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.

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.

The underlying mechanisms by which boron mitigates copper toxicity in Citrus sinensis leaves revealed by integrated analysis of transcriptome, metabolome and physiology.

Both copper (Cu) excess and boron (B) deficiency are often observed in some citrus orchard soils. The molecular mechanisms by which B alleviates excessive Cu in citrus are poorly understood. Seedlings of sweet orange (Citrus sinensis (L.) Osbeck cv. Xuegan) were treated with 0.5 (Cu0.5) or 350 (Cu350 or Cu excess) µM CuCl2 and 2.5 (B2.5) or 25 (B25) µM HBO3 for 24 wk. Thereafter, this study examined the effects of Cu and B treatments on gene expression levels revealed by RNA-Seq, metabolite profiles revealed by a widely targeted metabolome, and related physiological parameters in leaves. Cu350 upregulated 564 genes and 170 metabolites, and downregulated 598 genes and 58 metabolites in leaves of 2.5 µM B-treated seedlings (LB2.5), but it only upregulated 281 genes and 100 metabolites, and downregulated 136 genes and 40 metabolites in leaves of 25 µM B-treated seedlings (LB25). Cu350 decreased the concentrations of sucrose and total soluble sugars and increased the concentrations of starch, glucose, fructose and total nonstructural carbohydrates in LB2.5, but it only increased the glucose concentration in LB25. Further analysis demonstrated that B addition reduced the oxidative damage and alterations in primary and secondary metabolisms caused by Cu350, and alleviated the impairment of Cu350 to photosynthesis and cell wall metabolism, thus improving leaf growth. LB2.5 exhibited some adaptive responses to Cu350 to meet the increasing need for the dissipation of excessive excitation energy (EEE) and the detoxification of reactive oxygen species (reactive aldehydes) and Cu. Cu350 increased photorespiration, xanthophyll cycle-dependent thermal dissipation, nonstructural carbohydrate accumulation, and secondary metabolite biosynthesis and abundances; and upregulated tryptophan metabolism and related metabolite abundances, some antioxidant-related gene expression, and some antioxidant abundances. Additionally, this study identified some metabolic pathways, metabolites and genes that might lead to Cu tolerance in leaves.

Both hormones and energy-rich compounds play a role in the mitigation of elevated pH on aluminum toxicity in Citrus sinensis leaves.

The contribution of plant hormones and energy-rich compounds and their metabolites (ECMs) in alleviating aluminum (Al) toxicity by elevated pH remains to be clarified. For the first time, a targeted metabolome was applied to identify Al-pH-interaction-responsive hormones and ECMs in Citrus sinensis leaves. More Al-toxicity-responsive hormones and ECMs were identified at pH 4.0 [4 (10) upregulated and 7 (17) downregulated hormones (ECMs)] than those at pH 3.0 [1 (9) upregulated and 4 (14) downregulated hormones (ECMs)], suggesting that the elevated pH improved the adaptation of hormones and ECMs to Al toxicity in leaves. The roles of hormones and ECMs in reducing leaf Al toxicity mediated by elevated pH might include the following aspects: (a) improved leaf growth by upregulating the levels of jasmonoyl-L-isoleucine (JA-ILE), 6-benzyladenosine (BAPR), N6-isopentenyladenosine (IPR), cis-zeatin-O-glucoside riboside (cZROG), and auxins (AUXs), preventing Al toxicity-induced reduction of gibberellin (GA) biosynthesis, and avoiding jasmonic acid (JA)-mediated defense; (b) enhanced biosynthesis and accumulation of tryptophan (TRP), as well as the resulting increase in biosynthesis of auxin, melatonin and secondary metabolites (SMs); (c) improved ability to maintain the homeostasis of ATP and other phosphorus (P)-containing ECMs; and (d) enhanced internal detoxification of Al due to increased organic acid (OA) and SM accumulation and elevated ability to detoxify reactive oxygen species (ROS) due to enhanced SM accumulation. To conclude, the current results corroborate the hypotheses that elevated pH reduces Al toxicity by upregulating the ability to maintain the homeostasis of ATP and other P-containing ECMs in leaves under Al toxicity and (b) hormones participate in the elevated pH-mediated alleviation of Al toxicity by positively regulating growth, the ability to detoxify ROS, and the internal detoxification of Al in leaves under Al toxicity. Our findings provide novel insights into the roles of hormones and ECMs in mitigating Al toxicity mediated by the elevated pH.

Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding protein gene for canker formation.

Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1. This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620, which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1.

Metabolite Variations during the First Weeks of Growth of Immature Citrus sinensis and Citrus reticulata by Untargeted Liquid Chromatography-Mass Spectrometry/Mass Spectrometry Metabolomics.

Immature citruses are an important resource for the pharmaceutical industry due to their high levels of metabolites with health benefits. In this study, we used untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics to investigate the changes associated with fruit size in immature citrus fruits in the first weeks of growth. Three orange cultivars (Citrus sinensis 'Navel', Citrus sinensis 'Valencia', and Citrus sinensis 'Valencia Late') and a mandarin (Citrus reticulata Blanco 'Fremont') were separated into eight fruit sizes, extracted, and analyzed. Statistical analyses revealed a distinct separation between the mandarin and the oranges based on 56 metabolites, with an additional separation between the 'Navel' orange and the 'Valencia' and 'Valencia Late' oranges based on 21 metabolites. Then, metabolites that evolved significantly with fruit size growth were identified, including 40 up-regulated and 31 down-regulated metabolites. This study provides new insights into the metabolite modifications of immature Citrus sinensis and Citrus reticulata in the first weeks of growth and emphasizes the significance of including early sampled fruits in citrus maturation studies.

Transcriptome and weighted gene co-expression network analyses reveal key genes and pathways involved in early fruit ripening in Citrus sinensis.

BACKGROUND: The fruit ripening period is an important target trait in fruit tree crop breeding programs. Thus, citrus tree breeders seek to develop extreme early ripening cultivars that allow optimization of citrus maturation periods. In this study, we explored the regulatory network involved in fruit ripening in Citrus sinensis using the 'Newhall' navel orange variety and its early-ripening mutant, 'Gannanzao'. This research will provide a basis for further research on important signaling pathways, gene functions and variety breeding of Citrus sinensis related to fruit ripening period. RESULTS: Physiological analyses suggested that early fruit ripening in 'Gannanzao' is regulated by early accumulation of abscisic acid (ABA), persistently high levels of jasmonic acid (JA), and higher sucrose content in the pericarp. Pericarp samples from 'Gannanzao' and 'Newhall' navel oranges were sampled for RNA sequencing analysis at 180, 200, and 220 days after flowering; 1430 differentially expressed genes (DEGs) were identified. Functional enrichment analysis indicated that these DEGs were mainly enriched in the plant hormone signal transduction and sugar metabolism pathways, as well as other pathways related to fruit ripening. Important DEGs associated with fruit ripening in 'Gannanzao' included genes involved in ABA and JA metabolism and signal transduction, as well as sugar metabolism. Weighted gene co-expression network analysis showed that the deep pink module had the strongest correlations with ABA content, JA content, and early ripening. Based on gene functionality and gene expression analyses of 37 genes in this module, two candidate hub genes and two ethylene response factor 13 (ERF13) genes (Cs_ont_5g000690 and Cs_ont_5g000700) were identified as key genes regulated by ABA and JA signaling. These findings will help to clarify the mechanisms that underlie early citrus fruit ripening and will lead to the development of excellent genetic resources for further breeding of extreme early-ripening varieties. CONCLUSIONS: Through analyses of the 'Newhall' navel orange cultivar and its early-ripening mutant 'Gannanzao', we identified genes involved in ABA and JA metabolism, signal transduction, and sugar metabolism that were related to fruit ripening. Among these, two ERF13 genes were inferred to be key genes in the regulation of fruit ripening. These findings provide insights into the genetic architecture related to early fruit ripening in C. sinensis.

Adaptive Responses of Hormones to Nitrogen Deficiency in Citrus sinensis Leaves and Roots.

Some citrus orchards in China often experience nitrogen (N) deficiency. For the first time, targeted metabolomics was used to examine N-deficient effects on hormones in sweet orange (Citrus sinensis (L.) Osbeck cv. Xuegan) leaves and roots. The purpose was to validate the hypothesis that hormones play a role in N deficiency tolerance by regulating root/shoot dry weight ratio (R/S), root system architecture (RSA), and leaf and root senescence. N deficiency-induced decreases in gibberellins and indole-3-acetic acid (IAA) levels and increases in cis(+)-12-oxophytodienoic acid (OPDA) levels, ethylene production, and salicylic acid (SA) biosynthesis might contribute to reduced growth and accelerated senescence in leaves. The increased ethylene formation in N-deficient leaves might be caused by increased 1-aminocyclopropanecarboxylic acid and OPDA and decreased abscisic acid (ABA). N deficiency increased R/S, altered RSA, and delayed root senescence by lowering cytokinins, jasmonic acid, OPDA, and ABA levels and ethylene and SA biosynthesis, increasing 5-deoxystrigol levels, and maintaining IAA and gibberellin homeostasis. The unchanged IAA concentration in N-deficient roots involved increased leaf-to-root IAA transport. The different responses of leaf and root hormones to N deficiency might be involved in the regulation of R/S, RSA, and leaf and root senescence, thus improving N use efficiency, N remobilization efficiency, and the ability to acquire N, and hence conferring N deficiency tolerance.

