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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.

The orange subfamily (Aurantioideae) contains several Citrus species cultivated worldwide, such as sweet orange and lemon. The origin of Citrus species has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus -related genera and that South Central China is the primary center of origin of the Citrus genus. We found substantial variations in the sequence and expression of the PH4 gene in Citrus relative to Citrus -related genera. Gene editing and biochemical experiments demonstrate a central role for PH4 in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste. De novo genome assemblies of 12 species from Aurantioideae and pangenome analyses provide insights into the origin and evolution of the orange subfamily and the genetic basis of flavor in citrus fruits.

Seedlings of ‘Shatian pummelo’ ( Citrus grandis ) and ‘Xuegan’ ( Citrus sinensis ) were supplied daily with nutrient solution at a concentration of 0.5 (control), 100, 200, 300, 400, or 500 μM CuCl 2 for 6 months. Thereafter, seedling growth; leaf, root, and stem levels of nutrients; leaf gas exchange; levels of pigments; chlorophyll a fluorescence (OJIP) transients and related parameters; leaf and root relative water content; levels of nonstructural carbohydrates; H 2 O 2 production rate; and electrolyte leakage were comprehensively examined ( a ) to test the hypothesis that Cu directly damages root growth and function, thus impairing water and nutrient uptake and hence inhibiting shoot growth; ( b ) to establish whether the Cu-induced preferential accumulation of Cu in the roots is involved in Cu tolerance of Citrus ; and ( c ) to elucidate the possible causes for the Cu-induced decrease in photosynthesis. Most of the growth and physiological parameters were greatly altered only at 300–500 μM (excess) Cu-treated seedlings. Cu supply increased the level of Cu in the roots, stems, and leaves, with a greater increase in the roots than that in the stems and leaves. Many of the fibrous roots became rotten and died under excess Cu. These findings support the hypothesis that Cu directly damages root growth and function, thus impairing water and nutrient uptake and hence inhibiting shoot growth, and the conclusion that the preferential accumulation of Cu in the roots under excess Cu is involved in the tolerance of Citrus to Cu toxicity. The lower CO 2 assimilation in excess Cu-treated leaves was caused mainly by nonstomatal factors, including structural damage to thylakoids, feedback inhibition due to increased accumulation of nonstructural carbohydrates, decreased uptake of water and nutrients, increased production of reactive oxygen species, and impaired photosynthetic electron transport chain. Also, we discussed the possible causes for the excess Cu-induced decrease in leaf pigments and accumulation of nonstructural carbohydrates in the roots and leaves.

Background Citrus rootstock selection plays a critical role in modulating sugar-acid metabolism, thereby influencing fruit flavor quality. This study systematically investigated the dynamic changes in sugar and acid components, key enzyme activities, and related gene expression in ‘Newhall’ navel orange ( Citrus sinensis ) grafted onto Citrus reticulata (Rt), Citrus junos (Cj), and Poncirus trifoliata (Pt) rootstocks during fruit development (150–240 days after flowering, DAF). Results Pt significantly enhanced late-stage sucrose accumulation (48.86 ± 2.63 mg·g⁻¹), surpassing the means of Rt and Cj by 26.78% and 60.72% at 240 DAF, respectively. This was associated with elevated sucrose synthase (SS) activity and upregulated CsSS2 expression. In contrast, Rt rootstock promoted malic and quinic acid retention and exhibited the highest citrate synthase (CS), isocitrate dehydrogenase (IDH), and isocitrate lyase (ICL) activities, supported by significantly increased upregulation of acid metabolism genes ( CsCS1 , CsNADP-IDH1/2/3 ). Comprehensive evaluation using the TOPSIS model, weighted by entropy analysis (with tartaric acid, fructose, and glucose identified as primary determinants), ranked fruit quality as Pt > Rt > Cj at maturity. Conclusions These findings establish a mechanistic framework linking rootstock-specific regulation of enzymatic activities and gene expression with dynamic sugar-acid profiles, providing valuable insights for optimizing rootstock-scion combinations to enhance citrus flavor quality.

