Synergistic effect of seaweed extract and boric acid and/or calcium chloride on productivity and physico-chemical properties of Valencia orange.

Many citrus species and cultivars are grown successfully in tropical and subtropical countries, as well as in arid and semi-arid regions with low levels of organic matter and low cation exchange, resulting in lower nutrient uptake by the plant. The essential nutrients needed for citrus flowering and fruit set are limited in winter due to a reduction in transpiration rate, negatively effecting vegetative growth, flowering, yield, and fruit quality. The present investigation was carried out to assess the nutritional status, fruit yield parameters, and fruit quality of Valencia orange trees after foliar spraying of seaweed extract (SW) combined with calcium chloride and boric acid and their combinations in the 2020/2021 and 2021/2022 seasons. The treatments were arranged in a split-plot design (three levels spraying seaweed extract × four levels spraying calcium chloride and boric acid and their combinations × four replicates × one tree/replicate). The results indicated that all of the characteristics measured, including leaf chlorophyll, leaf mineral contents, fruit yield parameters, fruit physical properties, and fruit chemical properties, were significantly affected by the foliar spraying of seaweed extract (SW) combined with calcium chloride and boric acid and their combinations. Although all treatments increased the productivity and the physical and chemical properties of Valencia orange fruits compared to the control, a treatment of 10 g/L SW combined with 0.5 g/L boric acid and 1 g/L calcium chloride produced superior results. This ratio of SW, boric acid, and calcium chloride is therefore recommended to enhance productivity and improve the physico-chemical properties of Valencia orange for greater fruit yield.

Chronic consumption of orange juice modifies urinary excretion of flavanone gut-derived metabolites through gut microbiota modulation.

The metabolism and absorption of citrus flavanones are intrinsically linked to the gut microbiota, creating a bidirectional relationship where these compounds influence the microbiome, and in turn, the microbiota affects their metabolism. This study evaluates the effect of acute and chronic consumption of orange juice (OJ) on the urinary excretion of gut-derived flavanone metabolites and the gut microbiota. Health volunteers ingested 500 mL of OJ for 60 days in a single-arm human intervention study. Blood and feces were collected at baseline and after 60 days, with an additional 24-hour urine collection after a single dose on day 1 and day 63. LC-MS/MS analyzed urinary flavanone metabolites, while 16S rRNA sequencing characterized gut microbiota. Total urinary hesperetin conjugates excretion significantly decreased over 60 days, while gut-derived total phenolic acids, particularly three hydroxybenzoic acids, increased. Moreover, the heterogeneity of the total amount of flavanone conjugates, initially categorizing individuals into high-, medium- and low- urinary excretor profiles, shifted towards medium-excretor, except for five individuals who remained as low-excretors. This alteration was accompanied by a decrease in intestinal ß-glucosidase activity and a shift in the relative abundance of specific genera, such as decreases in Blautia, Eubacterium hallii, Anaerostipes, and Fusicatenibacter, among which, Blautia was associated with higher urinary flavanone conjugates excretion. Conversely, an increase in Prevotella was observed. In summary, chronic OJ consumption induced transient changes in gut microbiota and altered the metabolism of citrus flavanones, leading to distinct urinary excretion profiles of flavanone metabolites.

A high-quality chromosome-scale genome assembly of blood orange, an important pigmented sweet orange variety.

Blood orange (BO) is a rare red-fleshed sweet orange (SWO) with a high anthocyanin content and is associated with numerous health-related benefits. Here, we reported a high-quality chromosome-scale genome assembly for Neixiu (NX) BO, reaching 336.63 Mb in length with contig and scaffold N50 values of 30.6 Mb. Furthermore, 96% of the assembled sequences were successfully anchored to 9 pseudo-chromosomes. The genome assembly also revealed the presence of 37.87% transposon elements and 7.64% tandem repeats, and the annotation of 30,395 protein-coding genes. A high level of genome synteny was observed between BO and SWO, further supporting their genetic similarity. The speciation event that gave rise to the Citrus species predated the duplication event found within them. The genome-wide variation between NX and SWO was also compared. This first high-quality BO genome will serve as a fundamental basis for future studies on functional genomics and genome evolution.

