Integrated Metabolomic-Transcriptomic Analyses of Flavonoid Accumulation in Citrus Fruit under Exogenous Melatonin Treatment.

The flavonoids in citrus fruits are crucial physiological regulators and natural bioactive products of high pharmaceutical value. Melatonin is a pleiotropic hormone that can regulate plant morphogenesis and stress resistance and alter the accumulation of flavonoids in these processes. However, the direct effect of melatonin on citrus flavonoids remains unclear. In this study, nontargeted metabolomics and transcriptomics were utilized to reveal how exogenous melatonin affects flavonoid biosynthesis in "Bingtangcheng" citrus fruits. The melatonin treatment at 0.1 mmol L-1 significantly increased the contents of seven polymethoxylated flavones (PMFs) and up-regulated a series of flavonoid pathway genes, including 4CL (4-coumaroyl CoA ligase), FNS (flavone synthase), and FHs (flavonoid hydroxylases). Meanwhile, CHS (chalcone synthase) was down-regulated, causing a decrease in the content of most flavonoid glycosides. Pearson correlation analysis obtained 21 transcription factors co-expressed with differentially accumulated flavonoids, among which the AP2/EREBP members were the most numerous. Additionally, circadian rhythm and photosynthesis pathways were enriched in the DEG (differentially expressed gene) analysis, suggesting that melatonin might also mediate changes in the flavonoid biosynthesis pathway by affecting the fruit's circadian rhythm. These results provide valuable information for further exploration of the molecular mechanisms through which melatonin regulates citrus fruit metabolism.

Nobiletin as a chemopreventive natural product against cancer, a comprehensive review.

As a leading cause of death, second only to heart disease, cancer has always been one of the burning topics in medical research. When targeting multiple signal pathways in tumorigenesis chemoprevention, using natural or synthetic anti-cancer drugs is a vital strategy to reduce cancer damage. However, toxic effects, multidrug resistance (MDR) as well as cancer stem cells (CSCs) all prominently limited the clinical application of conventional anticancer drugs. With low side effects, strong biological activity, unique mechanism, and wide range of targets, natural products derived from plants are considered significant sources for new drug development. Nobiletin is one of the most attractive compounds, a unique flavonoid primarily isolated from the peel of citrus fruits. Numerous studies in vitro and in vivo have suggested that nobiletin and its derivatives possess the eminent potential to become effective cancer chemoprevention agents through various cellular and molecular levels. This article aims to comprehensively review the anticancer efficacy and specific mechanisms of nobiletin, enhancing our understanding of its chemoprevention properties and providing the latest research findings. At the end of this review, we also give some discussion and future perspectives regarding the challenges and opportunities in nobiletin efficient exploitation.

Citrus flavonoids and their antioxidant evaluation.

The antioxidant ability is the link and bridge connecting a variety of biological activities. Citrus flavonoids play an essential role in regulating oxidative stress and are an important source of daily intake of antioxidant supplements. Many studies have shown that citrus flavonoids promote health through antioxidation. In this review, the biosynthesis, composition and distribution of citrus flavonoids were concluded. The detection methods of antioxidant capacity of citrus flavonoids were divided into four categories: chemical, cellular, animal and clinical antioxidant capacity evaluation systems. The modeling methods, applicable scenarios, and their relative merits were compared based on these four systems. The antioxidant functions of citrus flavonoids under different evaluation systems were also discussed, especially the regulation of the Nrf2-antioxidases pathway. Some shortcomings in the current research were pointed out, and some suggestions for progress were put forward.

Three AP2/ERF family members modulate flavonoid synthesis by regulating type IV chalcone isomerase in citrus.