Molecular basis of one-step methyl anthranilate biosynthesis in grapes, sweet orange, and maize.

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate (MeAA) is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase (AAMT), grapes have been thought to use a two-step pathway for MeAA biosynthesis. By mining available transcriptomics data, we identified two AAMTs in Vitis vinifera (wine grape), as well as one ortholog in "Concord" grape. Many angiosperms methylate the plant hormone salicylic acid (SA) to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) SA methyltransferase can methylate both anthranilate (AA) and SA, we used this enzyme to examine the molecular basis of AA activity by introducing rational mutations, which identified several active site residues that increase activity with AA. Reversing this approach, we introduced mutations that imparted activity with SA in the maize AAMT, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis AAMTs shares an ancestor with jasmonic acid methyltransferases, similar to the AAMT from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning AA activity across methyltransferases and identify one-step enzymes by which grapes synthesize MeAA.

Deciphering the drug delivery potential of Type1 lipid transfer protein from Citrus sinensis for enhancing the therapeutic efficacy of drugs.

Type1 Non-specific Lipid Transfer Protein (CsLTP1) from Citrus sinensis is a small cationic protein possessing a long tunnel-like hydrophobic cavity. CsLTP1 performing membrane trafficking of lipids is a promising candidate for developing a potent drug delivery system. The present work includes in-silico studies and the evaluation of drugs binding to CsLTP1 using biophysical techniques along with the investigation of CsLTP1's ability to enhance the efficacy of drugs employing cell-based bioassays. The in-silico investigations identified Panobinostat, Vorinostat, Cetylpyridinium Chloride, and Fulvestrant with higher affinities and stability of binding to the hydrophobic pocket of CsLTP1. SPR studies revealed strong binding affinities of anticancer drugs, Panobinostat (KD = 1.40 µM) and Vorinostat (KD = 2.17 µM) to CsLTP1 along with the binding and release kinetics. CD and fluorescent spectroscopy revealed drug-induced conformational changes in CsLTP1. CsLTP1-associated drug forms showed remarkably enhanced efficacy in MCF-7 cells, representing increased cell cytotoxicity, intracellular ROS, reduced mitochondrial membrane potential, and up-regulation of proapoptotic markers than the free drugs employing qRT-PCR and western blot analysis. The findings demonstrate that CsLTP1 binds strongly to hydrophobic drugs to facilitate their transport, hence improving their therapeutic efficacy revealed by the in-vitro investigations. This study establishes an excellent foundation for developing CsLTP1-based efficient drug delivery system.

Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

Genome-wide characterization of heavy metal-associated isoprenylated plant protein gene family from Citrus sinensis in response to huanglongbing.

Introduction: Heavy metal-associated isoprenylated plant proteins (HIPPs) play vital roles in maintaining heavy metal balance and responding to both biotic and abiotic stresses in vascular plants. However, the role of HIPPs in the response to Huanglongbing (HLB), a harmful disease of citrus caused by the phloem-colonizing bacterium Candidatus Liberibacter asiaticus (CLas), has not been examined. Methods and results: In this study, a total of 26 HIPP genes were identified in Citrus sinensis, and they were grouped into 5 clades. The CsHIPP genes are distributed on 8 chromosomes and exhibited considerable synteny with HIPPs found in Arabidopsis thaliana. Additionally, we analyzed the gene structure, conserved motifs and domains of the CsHIPPs. Various cis-acting elements related to plant hormones and stress responses were identified in the promoters of CsHIPPs. Public transcriptome data and RT-qPCR analysis showed that the expression level of CsHIPP03 was significantly reduced in samples infected by CLas and Xanthomonas citri ssp. citri (Xcc). Furthermore, silencing the homologous gene of CsHIPP03 in Nicotiana benthamiana increased the disease resistance of plants to bacteria. Discussion: Our results provide a basis for functional studies of HIPP gene family in C. sinensis, highlighting their functions in bacterial resistance, and improve our understanding to the susceptibility mechanism of HLB.