Sensitivity of Salmonella Typhimurium to nisin in vitro and in orange juice under refrigeration.

The influence of environmental factors on Salmonella sensitivity to nisin in vitro and in refrigerated orange juice were investigated. Nisin activity was observed in the different conditions, but the highest efficiency was achieved at lower pH (4.0) and with higher bacteriocin concentration (174 µM). Moreover, the bactericidal action was directly proportional to the incubation period. When tested in orange juice, nisin caused a reduction of up to 4.05 logarithm cycles in the Salmonella population. So, environmental factors such as low pH and low temperature favored the sensitization of Salmonella cells to the bactericidal action of nisin. Therefore, this may represent an alternative to control Salmonella in refrigerated foods.

An antifouling electrochemical biosensor using self-signal for Salmonella typhimurium direct detection in food sample.

Eating food contaminated by foodborne pathogens can lead to illness. The development of electrochemical sensors for pathogen detection has received widespread attention. However, the analytical performance of electrochemical sensors is inevitably affected by the non-specific adsorption of molecules in the sample. Moreover, the external signal probes might be affected by the complex components in the sample accompanied with signal suppression. This work presents an electrochemical aptasensor for Salmonella typhimurium detection based on the self-signal of poly-xanthurenic acid and the antifouling ability of chondroitin sulfate. The detection time was 60 min. The linear range was from 101 to 107 CFU/mL, and the detection limit was 3 CFU/mL. The biosensors presented good repeatability and storage stability. And the biosensors has been successfully applied in milk and orange juice. This strategy is expected to be applied in the design of other antifouling biosensors, to achieve rapid detection of pathogens and ensure food safety.

Two-Month Consumption of Orange Juice Enriched with Vitamin D3 and Probiotics Decreases Body Weight, Insulin Resistance, Blood Lipids, and Arterial Blood Pressure in High-Cardiometabolic-Risk Patients on a Westernized Type Diet: Results from a Randomized Clinical Trial.

This study examined the effects of orange juice (OJ) supplemented with vitamin D3 (2000 IU) and probiotics (Lacticaseibacillus casei Shirota and Lacticaseibacillus rhamnosus GG, 108 cfu/mL) on cardiometabolic risk factors in overweight and obese adults following a Westernized-type diet. Fifty-three high-risk individuals were randomly assigned to one of two groups. Over 8 weeks, one group consumed a vitamin D3 and probiotic-enriched OJ and the other regular OJ (control). Diets remained unchanged and were documented through food diaries. Measures of metabolic and inflammatory markers and blood pressure were measured at the start and end of the study. Post-intervention, the enriched OJ group showed the following significant metabolic improvements (without changes in triglycerides, inflammation, or central blood pressure): reduced fasting insulin, peripheral blood pressure, body weight (-1.4 kg 95% CI: -2.4, -0.4), energy (-270 kcal 95% CI: -553.2, -13.7), macronutrient (dietary fat -238 kcal 95% CI: -11.9, -1.0; carbohydrates -155 kcal 95% CI: -282.4, -27.3; sugars -16.1 g 95% CI: -11.9, -1.0) intake, and better lipid profiles (total cholesterol -10.3 mg/dL 95% CI: -21.4, 0.9; LDL-C -7 mg/dL 95% CI: -13.5, -0.5). The enriched OJ led to weight loss, less energy/macronutrient consumption, improved lipid profiles, and increased insulin sensitivity after 8 weeks in those following a Westernized diet, thus indicating potential benefits for cardiometabolic risk. This study was a part of FunJuice-T2EDK-01922, which was funded by the EU Regional Development Fund and Greek National Resources.

Evaluation of the effect of Torulaspora delbrueckii on important volatile compounds in navel orange original brandy using E-nose combined with HS-SPME-GC-MS.