Flavanones and flavones are excellent source of bioactive compounds but the molecular basis of their highly efficient production remains elusive. Chalcone isomerase (CHI) family proteins play essential roles in flavonoid biosynthesis but little are known about the transcription factors controlling their gene expression. Here, we identified a type IV CHI (designated as CitCHIL1) from citrus which enhances the accumulation of citrus flavanones and flavones (CFLs). CitCHIL1 participates in a CFL biosynthetic metabolon and assists the cyclization of naringenin chalcone to (2S)-naringenin, which leads to the efficient influx of substrates to chalcone synthase (CHS) and improves the catalytic efficiency of CHS. Overexpressing CitCHIL1 in Citrus and Arabidopsis significantly increased flavonoid content and RNA interference-induced silencing of CitCHIL1 in citrus led to a 43% reduction in CFL content. Three AP2/ERF transcription factors were identified as positive regulators of the CitCHIL1 expression. Of these, two dehydration-responsive element binding (DREB) proteins, CitERF32 and CitERF33, activated the transcription by directly binding to the CGCCGC motif in the promoter, while CitRAV1 (RAV: related to ABI3/VP1) formed a transcription complex with CitERF33 that strongly enhanced the activation efficiency and flavonoid accumulation. These results not only illustrate the specific function that CitCHIL1 executes in CFL biosynthesis but also reveal a new DREB-RAV transcriptional complex regulating flavonoid production.

Characterization of a caffeoyl-CoA O-methyltransferase-like enzyme involved in biosynthesis of polymethoxylated flavones in Citrus reticulata.

Polymethoxylated flavones (PMFs), which accumulate exclusively in fruit peel of citrus, play important physiological and pharmacological roles but the genetic basis for the methylation of flavonoids has not been fully elucidated in citrus. Here we characterize a caffeoyl-CoA O-methyltransferase-like enzyme, designated CrOMT1. The expression pattern of CrOMT1 was highly correlated with the concentration of the three major PMFs in two different citrus fruit tissues during fruit maturation. Exposure of fruit to UV-B radiation sharply increased the level of CrOMT1 transcripts and also led to the accumulation of three PMFs. The potential role of CrOMT1 was studied by testing the catalytic activity of recombinant CrOMT1 with numerous possible substrates in vitro. The enzyme could most efficiently methylate flavones with neighboring hydroxy moieties, with high catalytic efficiencies found with 6-OH- and 8-OH-containing compounds, preferences that correspond precisely with the essential methylation sites involved in the synthesis of the three naturally occurring PMFs in Citrus reticulata. This indicates that CrOMT1 is capable of in vitro methylation reactions required to synthesize PMFs in vivo. Furthermore, transient overexpression of CrOMT1 increased levels of the three major PMFs in fruit, indicating that CrOMT1 is likely to play an essential role in the biosynthesis of PMFs in citrus.

Analysis of Phenolic Components and Related Biological Activities of 35 Apple (Malus pumila Mill.) Cultivars.

Apple (Malus pumila Mill.) is a popular fruit with high economic values and various biological activities that are beneficial to human health. In this study, 35 apple cultivars were collected and were evaluated for their basic quality indexes, phenolic compositions, antioxidant activity, anti-tumour, and anti-diabetic activities. The compositions of phenolics were detected by using high-performance liquid chromatography (HPLC) and high-resolution mass spectroscopy (HRMS) assays. The antioxidant activities of peel and pulp extracts from 35 apple cultivars were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and ferric reducing antioxidant power (FRAP) assay. Results showed that the contents of phenolic acids and proanthocyanidins showed significant correlations with the antioxidant activities. Phenolic-rich extracts significantly inhibited HepG2 cell proliferation, with the inhibition activity varied significantly between cultivars. 'Gold Delicious' pulp extract, 'Xiboliyabaidian' peel and pulp extracts showed protective effects on H2O2-induced injury of human umbilical vein endothelial cells (HUVEC). 'Red Fuji' peel extract, 'Xiboliyabaidian' peel and pulp extracts, as well as 'Gold Delicious' peel extract, significantly increased glucose consumption of HepG2 cells, in a dose-dependent manner. This research may provide theoretical guidance for further nutritional investigation of the apple resources.

