germination and Callogenesis.

The plant extract-mediated method is eco-friendly, simple, safe, and low-cost, using biomolecules as a reducing agent to separate nanoparticles. Lanthanum (La) is a rare earth metal that positively affects plant growth and agriculture. Citrus limon is a leading citrus fruit with many varieties. Conventional vegetative propagation methods depend on season, availability of plant material and are time-consuming. It is the main reason for limiting the acceptance of new varieties. So, In-vitro propagation of the lemon method is practiced overcoming all these problems. Lanthanum oxide nanoparticles (La2O3-NPs) were synthesized using plant extract of C. aurantium. Ultraviolet (UV)-Visible Spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Fourier Transform Infrared (FTIR) spectroscopy, and Thermal Gravimetric Analysis (TGA) were used to characterize the synthesized La2O3-NPs. Fabricated La2O3-NPs were oval and spherical, with an average size of 51.1 nm. UV-visible absorption spectra of La2O3-NPs were shown at a sharp single peak at 342 nm and FTIR showed stretching frequency at 455 cm-1-516 cm-1. In the TGA outcome, mass loss was 9.1%. In vitro experiments demonstrated that La2O3-NPs significantly enhanced the germination and growth of C. limon seeds, achieving an 83% germination rate at 5 mg/L concentration, with uncoated seeds showing root initiation at 10 days and shoot formation at 15 days. Furthermore, La2O3-NPs effectively stimulated callus induction and maturation, with optimal responses observed in media containing MS and 2 mg/L 2,4-D, resulting in a maximum callus frequency of 100% from leaves and 87.5% from shoots at 5 mg/L concentration. These findings underscore the potential of La2O3-NPs to improve seed germination rates, seedling vigor, and callogenesis efficiency, suggesting their promising integration into agricultural practices for sustainable crop production, especially in suboptimal growing conditions. Future research is recommended to explore the mechanisms and broader applications of La2O3-NPs across various plant species and environments.

Systematic analysis and functional verification of citrus ascorbate peroxidases reveal that CsAPX01 and CsAPX02 negatively regulate citrus bacterial canker through the hydrogen peroxide regulation.

Ascorbate peroxidases (APXs) are antioxidant enzymes that play vital roles in redox homeostasis in plants. Citrus is susceptible to infection by Xanthomonas citri subsp. citri (Xcc), resulting in citrus bacterial canker (CBC). The present study used bioinformatic and expression analyses to investigate the APX family in Citrus sinensis. Bioinformatic research revealed the chromosomal locations, phylogeny, gene structure, promoter elements, functional domains, conserved motifs, and most likely physicochemical properties of the sequences. Six APXs clustered in three groups were identified, with each protein containing a single peroxidase domain. The promoter regions contained a variety of transcription factor-binding and hormone-response components. Xcc infection induced different CsAPX01 and CsAPX02 expressions in the CBC-susceptible Wanjincheng and CBC-resistant Kumquat varieties. Subcellular localization and transient expression showed that CsAPX01 and CsAPX02 were expressed in the cytoplasm and nucleus and had hydrogen peroxide (H2O2)-scavenging activity. Virus-induced gene silencing (VIGS) of CsAPX01 and CsAPX02 resulted in strong resistance to CBC and H2O2 bursts without effects on the plant phenotype. The current study focused on investigating and characterizing the citrus APX family. It was found that CsAPX01 and CsAPX02 exacerbated CBC by altering the balance of H2O2. These findings emphasize the importance of APXs in enhancing plant resistance to pathogens.

Rootstock selection for 'Swatow' Mandarin trees grown at different locations throughout the Brazilian subtropics.