Simultaneous inoculation of non-Saccharomyces cerevisiae during the alcoholic fermentation process has been found to be an effective strategy for enhancing wine flavor. This study aimed to investigate the effect of Torulaspora delbrueckii NCUF305.2 on the flavor of navel orange original brandy (NOOB) using E-nose combined with HS-SPME-GC-MS. The results showed a significant increase (p < 0.05) in the sensitivity of NOOB to W5C, W3C, W1S, and W3S sensors by mixed fermentation (MF). Esters in NOOB increased by 4.13%, while higher alcohols increased by 21.93% (p < 0.001), terpenes and others increased by 52.07% and 40.99% (p < 0.01), respectively. Notably, several important volatile compounds with relative odor activity values above 10 showed an increase. Sensory analysis revealed that a more pronounced citrus-like flavor and higher overall appearance scores were found in MF than in pure fermentation (PF). These findings offer valuable theoretical guidance for enhancing the quality of fruit brandies.

Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

A critical evaluation on the valorization strategies to reduce and reuse orange waste in bakery industry.

Tons of orange by-products (OBPs) are generated during industrial orange processing. Currently, OBPs management is challenging due to their high amounts, physico-chemical characteristics (high water content, low pH, presence of essential oils) and seasonal nature of the production. Whereas agro-industrial OBPs can be highly valuable due to their abundant sources of bioactive compounds, which can add value to novel bakery products (e.g. bread, biscuits, cakes). This review covers the most recent research issues linked to the use of OBPs in bakery products, with a focus on available stabilization methods and on the main challenges to designing improved products. The application of OBPs improved the nutritional quality of bakery products, offering interesting sustainability benefits but also critical challenges. The valorization of OBPs may open new routes for the development of new natural ingredients for the food industry and lower food processing waste.

Synergistic efficacy of ultrasound and ammonium persulfate in inactivating Escherichia coli O157:H7 in buffered peptone water and orange juice.

This study investigated the synergistic effects of ammonium persulfate (PS) and ultrasound (US) on the inactivation of Escherichia coli O157:H7 in buffered peptone water (BPW) and orange juice products. A comprehensive assessment of PS concentrations ranging from 1 to 300 mM, considering not only the statistical significance but also the reliability and stability of the experimental outcomes, showed that 150 mM was the optimal PS concentration for the inactivation of E. coli O157:H7. Additionally, US output intensities varying from 30 % to 60 % of the maximum US intensity were evaluated, and 50 % US amplitude was found to be the optimal US condition. A 50 % amplitude setting on the sonicator corresponds to half of its maximum displacement, approximately 60 µm, based on a maximum amplitude of 120 µm. The inactivation level of E. coli O157:H7 was significantly enhanced by the combined treatment of PS and US, compared to each treatment of PS and US alone. In the BPW, a 10-min treatment with the combination of PS and US resulted in a significant synergistic inactivation, achieving up to a log reduction of 3.86 log CFU/mL. Similarly, in orange juice products, a 5-min treatment with the combination of PS and US yielded a significant synergistic inactivation, with a reduction reaching 5.90 log CFU/mL. Although the treatment caused a significant color change in the sample, the visual differences between the treated and non-treated groups were not pronounced. Furthermore, the combined treatment in orange juice demonstrated significantly enhanced antimicrobial efficacy relative to BPW. Despite identical 5-min treatment periods, the application in orange juice resulted in a substantially higher log reduction of E. coli O157:H7, achieving 7.16 log CFU/mL at a reduced PS concentration of 30 mM, whereas the same treatment in BPW yielded only a 2.89 log CFU/mL reduction at a PS concentration of 150 mM, thereby highlighting its significantly superior antimicrobial performance in orange juice. The mechanism underlying microbial inactivation, induced by the combined treatment of PS and US, was identified as significant cell membrane damage. This damage is mediated by sulfate radicals, generated through the sono-activation of persulfate. In addition, the low pH of orange juice, measured at 3.7, is likely to have further deteriorated the E. coli O157:H7 cells compared to BPW (pH 7.2), by disrupting their cell membranes, proton gradients, and energy metabolism. These findings underscore the effectiveness of PS and US integration as a promising approach for non-thermal pasteurization in the food industry. Further research is needed to optimize treatment parameters and fully explore the practical application of this technique in large-scale food processing operations. Sensory evaluation and nutritional assessment are also necessary to address the limitations of PS.