Characterization of a Flavonoid 3'/5'/7-O-Methyltransferase from Citrus reticulata and Evaluation of the In Vitro Cytotoxicity of Its Methylated Products.

O-methylation of flavonoids is an important modification reaction that occurs in plants. O-methylation contributes to the structural diversity of flavonoids, which have several biological and pharmacological functions. In this study, an O-methyltransferase gene (CrOMT2) was isolated from the fruit peel of Citrus reticulata, which encoding a multifunctional O-methyltransferase and could effectively catalyze the methylation of 3'-, 5'-, and 7-OH of flavonoids with vicinal hydroxyl substitutions. Substrate preference assays indicated that this recombinant enzyme favored polymethoxylated flavones (PMF)-type substrates in vitro, thereby providing biochemical evidence for the potential role of the enzyme in plants. Additionally, the cytotoxicity of the methylated products from the enzymatic catalytic reaction was evaluated in vitro using human gastric cell lines SGC-7901 and BGC-823. The results showed that the in vitro cytotoxicity of the flavonoids with the unsaturated C2-C3 bond was increased after being methylated at position 3'. These combined results provide biochemical insight regarding CrOMT2 in vitro and indicate the in vitro cytotoxicity of the products methylated by its catalytic reaction.

Transcriptomic Analyses of Root Restriction Effects on Phytohormone Content and Signal Transduction during Grape Berry Development and Ripening.

Phytohormones strongly influence growth, development and nutritional quality of agricultural products by modulating molecular and biochemical changes. The purpose of the present study was to investigate the influence of root restriction (RR) treatment on the dynamic changes of main phytohormones during the berry development and ripening of "Summer Black" early ripening seedless grape (Vitis vinifera × V. labrusca), and to analyze the changes in the biosynthesis and signal transduction pathways of phytohormones by transcriptomics. Enzyme-linked immunosorbent assay (ELISA) and Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS) were used to quantify the phytohormone levels, and RNA-Seq was used to analyze the transcript abundance. The results showed that 23 transcripts involved in the phytohormone biosynthesis and 34 transcripts involved in the signal transduction pathways were significantly changed by RR treatment. RR also increased abscisic acid, brassinosteroid, ethylene, jasmonic acid and salicylic acid levels, while decreasing auxin, cytokinin, and gibberellin contents. The results of the present study suggest that RR treatment can accelerate the grape ripening process, and specific candidate genes were identified for further functional analysis.

Antioxidant Capacity, Anticancer Ability and Flavonoids Composition of 35 Citrus (Citrus reticulata Blanco) Varieties.

Citrus (Citrus reticulate Blanco) is one of the most commonly consumed and widely distributed fruit in the world, which is possessing extensive bioactivities. Present study aimed to fully understand the flavonoids compositions, antioxidant capacities and in vitro anticancer abilities of different citrus resources. Citrus fruits of 35 varieties belonging to 5 types (pummelos, oranges, tangerines, mandarins and hybrids) were collected. Combining li quid chromatography combined with electrospray ionization mass spectrometry (LC-ESI-MS/MS) and ultra-performance liquid chromatography combined with diode array detector (UPLC-DAD), a total of 39 flavonoid compounds were identified, including 4 flavones, 9 flavanones and 26 polymethoxylated flavonoids (PMFs). Each citrus fruit was examined and compared by 4 parts, flavedo, albedo, segment membrane and juice sacs. The juice sacs had the lowest total phenolics, following by the segment membrane. Four antioxidant traits including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and cupric reducing antioxidant capacity (CUPRAC) were applied for the antioxidant capacities evaluation. Three gastric cancer cell lines, SGC-7901, BGC-823 and AGS were applied for the cytotoxicity evaluation. According to the results of correlation analysis, phenolics compounds might be the main contributor to the antioxidant activity of citrus extracts, while PMFs existing only in the flavedo might be closely related to the gastric cancer cell line cytotoxicity of citrus extracts. The results of present study might provide a theoretical guidance for the utilization of citrus resources.

