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Summary: Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA‐mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism‐related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid‐driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5‐CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5‐CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5‐CsbZIP44 precisely modulates ABA signal‐mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops. [ABSTRACT FROM AUTHOR]

SUMMARY: Chromoplasts act as a metabolic sink for carotenoids, in which plastoglobules serve as versatile lipoprotein particles. PGs in chloroplasts have been characterized. However, the features of PGs from non‐photosynthetic plastids are poorly understood. We found that the development of chromoplast plastoglobules (CPGs) in globular and crystalloid chromoplasts of citrus is associated with alterations in carotenoid storage. Using Nycodenz density gradient ultracentrifugation, an efficient protocol for isolating highly purified CPGs from sweet orange (Citrus sinensis) pulp was established. Forty‐four proteins were defined as likely comprise the core proteome of CPGs using comparative proteomics analysis. Lipidome analysis of different chromoplast microcompartments revealed that the nonpolar microenvironment within CPGs was modified by 35 triacylglycerides, two sitosterol esters, and one stigmasterol ester. Manipulation of the CPG‐localized gene CsELT1 (esterase/lipase/thioesterase) in citrus calli resulted in increased lipids and carotenoids, which is further evidence that the nonpolar microenvironment of CPGs contributes to carotenoid accumulation and storage in the chromoplasts. This multi‐feature analysis of CPGs sheds new light on the role of chromoplasts in carotenoid metabolism, paving the way for manipulating carotenoid content in citrus fruit and other crops. [ABSTRACT FROM AUTHOR]

Deciphering the genetic basis of organoleptic traits is critical for improving the quality of fruits, which greatly shapes their appeal to consumers. Here, we characterize the citrus R3-MYB transcription factor TRIPTYCHON-LIKE (CitTRL), which is closely associated with the levels of citric acid, proanthocyanidins (PAs), and anthocyanins. Overexpression of CitTRL lowered acidity levels and PA contents in citrus calli as well as anthocyanin and PA contents in Arabidopsis leaves and seeds. CitTRL interacts with the two basic helix–loop–helix (bHLH) proteins CitbHLH1 and ANTHOCYANIN 1 (CitAN1) to regulate fruit quality. We show that CitTRL competes with the R2R3-MYB CitRuby1 for binding to CitbHLH1 or CitAN1, thereby repressing their activation of anthocyanin structural genes. CitTRL also competes with a second R2R3-MYB, CitPH4, for binding to CitAN1, thus altering the expression of the vacuolar proton-pump gene PH5 and Leucoanthocyanidin reductase , responsible for vacuolar acidification and proanthocyanidins biosynthesis, respectively. Moreover, CitPH4 activates CitTRL transcription, thus forming an activator–repressor loop to prevent the overaccumulation of citric acid and PAs. Overall, this study demonstrates that CitTRL acts as a repressor of the accumulation of citric acid, PAs, and anthocyanins by a cross-regulation mechanism. Our results provide an opportunity to simultaneously manipulate these key traits as a means to produce citrus fruits that are both visually and organoleptically appealing. [ABSTRACT FROM AUTHOR]

Key message: CitWRKY28 and CitNAC029 are involved in cuticular wax synthesis as indicated by the comparative analysis of fruit aliphatic wax content between Citrus reticulata and Citrus trifoliata and gene co-expression analysis. Cuticular wax covers the fruit surface, playing important roles in reduction of fruit water loss and resistance to pathogen invasion. However, there is limited research on the synthesis and transcriptional regulation of cuticular wax in citrus fruit. In this study, we characterized the variations of aliphatic wax in HJ (Citrus reticulata) and ZK (Citrus trifoliata) from young fruit to mature fruit, as well as performed transcriptome sequencing on 27 samples at different fruit developmental stages. The results revealed that the ZK fruit always had a higher aliphatic wax content than the HJ fruit during development. qRT-PCR analysis demonstrated that two KCS genes, CitKCS1 and CitKCS12, had the most significant difference in expression between HJ and ZK. Furthermore, a heterologous expression assay in Arabidopsis indicated that CitKCS1 and CitKCS12 are involved in cuticular wax synthesis. Subsequently, gene co-expression network analysis screened CitWRKY28 and CitNAC029. Dual luciferase and EMSA assays indicated that CitWRKY28 might bind to the promoter of CitKCS1 and CitKCS12 and CitNAC029 might bind to that of CitKCS1 to activate their expression. Moreover, CitWRKY28 and CitNAC029 could promote the accumulation of cuticular wax in Arabidopsis leaves. Our findings provide new insights into the synthesis and regulation of cuticular wax and valuable information for further mining of wax-related genes in citrus fruit. [ABSTRACT FROM AUTHOR]