Bound polyphenols in insoluble dietary fiber of navel orange peel modulate LPS-induced intestinal-like co-culture inflammation through CSF2-mediated NF-κB/JAK-STAT pathway.

Our laboratory previously extracted bound polyphenols (BPP) in insoluble dietary fiber from navel orange peel (NOP-IDF), and the aim of this study was to investigate the anti-inflammatory activity and potential molecular mechanisms of BPP by establishing an LPS-induced intestinal-like Caco-2/RAW264.7 co-culture inflammation model. The results demonstrated that BPP reduced the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as the production of pro-inflammatory cytokines, nitric oxide (NO), and reactive oxidative species (ROS) during the inflammatory damage process. Furthermore, BPP alleviated the lipopolysaccharides (LPS)-induced intestinal barrier damage by attenuating the decrease in trans-epithelial electrical resistance (TEER), diamine oxidase (DAO) activity, and intestinal alkaline phosphatase (IAP) activity, as well as the downregulation of ZO-1, Occludin, and Claudin-1 protein expression levels. RNA-seq results on RAW264.7 cells in the co-culture model showed that the NF-κB and JAK-STAT pathways belonged to the most significantly affected signaling pathways in the KEGG analysis, and western blot confirmed that they are essential for the role of BPP in intestinal inflammation. Additionally, overexpression of the granulocyte-macrophage colony-stimulating factor (CSF2) gene triggered abnormal activation of the NF-κB and JAK-STAT pathways and high-level expression of inflammatory factors, while BPP effectively improved this phenomenon. The above results suggested that BPP could inhibit intestinal inflammatory injury and protect intestinal barrier integrity through CSF2-mediated NF-κB and JAK-STAT pathways.

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

Plackett-Burman screening of physico-chemical variables affecting Citrus peel-mediated synthesis of silver nanoparticles and their antimicrobial activity.

With the growing resistance of pathogenic microbes to traditional drugs, biogenic silver nanoparticles (SNPs) have recently drawn attention as potent antimicrobial agents. In the present study, SNPs synthesized with the aid of orange (Citrus sinensis) peel were engineered by screening variables affecting their properties via Plackett-Burman design. Among the variables screened (temperature, pH, shaking speed, incubation time, peel extract concentration, AgNO3 concentration and extract/AgNO3 volume ratio), pH was the only variable with significant effect on SNPs synthesis. Therefore, SNPs properties could be enhanced to possess highly regular shape with zeta size of 11.44 nm and zeta potential of - 23.7 mV. SNPs purified, capped and stabilized by cloud point extraction technique were then checked for their antimicrobial activity against Bacillus cereus, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium and Candida albicans. The maximum antimicrobial activity of SNPs was recorded against E. coli, L. monocytogenes and C. albicans with clear zone diameter of 33.2, 31.8 and 31.7 mm, respectively. Based on minimum inhibition concentration and minimum bactericidal concentration of SNPs (300 mg/l) as well as their effect on respiratory chain dehydrogenases, cellular sugar leakage, protein leakage and lipid peroxidation of microbial cells, E. coli was the most affected. Scanning electron microscopy, protein banding and DNA fragmentation proved obvious ultrastructural and molecular alterations of E. coli treated with SNPs. Thus, biogenic SNPs with enhanced properties can be synthesized with the aid of Citrus peel; and such engineered nanoparticles can be used as potent antimicrobial drug against E. coli.

Discovery of a Novel Bioactive Compound in Orange Peel Polar Fraction on the Inhibition of Trimethylamine and Trimethylamine N-Oxide through Metabolomics Approaches and In Vitro and In Vivo Assays: Feruloylputrescine Inhibits Trimethylamine via Suppressing cntA/B Enzyme.