Treatment of Donor Cells with Oxidative Phosphorylation Inhibitor CPI Enhances Porcine Cloned Embryo Development.

Somatic cell nuclear transfer (SCNT) technology holds great promise for livestock industry, life science and human biomedicine. However, the development and application of this technology is limited by the low developmental potential of SCNT embryos. The developmental competence of cloned embryos is influenced by the energy metabolic status of donor cells. The purpose of this study was to investigate the effects of CPI, an oxidative phosphorylation inhibitor, on the energy metabolism pathways of pig fibroblasts and the development of subsequent SCNT embryos. The results showed that treatment of porcine fibroblasts with CPI changed the cellular energy metabolic pathways from oxidative phosphorylation to glycolysis and enhanced the developmental ability of subsequent SCNT embryos. The present study establishes a simple, new way to improve pig cloning efficiency, helping to promote the development and application of pig SCNT technology.

Metabolome and Transcriptome Analysis Revealed the Basis of the Difference in Antioxidant Capacity in Different Tissues of Citrus reticulata 'Ponkan'.

Citrus is an important type of fruit, with antioxidant bioactivity. However, the variations in the antioxidant ability of different tissues in citrus and its metabolic and molecular basis remain unclear. Here, we assessed the antioxidant capacities of 12 tissues from Citrus reticulata 'Ponkan', finding that young leaves and root exhibited the strongest antioxidant capacity. Secondary metabolites accumulated differentially in parts of the citrus plant, of which flavonoids were enriched in stem, leaf, and flavedo; phenolic acids were enriched in the albedo, while coumarins were enriched in the root, potentially explaining the higher antioxidant capacities of these tissues. The spatially specific accumulation of metabolites was related to the expression levels of biosynthesis-related genes such as chalcone synthase (CHS), chalcone isomerase (CHI), flavone synthase (FNS), O-methyltransferase (OMT), flavonoid-3'-hydroxylase (F3'H), flavonoid-6/8-hydroxylase (F6/8H), p-coumaroyl CoA 2'-hydroxylase (C2'H), and prenyltransferase (PT), among others, in the phenylpropane pathway. Weighted gene co-expression network analysis (WGCNA) identified modules associated with flavonoids and coumarin content, among which we identified an OMT involved in coumarin O-methylation, and related transcription factors were predicted. Our study identifies key genes and metabolites influencing the antioxidant capacity of citrus, which could contribute to the enhanced understanding and utilization of bioactive citrus components.

Supplementation of SkQ1 Increases Mouse In Vitro Oocyte Maturation and Subsequent Embryonic Development by Reducing Oxidative Stress.

In vitro oocyte maturation (IVM) technology is important for assisted animal and human reproduction. However, the maturation rates and developmental potential of in vitro-matured oocytes are usually lower than those of in vivo-matured oocytes. Oxidative stress is a main factor that causes the lower maturation rates and quality of in vitro-matured oocytes. The purpose of this study was to investigate the effects of treatment with SkQ1, a mitochondria-targeted antioxidant, on mouse IVM and subsequent embryonic development. The results demonstrated that the supplementation of SkQ1 during IVM improves the maturation rates of mouse oocytes and the subsequent developmental competence of in vitro-fertilized embryos. The addition of SkQ1 to the IVM medium also decreased oxidative stress and apoptosis, and increased mitochondrial membrane potential in matured mouse oocytes. This study provides a new method through which to enhance the maturation rates and the quality of in vitro-matured mouse oocytes, thus promoting the application and development of assisted animal and human reproductive technology.

Hepatoprotective potential of four fruit extracts rich in different structural flavonoids against alcohol-induced liver injury via gut microbiota-liver axis.