It is known that nutrient deprivation following detachment can cause non-chilling peel pitting (NCPP) in citrus fruits when stored under a non-stressful environment and that this damage is reduced by pretreating the fruit with ethylene (ETH) (4 d, 10 µL L−1). The present work investigates the effect of this pretreatment on jasmonate (JA) accumulation and transcriptional regulation in mature Navelate oranges (Citrus sinensis L. Osbeck) stored under non-stressful conditions. ETH increased the expression of abundant genes participating in the synthesis of cis-(+)-12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and methyl jasmonate (MeJA). ETH also upregulated genes involved in jasmonoyl–isoleucine (JAIle) synthesis (CsJAR1) and decrease (CsCYP94B3 and CYP94C1), and CsSTA2, related to JA sulfation. The levels of these JA metabolites increased during fruit holding in ETH and after shifting them to air, with MeJA accumulation being especially remarkable. Overall, the beneficial effect of ETH on reducing NCPP appears to be related not only to this redirection of OPDA and JA metabolism towards the formation of JA derivatives but also to the regulation of JA signalling. Indeed, the repression of the receptor CsCOI1 and upregulation of various CsJAZs repressors caused by nutrient deprivation, together with the ETH-mediated induction of CsCOI1, CsTOPLESS, and abundant CsJAZs during long-term storage, suggests the occurrence of an ETH-enhanced negative transcriptional regulatory feedback loop in JA metabolism and signalling, by which the susceptibility of detached Navelate oranges to NCPP might be reduced.

Background. Conscious patients admitted to intensive care units (ICU) suffer from anxiety and agitation for various reasons, which can affect their recovery processes. Aims. To compare the effects of lavender and Citrus aurantium essential oils on anxiety and agitation of conscious patients admitted to ICUs. Design. A randomized parallel placebo-controlled trial. Methods. One hundred and fifty conscious patients admitted to ICUs were selected by convenience sampling and were randomly divided into three groups, groups of lavender aromatherapy and Citrus aurantium aromatherapy, in addition to the routine care and inhalation of five drops of lavender or Citrus aurantium essential oils for 30 minutes. The placebo group, in addition to routine care, was provided with 5 drops of normal saline for 30 minutes. Anxiety was assessed with the state subscale of State-Trait Anxiety Inventory, and agitation was examined with Richmond Agitation-Sedation Scale before, immediately, one hour, and three hours after the intervention. Results. All three groups suffered from relatively severe state anxiety before the intervention. The level of anxiety in the lavender and Citrus aurantium groups was significantly lower than that of the placebo group immediately and three hours after the intervention (P<0.05). No significant difference was observed between the two groups of lavender and Citrus aurantium. The majority of the samples in all three groups were agitated before the intervention, but agitation of all three groups decreased after the intervention. Restless/agitation reduced significantly in all three groups. Although restless/agitation of the lavender and Citrus aurantium groups reduced more than that of the placebo, no significant difference was found between the three groups. Conclusion. The results of the present study showed the positive effects of lavender aromatherapy and Citrus aurantium aromatherapy on reducing the anxiety of patients admitted to ICUs. Relevance to Clinical Practice. Aromatherapy can be used as an effective and safe intervention to reduce anxiety in ICUs.

Background Limonoids are major bioactive compounds that are produced by the triterpenoid metabolic pathway. The detailed biochemical process of limonoid biosynthesis and the mechanism of its molecular regulation remain elusive. The identification of transcription factors that regulate limonoid biosynthetic pathways is very important for understanding the underlying regulatory mechanisms. This information could also provide tools for manipulating biosynthesis genes to modulate limonoid production. Results In this study, the CiMYB42 transcription factor was isolated to identify its role in limonoid biosynthesis. Multiple alignment analysis and phylogenetic analysis demonstrated that CiMYB42 is a typical R2R3MYB transcription factor that shares high similarity of its amino acid sequence with AtMYB42 . Limonoids contents were higher in Citrus sinensis and Citrus grandis than in other species. Limonoid accumulation during leaf development also showed diverse trends in different genotypes. The expression of CiMYB42 was significantly related to the limonoid content and the expression of CiOSC in some citrus accessions. The overexpression of CiMYB42 in sweet orange resulted in significant accumulation of limonin, whereas the downregulation of CiMYB42 by RNAi resulted in a dwarf phenotype and less nomilin accumulation. Furthermore, the results of a yeast one-hybrid assay and EMSA indicated that CiMYB42 binds exclusively to the TTGTTG sequence (type II MYB core) in the promoter of CiOSC . Together, these results suggest that CiMYB42 positively regulates limonoid biosynthesis by regulating the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core) of its promoter. Conclusions CiMYB42 is an important transcription activator involved in limonoid biosynthesis that regulates the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core).