A Citrus sinensis R2R3 MYB transcription factor (CsMYB96) has previously been shown to be strongly associated with the expression of many genes related to wax biosynthesis in the fruit. In this study, CsMYB96 was found to alleviate water loss by simultaneously regulating the expression of genes encoding plasma membrane intrinsic proteins (CsPIP s) and wax-related genes. Expression profiling indicated that CsPIP1;1 and CsPIP2;4 had high expression that was representative of other aquaporins, and they were down-regulated in the peel of post-harvest citrus fruit. CsPIP2;4 was further characterized as the predominant CsPIP, with high expression and high-water channel activity. Transient overexpression of CsPIP2;4 accelerated water loss in citrus fruit. In silico analysis further indicated that the expression of CsMYB96 had a significant negative correlation with that of CsPIP s. In vivo and in vitro experiments confirmed that CsMYB96 was able to directly repress the expression of CsPIP s. In addition, CsMYB96 was able to activate wax-related genes and promote wax biosynthesis for defense against water loss. Transient and stable overexpression of CsMYB96 reduced water loss from both citrus fruit and Arabidopsis. [ABSTRACT FROM AUTHOR]

Carotenoids play vital roles in the coloration of plant tissues and organs, particularly fruits; however, the regulation of carotenoid metabolism in fruits during ripening is largely unknown. Here, we show that red light promotes fruit coloration by inducing accelerated degreening and carotenoid accumulation in kumquat fruits. Transcriptome profiling revealed that a NAC (NAM/ATAF/CUC2) family transcription factor, FcrNAC22, is specifically induced in red light-irradiated fruits. FcrNAC22 localizes to the nucleus, and its gene expression is up-regulated as fruits change color. Results from dual luciferase, yeast one-hybrid assays and electrophoretic mobility shift assays indicate that FcrNAC22 directly binds to, and activates the promoters of three genes encoding key enzymes in the carotenoid metabolic pathway. Moreover, FcrNAC22 overexpression in citrus and tomato fruits as well as in citrus callus enhances expression of most carotenoid biosynthetic genes, accelerates plastid conversion into chromoplasts, and promotes color change. Knock down of FcrNAC22 expression in transiently transformed citrus fruits attenuates fruit coloration induced by red light. Taken together, our results demonstrate that FcrNAC22 is an important transcription factor that mediates red light-induced fruit coloration via up-regulation of carotenoid metabolism. [ABSTRACT FROM AUTHOR]

Carotenoids in citrus contribute to the quality of the fruit, but the mechanism of its transcriptional regulation is fairly unknown. Here, we characterized a citrus FRUITFULL sub-clade MADS gene, CsMADS5 , that was ripening-inducible and acted as a nucleus-localized trans-activator. Transient overexpression of CsMADS5 in citrus induced fruit coloration and enhanced carotenoid concentrations. The expression of carotenogenic genes including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene β-cyclase 1 (LCYb1) was increased in the peels of fruits overexpressing CsMADS5. Similar results were observed from stable overexpression of CsMADS5 in tomato fruits and citrus calli, even though the effect of CsMADS5 on carotenoid metabolism in transgenic citrus calli was limited. Further biochemical analyses demonstrated that CsMADS5 activated the transcription of PSY , PDS , and LCYb1 by directly binding to their promoters. We concluded that CsMADS5 positively regulates carotenoid biosynthesis in fruits by directly activating the transcription of carotenogenic genes. Moreover, CsMADS5 physically interacted with a positive regulator CsMADS6, indicating that CsMADS5 may form an enhancer complex with CsMADS6 to synergistically promote carotenoid accumulation. These findings expand our understanding of the complex transcriptional regulatory hierarchy of carotenoid biosynthesis during fruit ripening. [ABSTRACT FROM AUTHOR]