Evaluating citrus rootstocks is of paramount importance in determining their suitability for a certain region and promoting resilience in orchards by increasing the genetic pool, thereby potentially contributing to a more strategic establishment of new plantings. This long-term field study (2000-2013) aimed to evaluate different rootstocks for 'Swatow' mandarin grown at two locations (Paranavaí and Londrina) in the Brazilian subtropics. Nine rootstocks were evaluated, including 'Rangpur' lime, 'Swingle' citrumelo, 'Volkamer' lemon, 'Caipira DAC' sweet orange, 'Cleopatra' and 'Sunki' mandarins, 'Trifoliate' orange, 'Carrizo', and 'Fepagro C-13' citranges. Trees were assessed for vegetative growth, yield, fruit quality, density, and yield estimates. The experimental design was a randomized block arranged in a 9 × 2 setting (rootstock × location) with 6 replicates and 4 trees per plot. 'Swatow' trees grew more vigorously in Londrina than Paranavaí, particularly for 'Cleopatra' and 'Sunki' pairings. Tree vigor was reduced with 'Trifoliate', resulting in higher tree density estimates and yield efficiency. This rootstock, along with 'Rangpur', 'Swingle', and 'Carrizo' provided superior yield to the scion. All tested rootstocks conferred good fruit quality. Fruits were larger and heavier in 'Sunki' pairings, showing higher soluble solids (SS) content, along with 'Caipira DAC', 'Trifoliate', 'Swingle', and 'Carrizo' at both locations. Our findings confirm the suitability of 'Trifoliate' orange, 'Carrizo' citrange, or 'Caipira DAC' orange rootstocks as promising candidates for 'Swatow' mandarin cultivation in humid subtropical and analogous regions. Further investigations are invoked to improve the horticultural performance of 'Swatow' mandarin trees grafted onto these rootstocks.

Biocontrol of citrus fungal pathogens by lipopeptides produced by Bacillus velezensis TZ01.

Citrus diseases caused by fungal pathogens drastically decreased the yield and quality of citrus fruits, leading to huge economic losses. Given the threats of chemical pesticides on the environment and human health, biocontrol agents have received considerable attention worldwide as ecofriendly and sustainable alternative to chemical fungicides. In the present study, we isolated a Bacillus velezensis strain TZ01 with potent antagonistic effect against three citrus pathogenic fungi: Diaporthe citri, Colletotrichum gloeosporioides and Alternaria alternata. The culture supernatant of this strain exhibited remarkable antifungal activity on potato dextrose agar plates and detached leaves of five citrus varieties. Treatment with TZ01 culture supernatant obviously affected the hyphal morphology and caused nucleic acid leakage. The crude lipopeptides (LPs) extracted from the culture supernatant were found as the major active ingredients, and could maintain the activity under a wide range of temperature and pH and ultraviolet radiation. Furthermore, the type of LPs, produced in vitro, were explored. Whole-genome sequencing of TZ01 revealed secondary metabolite gene clusters encoding synthetases for non-ribosomal peptides and polyketide production, and gene clusters responsible for the synthesis of three important LPs (surfactin, iturin, and fengycin) were identified in the genome. The liquid chromatography-mass spectrometry analysis confirmed the presence of various homologs of surfactin A, bacillomycin D, and fengycin A in the extracted LPs. Taken together, these results contribute to the possible biocontrol mechanisms of B. velezensis strain TZ01, as well as providing a promising new candidate strain as a biological control agent for controlling citrus fungal pathogens.

Effect of excitation voltage in a magnetically induced electric field on the physicochemical, structural and functional properties of citrus pectin.

Treatment with a magnetic induced electric field (MIEF) under acidic conditions has proven to be an effective method for modifying pectin, enhancing its functional attributes. In this study, the effects of varying excitation voltages of MIEF under acidic conditions on the physicochemical, structural, and functional properties of citrus pectin (CP) were explored. The results demonstrated that compared to CP without MIEF treatment, MIEF-treated CP exhibited enhanced thermal stability, rheological behavior, emulsifying and gel-forming abilities, and antioxidant capacity. These improvements were attributed to higher degrees of esterification, reduced molecular weights, and increased levels of galacturonic acid and homogalacturonan in the structural backbone of the treated CP. Additionally, MIEF treatment under acidic conditions altered the surface morphology and crystalline structure of CP. Therefore, our findings suggest that applying moderate excitation voltages (150-200 V) during MIEF treatment can enhance the functional properties of CP, leading to the production of high-quality modified pectin.