This study compares the inhibitory effects of orange peel polar fraction (OPP) and orange peel nonpolar fraction (OPNP) on trimethylamine (TMA) and trimethylamine N-oxide (TMAO) production in response to l-carnitine treatment in vivo and in vitro. Metabolomics is used to identify bioactive compounds. The research demonstrates that the OPP effectively regulates atherosclerosis-related markers, TMA and TMAO in plasma and urine, compared to the OPNP. Our investigation reveals that these inhibitory effects are independent of changes in gut microbiota composition. The effects are attributed to the modulation of cntA/B enzyme activity and FMO3 mRNA expression in vitro. Moreover, OPP exhibits stronger inhibitory effects on TMA production than OPNP, potentially due to its higher content of feruloylputrescine, which displays the highest inhibitory activity on the cntA/B enzyme and TMA production. These findings suggest that the OPP containing feruloylputrescine has the potential to alleviate cardiovascular diseases by modulating cntA/B and FMO3 enzymes without directly influencing gut microbiota composition.

Bacterial community shifts of commercial apples, oranges, and peaches at different harvest points across multiple growing seasons.

Assessing the microbes present on tree fruit carpospheres as the fruit enters postharvest processing could have useful applications, as these microbes could have a major influence on spoilage, food safety, verification of packing process controls, or other aspects of processing. The goal of this study was to establish a baseline profile of bacterial communities associated with apple (pome fruit), peach (stone fruit), and Navel orange (citrus fruit) at harvest. We found that commercial peaches had the greatest bacterial richness followed by oranges then apples. Time of harvest significantly changed bacterial diversity in oranges and peaches, but not apples. Shifts in diversity varied by fruit type, where 70% of the variability in beta diversity on the apple carposphere was driven by the gain and loss of species (i.e., nestedness). The peach and orange carposphere bacterial community shifts were driven by nearly an even split between turnover (species replacement) and nestedness. We identified a small core microbiome for apples across and between growing seasons that included only Methylobacteriaceae and Sphingomonadaceae among the samples, while peaches had a larger core microbiome composed of five bacterial families: Bacillaceae, Geodermtophilaceae, Nocardioidaceae, Micrococcaeceae, and Trueperaceae. There was a relatively diverse core microbiome for oranges that shared all the families present on apples and peaches, except for Trueperaceae, but also included an additional nine bacterial families not shared including Oxalobacteraceae, Cytophagaceae, and Comamonadaceae. Overall, our findings illustrate the important temporal dynamics of bacterial communities found on major commercial tree fruit, but also the core bacterial families that constantly remain with both implications being important entering postharvest packing and processing.

Response of carbon fixation, allocation, and growth to source-sink manipulation by defoliation in vegetative citrus trees.

Source-sink balance in plants determines carbon distribution, and altering it can impact carbon fixation, transport, and allocation. We aimed to investigate the effect of altered source-sink ratios on carbon fixation, transport, and distribution in 'Valencia' sweet orange (Citrus x sinensis) by various defoliation treatments (0%, 33%, 66%, and 83% leaf removal). Gas exchange parameters were measured on 0 and 10 days after defoliation using A/Ci response curves, and leaf export was measured two days after defoliation using radioisotope tracer techniques. Greater defoliation increased the maximum rate of carboxylation (Vcmax), electron transport rate (J1200), and triose-phosphate utilization rate (TPU). Leaf export was unaffected by defoliation but increased in leaves closer to the shoot apex. Basipetal translocation velocity in the trunk remained unaltered, indicating that more photosynthates remained in the shoot rather than being transported directly to the root sink. Defoliated plants initiated more new flush shoots but accumulated less shoot biomass per plant after 8 weeks. Carbon allocation to fine roots was smaller in defoliated plants, suggesting defoliation led to retention of carbohydrates in aboveground organs such as the trunk and other shoots from previous growing cycles. In conclusion, the low source-sink ratio increased carbon fixation without impacting individual leaf export in citrus. The results suggest that intermediate sinks such as the aboveground perennial organs play a role in mediating the translocation velocity. Further research is necessary to better understand the dynamics of source-sink regulation in citrus trees.

Enhancing the Quality of Low-Alcohol Navel Orange Wine through Simultaneous Co-Fermentation Using Saccharomyces cerevisiae SC-125, Angel Yeast SY, and Lactiplantibacillus plantarum BC114.