Background Flavanone 3-hydroxylase (F3H), a key enzyme in the flavonoid biosynthetic pathway, plays an important role in the regulation of flavonols and anthocyanidins accumulation. Citrus fruit is a rich source of flavonoids with varied flavonoid compositions among different varieties. To date, the study on F3H is limited in citrus, and its roles in regulating flavonoid accumulation in citrus fruit are still unclear. Results In this study, we isolated a CitF3H from three different citrus varieties, Satsuma mandarin ( Citrus unshiu Marc.), Ponkan mandarin ( C . reticulata Blanco) and blood orange ‘Moro’ ( C . sinensis Osbeck). Functional analysis showed that CitF3H encoded a functional flavanone 3-hydroxylase. It catalyzed the hydroxylation of naringenin to yield dihydrokaempferol, which was a precursor of anthocyanins in flavonoid biosynthetic pathway. In the juice sacs, CitF3H was differentially expressed among the three citrus varieties, and its expression level was positively correlated with the accumulation of anthocyanins during the ripening process. In the juice sacs of Satsuma mandarin and Ponkan mandarin the expression of CitF3H kept constant at an extremely low level, and no anthocyanin was accumulated during the ripening process. In contrast, the expression of CitF3H increased rapidly along with the accumulation of anthocyanin in the juice sacs of blood orange ‘Moro’ during the ripening process. In addition, we found that blue light irradiation was effective to up-regulate the expression of CitF3H and improve anthocyanin accumulation in the juice sacs of blood orange ‘Moro’ in vitro. Conclusion CitF3H was a key gene regulating anthocyanin accumulation in the juice sacs of citrus fruit. The results presented in this study will contribute to elucidating anthocyanin biosynthesis in citrus fruit, and provide new strategies to improve the nutritional and commercial values of citrus fruit.

Background Citrus is one of the most important fresh fruit crops worldwide. Juice sac granulation is a physiological disorder, which leads to a reduction in soluble solid concentration, total sugar, and titratable acidity of citrus fruits. Pectin methylesterase (PME) catalyzes the de-methylesterification of homogalacturonans and plays crucial roles in cell wall modification during plant development and fruit ripening. Although PME family has been well investigated in various model plants, little is known regarding the evolutionary property and biological function of PME family genes in citrus. Results In this study, 53 non-redundant PME genes were identified from Citrus sinensis genome, and these PME genes were divided into four clades based on the phylogenetic relationship. Subsequently, bioinformatics analyses of gene structure, conserved domain, chromosome localization, gene duplication, and collinearity were performed on CsPME genes, providing important clues for further research on the functions of CsPME genes. The expression profiles of CsPME genes in response to juice sac granulation and low-temperature stress revealed that CsPME g enes were involved in the low temperature-induced juice sac granulation in navel orange fruits. Subcellular localization analysis suggested that CsPME genes were localized on the apoplast, endoplasmic reticulum, plasma membrane, and vacuole membrane. Moreover, yeast one-hybrid screening and dual luciferase activity assay revealed that the transcription factor CsRVE1 directly bound to the promoter of CsPME3 and activated its activity. Conclusion In summary, this study conducts a comprehensive analysis of the PME gene family in citrus, and provides a novel insight into the biological functions and regulation patterns of CsPME genes during juice sac granulation of citrus.

GABA shunt is an important metabolic hub that plays a key role in plant growth, development, and response. Although a functional connection between GABA shunt and the TCA cycle via a mitochondrial GABA permease ( gabP ) gene was reported previously from model plants, this connection in non-model plants, such as citrus, is poorly studied. Herein, we used bioinformatics and multi-omics for in-silico characterization, structural modeling, and ligand-receptor binding profile of GABA transport carrier ( CsgabP ) from the non-model plant, Valencia sweet orange ( Citrus sinensis ) and to explore their potential role(s) in GABA uptake and citrus response to Huanglongbing. Genome-wide analysis showed that the citrus genome encodes for two putative CsgabP proteins ( CsgabP -1 [521 aa] and CsgabP -2 [419 aa]) that were relatively highly homologous to each other and homologous to amino acid permease BAT1 proteins from other plant species. Primary structures of both CsgabPs showed high similarity and conserved sequences between them. CsgabPs are highly hydrophobic integral transmembrane transporter proteins with internal hydrophilic N- and C-terminal ends and depend on 9–12 transmembrane segments. The crystallographic three-dimensional (3D) structures of both CsgabP s were predicted and confirmed using AlphaFold as a monomer with 100% coverage, but not experimentally determined crystallographic structures. Targeted metabolomics showed that CsgabP was upregulated upon GABA supplementation in healthy and ‘ Candidatus Liberibacter asiaticus’-infected citrus plants. Likewise, CsgabP was involved in citrus response(s) to different biotic stress including infection with ‘ Ca . L. asiaticus’ or infestation with its vector, Diaphorina citri . These findings probably clarify the molecular and functional connection between the GABA shunt and the TCA cycle in non-model plants such as citrus.