Alcoholic liver injury (ALI) accounts for a major share of the global burden of non-viral liver disease. In the absence of specialized medications, research on using fruit flavonoids as a treatment is gaining momentum. This study investigated the hepatoprotective effects of four fruits rich in structurally diverse flavonoids: ougan (Citrus reticulata cv. Suavissima, OG), mulberry (Morus alba L., MB), apple (Malus × domestica Borkh., AP), and turnjujube (Hovenia dulcis Thunnb., TJ). A total of one flavanone glycoside, three polymethoxyflavones, two anthocyanins, one flavonol glycoside, and one dihydroflavonol were identified through UPLC analysis. In an acute ethanol-induced ALI mouse model, C57BL/6J mice were supplemented with 200 mg/kg·BW/day of different fruit extracts for three weeks. Our results showed that the four extracts exhibited promising benefits in improving lipid metabolism disorders, iron overload, and oxidative stress. RT-PCR and Western blot tests suggested that the potential mechanism may partially be attributed to the activation of the NRF2-mediated antioxidant response and the inhibition of ferroptosis pathways. Furthermore, fruit extracts administration demonstrated a specific regulatory role in intestinal microecology, with increases in beneficial bacteria such as Dubosiella, Lactobacillus, and Bifidobacterium. Spearman correlation analysis revealed strong links between intestinal flora, lipid metabolism, and iron homeostasis, implying that the fruit extracts mitigated ALI via the gut microbiota-liver axis. In vitro experiments reaffirmed the activity against ethanol-induced oxidative damage and highlighted the positive effects of flavonoid components. These findings endorse the prospective application of OG, MB, AP, and TJ as dietary supplements or novel treatments for ALI.

Detection and classification of volatile compounds emitted by three fungi-infected citrus fruit using gas chromatography-mass spectrometry.

Citrus fruit produced some characteristic volatile compounds when infected by fungi compared with the healthy fruit. In the present study, volatile metabolites of postharvest citrus fruit with three different diseases including stem-end rot, blue mold and green mold were detected. Multivariate analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to classify the volatile compounds between the infected and non-infected citrus fruit. The results indicated that volatile compounds of unrotten, unrotten-rotten junction, and rotten tissues were successfully classified. Importantly, eight volatile compounds as biomarkers for stem-end rot and one biomarker for green mold of citrus were screened to discriminate the infected citrus fruit. This study offers the application potential of odor profiling of volatile compounds for detecting the fungi infection in postharvest citrus fruit.

Genome-wide analysis of cytochrome P450 genes in Citrus clementina and characterization of a CYP gene encoding flavonoid 3'-hydroxylase.

Cytochrome P450s (CYPs) are the largest family of enzymes in plant and play multifarious roles in development and defense but the available information about the CYP superfamily in citrus is very limited. Here we provide a comprehensive genome-wide analysis of the CYP superfamily in Citrus clementina genome, identifying 301 CYP genes grouped into ten clans and 49 families. The characteristics of both gene structures and motif compositions strongly supported the reliability of the phylogenetic relationship. Duplication analysis indicated that tandem duplication was the major driving force of expansion for this superfamily. Promoter analysis revealed numerous cis-acting elements related to various responsiveness. RNA-seq data elucidated their expression patterns in citrus fruit peel both during development and in response to UV-B. Furthermore, we characterize a UV-B-induced CYP gene (Ciclev10019637m, designated CitF3'H) as a flavonoid 3'-hydroxylase for the first time. CitF3'H catalyzed numerous flavonoids and favored naringenin in yeast assays. Virus-induced silencing of CitF3'H in citrus seedlings significantly reduced the levels of 3'-hydroxylated flavonoids and their derivatives. These results together with the endoplasmic reticulum-localization of CitF3'H in plant suggest that this enzyme is responsible for the biosynthesis of 3'-hydroxylated flavonoids in citrus. Taken together, our findings provide extensive information about the CYP superfamily in citrus and contribute to further functional verification.

Phenolics and Terpenoids Profiling in Diverse Loquat Fruit Varieties and Systematic Assessment of Their Mitigation of Alcohol-Induced Oxidative Stress.