To date, there has been limited research on the interactive effects of yeast and lactic acid bacteria (LAB) on the sensory qualities of navel orange wine. In this study, using Jintang navel orange juice as the raw material, multi-microbial fermentation was conducted with Saccharomyces cerevisiae SC-125 and Angel yeast SY, as well as Lactiplantibacillus plantarum BC114. Single yeast and co-fermentation with Lactiplantibacillus plantarum were used as the control groups. The research aimed to investigate the physicochemical parameters of navel orange wine during fermentation. Additionally, headspace solid-phase microextraction gas chromatography-mass spectrometry (HP-SPME-GC-MS) was employed to determine and analyze the types and levels of flavor compounds in the navel orange wines produced through the different fermentation methods. The co-fermentation using the three strains significantly enhanced both the quantity and variety of volatile compounds in the navel orange wine, concomitant with heightened total phenol and flavonoid levels. Furthermore, a notable improvement was observed in the free radical scavenging activity. A sensory evaluation was carried out to analyze the differences among the various navel orange wines, shedding light on the impact of different wine yeasts and co-fermentation with LAB on the quality of navel orange wines.

Genome-wide identification and characterization of the sweet orange (Citrus sinensis) basic helix-loop-helix (bHLH) family reveals a role for CsbHLH085 as a regulator of citrus bacterial canker resistance.

Citrus bacterial canker (CBC) is a harmful bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), negatively impacting citrus production worldwide. The basic helix-loop-helix (bHLH) transcription factor family plays crucial roles in plant development and stress responses. This study aimed to identify and annotate bHLH proteins encoded in the Citrus sinensis genome and explore their involvement and functional importance in regulating CBC resistance. A total of 135 putative CsbHLHs TFs were identified and categorized into 16 subfamilies. Their chromosomal locations, collinearity, and phylogenetic relationships were comprehensively analyzed. Upon Xcc strain YN1 infection, certain CsbHLHs were differentially regulated in CBC-resistant and CBC-sensitive citrus varieties. Among these, CsbHLH085 was selected for further functional characterization. CsbHLH085 was upregulated in the CBC-resistant citrus variety, was localized in the nucleus, and had a transcriptional activation activity. CsbHLH085 overexpression in Citrus significantly enhanced CBC resistance, accompanied by increased levels of salicylic acid (SA), jasmonic acid (JA), reactive oxygen species (ROS), and decreased levels of abscisic acid (ABA) and antioxidant enzymes. Conversely, CsbHLH085 virus-induced gene silencing resulted in opposite phenotypic and biochemical responses. CsbHLH085 silencing also affected the expression of phytohormone biosynthesis and signaling genes involved in SA, JA, and ABA signaling. These findings highlight the crucial role of CsbHLH085 in regulating CBC resistance, suggesting its potential as a target for biotechnological-assisted breeding citrus varieties with improved resistance against phytopathogens.

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.

Climate changes altered the citrus fruit quality: A 9-year case study in China.

Global climate change has significantly impacted the production of various crops, particularly long-term fruit-bearing plants such as citrus. This study analyzed the fruit quality of 12 citrus orchards (Citrus Sinensis L.Osbeck cv. Bingtang) in a subtropical region in Yunnan, China from 2014 to 2022. The results indicated that high rainfall (>220 mm) and low cumulative temperature (<3150 °C) promoted increases in titratable acidity (>1.8 %) in young fruits. As the fruits further expanded (with a horizontal diameter increasing from 50 to 65 mm), excessive rainfall (300-400 mm), lower cumulative temperature (<2400 °C), and a reduced diurnal temperature range (<10 °C) hindered decreases in titratable acidity. Conversely, low rainfall (<220 mm), high cumulative temperature (>3150 °C), and a high diurnal temperature range (>14 °C) promoted the accumulation of soluble solids in young fruits (9 %) at 120 days after flowering (DAF). Furthermore, low rainfall (<100 mm) favored the accumulation of soluble solids (1.5 %) during fruit expansion (195-225DAF). To quantify the relationship between fruit acidity and climate variables at 120 DAF, we developed a regression model, which was further validated by actual measurements and accurately predicted fruit acidity in 2023. Our findings have the potential to assist citrus growers in optimizing cultivation techniques for the production of high-quality citrus under increasingly variable climatic conditions.