Analysis of huanglongbing-associated RNA-seq data reveals disturbances in biological processes within Citrus spp. triggered by Candidatus Liberibacter asiaticus infection.

Introduction: Huanglongbing (HLB), a disease that's ubiquitous worldwide, wreaks havoc on the citrus industry. The primary culprit of HLB is the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas) that infects the phloem, but its damaging mechanism is yet to be fully understood. Methods and results: In this study, a multitude of tools including weighted correlation network analysis (WGCNA), protein-protein interaction (PPI) network analysis and gene expression profiling are employed to unravel the intricacies of its pathogenesis. The investigation pinpoints various central genes, such as the ethylene-responsive transcription factor 9 (ERF9) and thioredoxin reductase 1 (TrxR1), that are associated with CLas invasion and resultant disturbances in numerous biological operations. Additionally, the study uncovers a range of responses through the detection of differential expressed genes (DEGs) across different experiments. The discovery of core DEGs leads to the identification of pivotal genes such as the sieve element occlusion (SEO) and the wall-associated receptor kinase-like 15 (WAKL15). PPI network analysis highlights potential vital proteins, while GO and KEGG pathway enrichment analysis illustrate a significant impact on multiple defensive and metabolic pathways. Gene set enrichment analysis (GSEA) indicates significant alterations in biological processes such as leaf senescence and response to biotic stimuli. Discussion: This all-encompassing approach extends valuable understanding into the pathogenesis of CLas, potentially aiding future research and therapeutic strategies for HLB.

N-acetylcysteine absorption and its potential dual effect improve fitness and fruit yield in Xylella fastidiosa infected plants.

BACKGROUND: Xylella fastidiosa is a multi-host bacterium that can be detected in hundreds of plant species including several crops. Diseases caused by X. fastidiosa are considered a threat to global food production. The primary method for managing diseases caused by X. fastidiosa involves using insecticides to control the vector. Hence, it is necessary to adopt new and sustainable disease management technologies to control not only the insect but also the bacteria and plant health. We demonstrated that N-acetylcysteine (NAC), a low-cost cysteine analogue, is a sustainable molecule that can be used in agriculture to decrease the damage caused by X. fastidiosa and improve plant health. RESULTS: Using 15N-NAC we proved that this analogue was absorbed by the roots and transported to different parts of the plant. Inside the plant, NAC reduced the bacterial population by 60-fold and the number of xylem vessels blocked by bacterial biofilms. This reflected in a recovery of 0.28-fold of the daily sap flow compared to health plants. In addition, NAC-treated citrus variegated chlorosis (CVC) plants decreased the oxidative stress by improving the activity of detoxifying enzymes. Moreover, the use of NAC in field conditions positively contributed to the increase in fruit yield of CVC-diseased plants. CONCLUSION: Our research not only advances the understanding of NAC absorption in plants, but also indicates its dual effect as an antimicrobial and antioxidant molecule. This, in turn, negatively affects bacterial survival while improving plant health by decreasing oxidative stress. Overall, the positive field-based evidence supports the viability of NAC as a sustainable agricultural application. © 2024 Society of Chemical Industry.

Comparison of Insecticide Sprays in Alternate Rows and in All Rows on Asian Citrus Psyllid Control and Huanglongbing Progress in Sweet Orange Orchards.

One strategy to reduce huanglongbing (HLB) is controlling its insect vector, the Asian citrus psyllid (ACP) Diaphorina citri, by preventive insecticide sprays. The recommendation is to spray insecticide in all rows (conventional spray [CONV]), but some growers empirically spray in alternate rows (ALT) to increase the spray frequency without increasing the operating cost. Therefore, this work compared the effect of ALT with CONV on the ACP population and HLB incidence. The spray deposition (amount of metallic copper per leaf area), coverage (percentage of water-sensitive paper area covered by spray), and efficacy (ACP mortality) of each treatment were also evaluated on both sides of the trees. Two field trials were performed: Trial #1 compared ALT every 7 days (ALT7) with CONV every 14 days (CONV14), and trial #2 compared different spray frequencies of ALT with CONV every 7 days (CONV7). In trial #1, no differences were observed in the ACP population or HLB progress between ALT7 and CONV14 after 5 years. In trial #2, ALT7 presented the highest percentage of ACP and cumulative HLB incidence than CONV7 and ALT every 3 to 4 days, after 2 years. Hence, when the frequency of ALT was half the frequency of CONV, similar results were observed. Spray deposition, coverage, and efficacy were similar between tree sides in CONV, but they were uneven in ALT, resulting in higher values on the tree side that directly received the spray. Insecticide spray should be performed with the frequency enough to keep new shoots protected during their growth.