Dynamic changes in the metabolome and microbiome during Citrus depressa Hayata liquid fermentation.

Citri Reticulatae Pericarpium (CRP) is a common traditional Chinese herbal medicine, valued for its multi-bioactivity. However, its processing time, environment, and microorganisms all affect its quality and bioactivity. To address this, the study replaced solid-state fermentation with liquid fermentation using microorganisms and isolated Bacillus amyloliquefaciens, respectively. This aimed to discover a more stable processing method and examine metabolite-micobiota correlations. Non-targeted metabolomics identified 70 differential metabolites, focusing on amino acids, polymethoxyflavones (PMFs), and carbohydrates. Long-read sequencing showed a shift in dominant bacterial genera from Lactobacillus to Pediococcus, then to Clostridium. Spearman analysis revealed a positive correlation between specific Clostridium species and PMFs production. B. amyloliquefaciens fermentation notably increased PMFs content without reducing hesperidin levels, suggesting its potential as an alternative processing method. This study offers valuable insights into metabolome-microbiome interactions for future biotransformation research.

Influence of pH on the emulsifying property of high methyl-esterified citrus pectin in the presence of calcium cations.

High methyl-esterified citrus pectin (HMCP) is often used as a thickness in food products and is considered a poor emulsifier, especially in neutral pH solutions. Our previous findings show that the emulsifying capacity of HMCP could be significantly enhanced by calcium cations. Besides, the pH of the solution decreased in the presence of calcium cations. However, the impact of solution pH on HMCP emulsifying capacity in the presence of calcium cations is unclear. In this study, the pH of the HMCP solution was adjusted from 3.00 to 8.00 before adding calcium cations. The solution properties and emulsifying properties were analyzed in light of the existence of calcium cations. The results showed that the pH of the HMCP solutions decreased after bringing calcium cations into them. Calcium cations could change the solution rheological properties, particle size distributions and morphologies, and the particle microenvironmental hydrophobic areas in HMCP solutions while increasing the pH of HMCP solutions, contributing to improving the emulsifying capacity of HMCP. HMCP had the best emulsifying ability when the pH of the HMCP solutions was kept at a neutral level. This research gives us new ideas to adjust the emulsifying property of HMCP.

Effect of pectin modification on the properties of cellulose nanofibrils prepared from citrus peel.

Cellulose nanofibrils (CNFs) from non-woody biomass, including citrus peel (CpCNFs), are promising naturally occurring nanomaterials; however, their properties depend on the composition of non-cellulosic components, including pectin. In this study, the effects of pectin modifications on CpCNFs were examined, including demethylesterification using alkaline treatment and enzymatic degradation of pectin using pectinase. CpCNFs could be redispersed in water with little aggregation after drying; however, the redispersibilities of both alkaline-treated (AT-CpCNFs) and pectinase-treated CpCNFs (PT-CpCNFs) were improved. Both AT-CpCNFs and PT-CpCNFs exhibited higher viscosity than untreated CpCNFs (UT-CpCNFs); redispersion in water after drying further increased the viscosity. A quartz crystal microbalance revealed that interactions between AT-CpCNFs were barely detectable, and interactions between PT-CpCNFs were stronger than those between UT-CpCNFs. The increase in the carboxylate groups of pectin due to demethylesterification in AT-CpCNF may have increased the viscosity and reduced the interactions between AT-CpCNFs, explaining the improved redispersibility. The increase in the viscosity of PT-CpCNFs may be attributed to the increased purity of CNFs, which is assumed to be more viscous than pectin. Our results show that the properties of CpCNFs are affected by the structure, properties, and content of pectin and can be controlled by pectin modification.