To compare and investigate the phenolic compounds in the peel and flesh of loquat (Eriobotrya japonica) and evaluate their ability to protect against alcohol-induced liver oxidative stress, we employed a combination of ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry (HRMS) to qualitatively and quantitatively analyze 22 phenolics and 2 terpenoid compounds in loquat peel and flesh extracts (extraction with 95% ethanol). Among these, six compounds were identified for the first time in loquat, revealing distinct distribution patterns based on variety and tissue. Various chemical models, such as DPPH, FRAP, ORAC, and ABTS, were used to assess free radical scavenging and metal ion reduction capabilities. The results indicate that peel extracts exhibited higher antioxidant capacity compared with flesh extracts. Using a normal mouse liver cell line, AML-12, we explored the protective effects of loquat extracts and individual compounds against ethanol-induced oxidative stress. The findings demonstrate the enhanced cell viability and the induction of antioxidant enzyme activity through the modulation of Nrf2 and Keap1 gene expression. In a C57/BL6 mouse model of alcohol-induced liver damage, loquat extract was found to alleviate liver injury induced by alcohol. The restoration of perturbed serum liver health indicators underscored the efficacy of loquat extract in reclaiming equilibrium. The culmination of these findings significantly bolsters the foundational knowledge necessary to explore the utilization of loquat fruit extract in the creation of health-focused products.

Functional characterization of two flavone synthase II members in citrus.

Polymethoxylated flavones (PMFs), the main form of flavones in citrus, are derived from the flavone branch of the flavonoid biosynthesis pathway. Flavone synthases (FNSs) are enzymes that catalyze the synthesis of flavones from flavanones. However, the FNS in citrus has not been characterized yet. Here, we identified two type II FNSs, designated CitFNSII-1 and CitFNSII-2, based on phylogenetics and transcriptome analysis. Both recombinant CitFNSII-1 and CitFNSII-2 proteins directly converted naringenin, pinocembrin, and liquiritigenin to the corresponding flavones in yeast. In addition, transient overexpression of CitFNSII-1 and CitFNSII-2, respectively, in citrus peel significantly enhanced the accumulation of total PMFs, while virus-induced CitFNSII-1 and CitFNSII-2 genes silencing simultaneously significantly reduced the expression levels of both genes and total PMF content in citrus seedlings. CitFNSII-1 and CitFNSII-2 presented distinct expression patterns in different cultivars as well as different developmental stages. Methyl salicylate (MeSA) treatment reduced the CitFNSII-2 expression as well as the PMFs content in the peel of Citrus sinensis fruit but did not affect the CitFNSII-1 expression. These results indicated that both CitFNSII-1 and CitFNSII-2 participated in the flavone biosynthesis in citrus while the regulatory mechanism governing their expression might be specific. Our findings improved the understanding of the PMFs biosynthesis pathway in citrus and laid the foundation for further investigation on flavone synthesis regulation.

Advances of section drying in citrus fruit: The metabolic changes, mechanisms and prevention methods.

Citrus fruit are consumed worldwide due to their excellent features, such as delicious taste and health-promoting compounds. However, section drying, a physiological disorder of citrus fruit, often occurs both in the preharvest and postharvest storage, causing a significant reduction in fruit quality and consumer acceptance. In this review, section drying of citrus fruit was divided into three types: granulation, vesicle collapse and both above. The main causes, metabolic changes and mechanisms of section drying were discussed, respectively. Furthermore, the prevention methods of section drying in citrus fruit, including preharvest and postharvest methods, were also summarized. Given the significant influence of section drying in citrus fruit production, the mechanisms and prevention methods of section drying are worth further exploration. A better understanding of section drying may provide guidance for the prevention of this disorder and future breeding of citrus fruit.

Selenium-enriched Polysaccharide: an Effective and Safe Selenium Source of C57 Mice to Improve Growth Performance, Regulate Selenium Deposition, and Promote Antioxidant Capacity.