A Calcium-Dependent Protein Kinase Regulates the Defense Response in Citrus sinensis.

Citrus Huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CLas), is one of the most destructive citrus diseases worldwide, and defense-related Citrus sinensis gene resources remain largely unexplored. Calcium signaling plays an important role in diverse biological processes. In plants, a few calcium-dependent protein kinases (CDPKs/CPKs) have been shown to contribute to defense against pathogenic microbes. The genome of C. sinensis encodes dozens of CPKs. In this study, the role of C. sinensis calcium-dependent protein kinases (CsCPKs) in C. sinensis defense was investigated. Silencing of CsCPK6 compromised the induction of defense-related genes in C. sinensis. Expression of a constitutively active form of CsCPK6 (CsCPK6CA) triggered the activation of defense-related genes in C. sinensis. Complementation of CsCPK6 rescued the defense-related gene induction in an Arabidopsis thaliana cpk4/11 mutant, indicating that CsCPK6 carries CPK activity and is capable of functioning as a CPK in Arabidopsis. Moreover, an effector derived from CLas inhibits defense induced by the expression of CsCPK6CA and autophosphorylation of CsCPK6, which suggests the involvement of CsCPK6 and calcium signaling in defense. These results support a positive role for CsCPK6 in C. sinensis defense against CLas, and the autoinhibitory regulation of CsCPK6 provides a potential genome-editing target for improving C. sinensis defense. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of Type1 Lipid Transfer Protein from Citrus sinensis: Unraveling its potential as an antimicrobial and insecticidal agent.

Lipid Transfer Protein1 (LTP1) is a cationic, multifaceted protein belonging to the pathogenesis-related protein (PR14) family. Despite being involved in diverse physiological processes and defense mechanisms, the precise in-vivo role of LTP1 remains undiscovered. This work presents the characterization of recombinant Citrus sinensis LTP1 (CsLTP1) along with lipid binding studies through in-silico and in-vitro approaches. CsLTP1 demonstrated great thermal and pH stability with a huge biotechnological potential. It showed in-vitro binding capacity with jasmonic acid and lipids involved in regulating plant immune responses. Gene expression profiling indicated a significant upregulation of CsLTP1 in Candidatus-infected Citrus plants. CsLTP1 disrupted the cell membrane integrity of various pathogens, making it a potent antimicrobial agent. Further, in-vivo antimicrobial and insecticidal properties of CsLTP1 have been explored. The impact of exogenous CsLTP1 treatment on rice crop metabolism for managing blight disease has been studied using GC-MS. CsLTP1 triggered crucial metabolic pathways in rice plants while controlling the blight disease. CsLTP1 effectively inhibited Helicoverpa armigera larvae by impeding mid-gut α-amylase activity and obstructing its developmental stages. This study highlights the pivotal role of CsLTP1 in plant defense by offering insights for developing multi-target therapeutic agent or disease-resistant varieties to comprehensively tackle the challenges towards crop protection.

Nigella sativa, Anthemis hyaline and Citrus sinensis extracts reduce SARS-CoV-2 replication by fluctuating Rho GTPase, PI3K-AKT, and MAPK/ERK pathways in HeLa-CEACAM1a cells.

Traditional remedies have long utilized Anthemis hyaline, Nigella sativa, and Citrus sinensis peel extracts as treatments for microbial infections. This study aimed to investigate the influence of Anthemis hyaline, Nigella sativa, and Citrus sinensis extracts on coronavirus replication and apoptosis-related pathways. HeLa-CEACAM1a cells were exposed to mouse hepatitis virus-A59. After viral inoculation, the mRNA levels of 36 genes were quantified using a Fluidigm Dynamic Array nanofluidic chip. IL-8 level and intracellular Ca2+ concentration was measured, and viral titer was assessed by the TCID50/ml assay to detect the extent of infection. Treatment with Nigella sativa extract surged the inflammatory cytokine IL-8 level at both 24 and 48-hour. Changes in gene expression were notable for RHOA, VAV3, ROCK2, CFL1, RASA1, and MPRIP genes following treatment with any of the extracts. The addition of Anthemis hyaline, Nigella sativa, or Citrus sinensis extracts to coronavirus-infected cells reduced viral presence, with Anthemis hyaline extract leading to a virtually undetectable viral load at 6- and 8-hours after infection. While all treatments influenced IL-8 production and viral levels, Anthemis hyaline extract displayed the most pronounced reduction in viral load. Consequently, Anthemis hyaline extract emerges as the most promising agent, harboring potential therapeutic compounds.