[Development of an Odor Evaluation Method for Citrus Unshiu Peel Using an Electronic Nose Based on the Intensity of per Unit Length (INPULTH)].

Although odor is an important indicator of herbal medicine quality, an objective odor evaluation method remains undiscovered. Quantitative measurement using previous methods is complicated as Citrus Unshiu Peel (Chimpi) emits an odor when broken. To establish odor evaluation methods for herbal medicines using chimpi as an example, we developed a reproducible method for breaking samples and an objective odor evaluation method using an electronic nose (e-nose). First, an odor-emitting device (OED) was fabricated by modifying a pill cutter, which suppressed the spread of odor components into the room air while cutting the samples. The odor was emitted from chimpi with an OED and measured using an e-nose. The cut length was then measured. The sensor intensity was positively correlated with the cut length (r = 0.840-0.927) in the same sample, and the intensity per unit length (INPULTH) calculated from the sensor intensity and cut length enables the comparison of the sensor intensity among different samples. In addition, average d-limonene emission level measured by GC-MS was positively correlated with average INPULTH (r = 0.999), which suggests that this OED and e-nose method enables the comparison of the sensor intensity and d-limonene emissions. INPULTH also positively correlated with other seven monoterpenes such as p-cymene, ß-myrcene, ß-phellandrene, α-pinene, ß-pinene, γ-terpinene, and α-terpinolene (r = 0.701-0.865). Therefore, monoterpene content can be evaluated by measuring the odor in the same way as d-limonene. In conclusion, we developed a simple odor intensity evaluation method optimized for chimpi to establish an odor evaluation method for herbal medicines.

A synergistic interaction between Citrus hystrix peel essential oil and tetracycline and evaluation of their antibacterial mechanism of action in vitro and in silico against Escherichia coli.

Citrus hystrix essential oil (CHEO) have shown various pharmacological properties including antibacterial activity. This EO also possessed antibacterial effect against foodborne pathogens. There is less information available about the synergy interaction between CHEO and tetracycline, as well as their mechanism of action. Therefore, this study was conducted to evaluate the synergistic effect of CHEO and tetracycline against clinical isolate of Escherichia coli. Antibiofilm, bacteriolytic, and efflux pump inhibitor activities were also performed. The chemical composition of CHEO was analysed using GC-MS. Three major compounds, D-limonene (25.02%), ß-pinene (23.37%), and ß-sabinene (22.20%) were identified. CHEO exhibited moderate antibacterial activity with MIC value of 250 µg/mL. The combination of CHEO (7.8 µg/mL) and tetracycline (62.5 µg/mL) produced a synergistic effect on E. coli with fractional inhibitory concentration index of 0.5. This mixture inhibited biofilm formation in E. coli. The combination of 7.8 µg/mL CHEO and 62.5 µg/mL tetracycline demonstrated bacteriolytic activity. In addition, the CHEO at 250 µg/mL showed a significant effect in inhibiting efflux pump. D-limonene has a binding free energy value of -20.13 kcal/mol with ompA transmembrane domain of E. coli. This finding indicates that CHEO has a potency to be developed as natural antibacterial against E. coli.

Antioxidant, anti-inflammatory and antibacterial activities of methanol extract and essential oil of two varieties of Citrus sinensis L. in Iran.

This study aimed to investigate the antioxidant, anti-inflammatory, antifungal, and antibacterial properties of skin and Flesh methanol extract and essential oil of two varieties of species of Citrus sinensis L in Iran (northern and southern oranges). This study evaluated total phenol and flavonoid contents and the antioxidant activity of methanol extracts at different concentrations (25, 50, 100, 200, and 400 µg/ml). Albumin denaturation inhibition and RBC membrane immobilisation assays were used as an in vitro model to investigate the anti-inflammatory effects. Based on the results, the highest phenol and flavonoid contents and antioxidant activity were related to the northern orange skin. This extract also had the most significant impact on albumin denaturation inhibition and RBC membrane immobilisation by IC50 of 365 ± 12 µg/ml and 940 ± 22 µg/ml, respectively. In antimicrobial assays, only the skin and flesh of northern orange positively affected Gram-positive bacteria.