Selenium-enriched polysaccharide (SeEPS) was prepared by reducing Se(IV) to elemental selenium and organic selenium in polysaccharide medium by the obtained Enterobacter cloacae strain Z0206 under aerobic conditions. In the present study, we focused on investigating the role of short-term supplementation of SeEPS at supernutritional doses in the regulation of growth performance, liver damage, antioxidant capacity, and selenium (Se) accumulation in C57 mice. Thirty-two C57 mice were randomly divided into four groups: the control group was gavaged with equal volume of phosphate-buffered saline, while the sodium selenite (Na2SeO3), selenomethionine (SeMet), and SeEPS groups were gavaged with 0.5 mg Se/kg BW of Na2SeO3, SeMet, and selenium-enriched polysaccharide (n = 8), respectively. We examined liver injury indicators, antioxidant capacity in the serum and liver, selenium deposition at different sites, selenoprotein levels, and selenocysteine-synthesizing and degradation-associated gene expression in mouse livers. SeEPS supplementation dramatically increased average daily weight gain but reduced the feed-to-gain ratio (F/G) of mice (P < 0.05). Compared to Na2SeO3 and SeMet supplementation, SeEPS supplementation at supernutritional doses did not cause the liver damage. SeEPS supplementation also markedly enhanced total antioxidant capacity (T-AOC), catalase (CAT), glutathione peroxidase (GSH-PX), and total superoxide dismutase (T-SOD) activities but reduced malondialdehyde (MDA) levels in the liver and serum (P < 0.05), while significantly increasing selenocysteine-synthesizing and degradation-related gene (SEPHS2, SEPSECS, Secisbp, Scly) expression at the mRNA level (P < 0.05), thus upregulating the mRNA levels of selenoproteins (SELENOP, SELENOK) (P < 0.05). We suggest that SeEPS could be a potential replacement for inorganic selenium to improve animals' growth performance, promote antioxidant capacity, and regulate selenium deposition.

Integrated Physiochemical, Hormonal, and Transcriptomic Analysis Revealed the Underlying Mechanisms for Granulation in Huyou (Citrus changshanensis) Fruit.

Juice sac granulation is a common internal physiological disorder of citrus fruit. In the present study, we compared the physiochemical characteristics and transcriptome profiles of juice sacs in different granulation levels from Huyou fruit (Citrus changshanensis). The accumulation of cell wall components, including the water-soluble pectin, protopectin, cellulose, and lignin, were significantly correlated with the granulation process, resulting in the firmness increase of the juice sac. The in situ labeling of the cell wall components indicated the early accumulation of cellulose and high-methylesterified pectin in the outer layer cells, as well as the late accumulation of lignin in the inner layer cells of the juice sac. Several phytohormones, including auxins, abscisic acids, cytokinins, jasmonic acid, salicylic acid, and/or their metabolites, were positively correlated to the granulation level, indicating an active and complex phytohormones metabolism in the granulation process. Combining the trend analysis by the Mfuzz method and the module-trait correlation analysis by the Weighted Gene Co-expression Network Analysis method, a total of 2940 differentially expressed genes (DEGs) were found to be positively correlated with the granulation level. Gene Ontology (GO) enrichment indicated that the selected DEGs were mainly involved in the cell wall organization and biogenesis, cell wall macromolecule metabolic process, carbohydrate metabolic process, and polysaccharide metabolic process. Among these selected genes, those encoding ß-1,4-xylosyltransferase IRX9, cellulose synthase, xyloglucan: xyloglucosyl transferase, xyloglucan galactosyltransferase MUR3, α-1,4-galacturonosyltransferase, expansin, polygalacturonase, pectinesterase, ß-glucosidase, ß-galactosidase, endo-1,3(4)-ß-glucanase, endoglucanase and pectate lyase that required for the biosynthesis or structural modification of cell wall were identified. In addition, NAC, MYB, bHLH, and MADS were the top abundant transcription factors (TFs) families positively correlated with the granulation level, while the LOB was the top abundant TFs family negatively correlated with the granulation level.