Brevipalpus yothersi Baker (Tenuipalpidae) development in sweet orange plants is influenced by previous mite infestation and the presence of shelters.

Citrus leprosis is the most important viral disease affecting citrus. The disease is caused predominantly by CiLV-C and is transmitted by Brevipalpus yothersi Baker mites. This study brings some insight into the colonization of B. yothersi in citrus [(Citrus × sinensis (L.) Osbeck (Rutaceae)] previously infested by viruliferous or non-viruliferous B. yothersi. It also assesses the putative role of shelters on the behavior of B. yothersi. Expression of PR1 and PR4 genes, markers of plant defense mechanisms, were evaluated by RT-qPCR to correlate the role of the plant hormonal changes during the tri-trophic virus-mite-plant interplay. A previous infestation with either non-viruliferous and viruliferous mites positively influenced oviposition and the number of adult individuals in the resulting populations. Mite populations were higher on branches that had received a previous mite infestation than branches that did not. There was an increase in the expression of PR4, a marker gene in the jasmonic acid (JA) pathway, in the treatment with non-viruliferous mites, indicating a response from the plant to their feeding. Conversely, an induced expression of PR1, a marker gene in the salicylic acid (SA) pathway, was observed mainly in the treatment with viruliferous mites, which suggests the activation of a plant response against the pathogen. The earlier mite infestation, as well as the presence of leprosis lesions and a gypsum mixture as artificial shelters, all fostered the growth of the B. yothersi populations after the second infestation, regardless of the presence or absence of CiLV-C. Furthermore, it is suggested that B. yothersi feeding actually induces the JA pathway in plants. At the same time, the CiLV-C represses the JA pathway and induces the SA pathway, which benefits the mite vector.

Boron Reduced Copper Excess-Induced Oxidative Damage in Citrus sinensis by Modulating Reactive Oxygen Species and Methylglyoxal Formation and Their Detoxification Systems.

Citrus is mainly cultivated in acid soil with low boron (B) and high copper (Cu). In this study, Citrus sinensis seedlings were submitted to 0.5 (control) or 350 µM Cu (Cu excess or Cu exposure) and 2.5, 10, or 25 µM B for 24 weeks. Thereafter, H2O2 production rate (HPR), superoxide production rate (SAPR), malondialdehyde, methylglyoxal, and reactive oxygen species (ROS) and methylglyoxal detoxification systems were measured in leaves and roots in order to test the hypothesis that B addition mitigated Cu excess-induced oxidative damage in leaves and roots by reducing the Cu excess-induced formation and accumulation of ROS and MG and by counteracting the impairments of Cu excess on ROS and methylglyoxal detoxification systems. Cu and B treatments displayed an interactive influence on ROS and methylglyoxal formation and their detoxification systems. Cu excess increased the HPR, SAPR, methylglyoxal level, and malondialdehyde level by 10.9% (54.3%), 38.9% (31.4%), 50.3% (24.9%), and 312.4% (585.4%), respectively, in leaves (roots) of 2.5 µM B-treated seedlings, while it only increased the malondialdehyde level by 48.5% (97.8%) in leaves (roots) of 25 µM B-treated seedlings. Additionally, B addition counteracted the impairments of Cu excess on antioxidant enzymes, ascorbate-glutathione cycle, sulfur metabolism-related enzymes, sulfur-containing compounds, and methylglyoxal detoxification system, thereby protecting the leaves and roots of Cu-exposed seedlings against oxidative damage via the coordinated actions of ROS and methylglyoxal removal systems. Our findings corroborated the hypothesis that B addition alleviated Cu excess-induced oxidative damage in leaves and roots by decreasing the Cu excess-induced formation and accumulation of ROS and MG and by lessening the impairments of Cu excess on their detoxification systems. Further analysis indicated that the pathways involved in the B-induced amelioration of oxidative stress caused by Cu excess differed between leaves and roots.