Toxicity and enzymatic mechanism of Citrus spp. essential oils and major constituents on Haemaphysalis longicornis (Acari: Ixodidae) and non-target Harmonia axyridis (Coleoptera: Coccinellidae).

Plant essential oils (EOs)-based acaricides have been recognized as environmentally-friendly alternatives to synthetic acaricides because of their low toxicity against non-target species. Despite this, there are knowledge gaps regarding the toxicity mechanisms of plant EOs against non-target species. Here, the toxicology and enzymatic mechanism of Citrus reticulata and Citrus lemon EOs were evaluated against the vector pest, Haemaphysalis longicornis, and non-target ladybird beetle, Harmonia axyridis. Both EOs were mainly composed of d-Limonene, followed by ß-Myrcene and γ-Terpinene in C. reticulata, and (-)-ß-Pinene and γ-Terpinene in C. lemon. Citrus reticulata and C. lemon EOs were toxic to Hae. longicornis, with 50 % lethal concentration (LC50) values estimated at 0.43 and 0.98 µL/mL via nymphal immersion test, and 42.52 and 46.38 µL/mL via spray application, respectively. Among the constituents tested, ß-Myrcene was the most effective, with LC50 values of 0.17 and 47.87 µL/mL via immersion and spray treatment, respectively. A significant mortality of non-target Har. axyridis was found when treated by the EOs at concentrations two times greater than LC50 estimated against H. longicornis. The biochemical assay revealed that the EOs induced changes in the antioxidant enzyme activity of superoxide dismutases, catalase, and glutathione peroxidase in Hae. longicornis and Har. axyridis. The results demonstrated the acaricidal potential of citrus EOs and their major constituents for tick control, revealed the risk of the EOs to non-target species, and provided relevant insights into the mechanisms underlying their toxicity.

A two-in-one molybdenum disulfide-chitosan nanoparticles system for activating plant defense mechanisms and reactive oxygen species to treat Citrus Huanglongbing.

Citrus Huanglongbing (HLB) poses an enormous challenge to Citrus cultivation worldwide, necessitating groundbreaking interventions beyond conventional pharmaceutical methods. In this study, we propose molybdenum disulfide-chitosan nanoparticles (MoS2-CS NPs) through electrostatic adsorption, preserving the plant-beneficial properties of molybdenum disulfide (MoS2), while enhancing its antibacterial effectiveness through chitosan modification. MoS2-CS NPs exhibited significant antibacterial efficacy against RM1021, and the closest relatives to Candidatus Liberibacter asiaticus (CLas), Erwinia carotovora, and Xanthomonas citri achieved survival rates of 7.40 % ±â€¯1.74 %, 8.94 % ±â€¯1.40 %, and 6.41 % ±â€¯0.56 %, respectively. In vivo results showed, CLas survival rate of 10.42 % ±â€¯3.51 %. Furthermore, treatment with MoS2-CS NPs resulted in an increase in chlorophyll and carotenoid content. Concomitantly, a significant reduction in malondialdehyde (MDA), soluble sugar, hydrogen peroxide (H2O2), and starch contents was also observed. Mechanistically, MoS2-CS NPs enhanced the activity of antioxidant-related enzymes by upregulating the expression of antioxidant genes, thereby galvanizing the antioxidant system to alleviate oxidative stress. Collectively, this dual functionality-combining direct antibacterial action with the activation of plant defense mechanisms-marks a promising strategy for managing Citrus Huanglongbing and suggests potential agricultural applications for MoS2-based antibacterial treatments.

Citrus Fruits Produce Direct Defense Responses against Oviposition by Bactrocera minax (Diptera: Tephritidae).

Plants perceive and orchestrate defense responses when herbivorous insects are ovipositing. Fruits, as a crucial reproductive organ in plants, have rarely been researched on the responses to insect eggs. Here, we found that oviposition by the specialist insect Bactrocera minax in navel oranges activated the lignin synthesis pathway and cell division, causing mechanical pressure that crushed the eggs. Transcriptome and metabolome analyses revealed an enrichment of oviposition-induced genes and metabolites within the lignin synthesis pathway, which was confirmed by histochemical staining. Furthermore, hydrogen peroxide (H2O2) accumulation was observed at the oviposition sites. Plant defense-related hormones jasmonic acid (JA) and salicylic acid (SA) exhibited rapid induction after oviposition, while indole-3-acetic acid (IAA) activation occurred in the later stages of oviposition. Additionally, secondary metabolites induced by prior egg deposition were found to influence larval performance. Our studies provide molecular evidence that host fruits have evolved defense mechanisms against insect eggs and pave the way for future development of insect-resistant citrus varieties.

Citrus pectin modulates chicken peripheral blood mononuclear cell proteome in vitro.

Citrus pectin (CP) is a dietary fiber used in animal nutrition with anti-inflammatory properties. CP downregulates chicken immunoregulatory monocytes' functions, like chemotaxis and phagocytosis, in vitro. The molecular underlying background is still unknown. This study investigated the activity of CP on chicken peripheral blood mononuclear cells (PBMC) proteome. An overall number of 1503 proteins were identified and quantified. The supervised sparse variant partial least squares-discriminant analysis (sPLS-DA) for paired data highlighted 373 discriminant proteins between CP-treated and the control group, of which 50 proteins with the highest abundance in CP and 137 in the control group were selected for Gene Ontology (GO) analyses using ProteINSIDE. Discriminant Protein highly abundant in CP-treated cells were involved in actin cytoskeleton organization and negative regulation of cell migration. Interestingly, MARCKSL1, a chemotaxis inhibitor, was upregulated in CP-treated cells. On the contrary, CP incubation downregulated MARCKS, LGALS3, and LGALS8, which are involved in cytoskeleton rearrangements, cell migration, and phagocytosis. In conclusion, these results provide a proteomics background to the anti-inflammatory activity of CP, demonstrating that the in vitro downregulation of phagocytosis and chemotaxis is related to changes in proteins related to the cytoskeleton.

CgLS mediates limonene synthesis of main essential oil component in secretory cavity cells of Citrus grandis 'Tomentosa' fruits.

d-Limonene is the predominant component of essential oil from Exocarpium Citri Grandis, known for its antibacterial, antioxidant, insecticidal, and anti-inflammatory properties. The synthesis, transport, and accumulation of d-limonene in Citrus grandis 'Tomentosa' fruits are regulated by limonene synthetase (LS) and its associated regulatory genes. This study addresses the gap in understanding the spatiotemporal cytological changes of LS and its regulatory genes. Through cytochemical techniques, we investigated the distribution of essential oil in the plastids, endoplasmic reticulum, and vacuoles of secretory cavity cells. We identified the LS-encoding gene CgLS via transcriptomics and demonstrated in vitro that CgLS catalyzes the formation of d-limonene from geranyl diphosphate (GPP). Transient overexpression of CgLS increased monoterpene limonene accumulation, while TRV virus-induced gene silencing reduced it. CgLS expression levels and d-limonene content showed spatiotemporal consistency with fruit development, with in situ hybridization revealing predominant expression in secretory cavity cells. Immunocytochemical localization indicated that CgLS is primarily located in the endoplasmic reticulum, plastids, and vacuoles. Our findings suggest that CgLS is translated in the endoplasmic reticulum and transported to plastids and vacuoles where d-limonene synthesis occurs. This study provides comprehensive insights into the characteristics of CgLS and its role in d-limonene synthesis at the tissue, cellular, and molecular levels in C. grandis 'Tomentosa'.