Mitochondria are crucial for the production of primary and secondary metabolites, which largely determine the quality of fruit. However, a method for isolating high-quality mitochondria is currently not available in citrus fruit, preventing high-throughput characterization of mitochondrial functions. Here, based on differential and discontinuous Percoll density gradient centrifugation, we devised a universal protocol for isolating mitochondria from the pulp of four major citrus species, including satsuma mandarin, ponkan mandarin, sweet orange, and pummelo. Western blot analysis and microscopy confirmed the high purity and intactness of the isolated mitochondria. By using this protocol coupled with a label-free proteomic approach, a total of 3353 nonredundant proteins were identified. Comparison of the four mitochondrial proteomes revealed that the proteins commonly detected in all proteomes participate in several typical metabolic pathways (such as tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation) and pathways closely related to fruit quality (such as γ-aminobutyric acid (GABA) shunt, ascorbate metabolism, and biosynthesis of secondary metabolites). In addition, differentially abundant proteins (DAPs) between different types of species were also identified; these were found to be mainly involved in fatty acid and amino acid metabolism and were further confirmed to be localized to the mitochondria by subcellular localization analysis. In summary, the proposed protocol for the isolation of highly pure mitochondria from different citrus fruits may be used to obtain high-coverage mitochondrial proteomes, which can help to establish the association between mitochondrial metabolism and fruit storability or quality characteristics of different species and lay the foundation for discovering novel functions of mitochondria in plants. [ABSTRACT FROM AUTHOR]

Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the positive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demonstrated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5. [ABSTRACT FROM AUTHOR]

Proanthocyanidins (PAs; or condensed tannins) are a major class of flavonoids that contribute to citrus fruit quality. However, the molecular mechanism responsible for PA biosynthesis and accumulation in citrus remains unclear. Here, we identify a PH4–Noemi regulatory complex that regulates proanthocyanidin biosynthesis in citrus. Overexpression of PH4 or Noemi in citrus calli activated the expression of PA biosynthetic genes and significantly increased the PA content. Interestingly, Noemi was also shown to be up-regulated in CsPH4 -overexpressing lines compared with wild-type calli. Simultaneously, CsPH4 partially complemented the PA-deficient phenotype of the Arabidopsis tt2 mutant and promoted PA accumulation in the wild-type. Further analysis revealed that CsPH4 interacted with Noemi, and together these proteins synergistically activated the expression of PA biosynthetic genes by directly binding to the MYB-recognizing elements (MRE) of the promoters of these genes. Moreover, CsPH4 could directly bind to the promoter of Noemi and up-regulate the expression of this gene. These findings explain how the CsPH4–Noemi regulatory complex contributes to the activation of PA biosynthetic genes via a positive feedback loop and provide new insights into the molecular mechanisms underlying PA biosynthesis, which can be effectively employed for metabolic engineering to improve citrus fruit quality. [ABSTRACT FROM AUTHOR]

Background: Citrus fruits are consumed freshly or as juice to directly provide various dietary flavonoids to humans. Diverse metabolites are present among Citrus genera, and many flavonoids biosynthetic genes were induced after abiotic stresses. To better understand the underlying mechanism, we designed experiments to overexpress a UDP-GLUCOSYL TRANSFERASE gene from sweet orange (Citrus sinensis) to evaluate its possible function in metabolism and response to stress. Results: Our results demonstrated that overexpression of Cs-UGT78D3 resulted in high accumulation of proanthocyanidins in the seed coat and a dark brown color to transgenic Arabidopsis seeds. In addition, the total contents of flavonoid and anthocyanin were significantly enhanced in the leaves of overexpressed lines. Gene expression analyses indicated that many flavonoid (flavonol) and anthocyanin genes were up-regulated by 4–15 folds in transgenic Arabidopsis. Moreover, after 14 days of high light stress, the transgenic Arabidopsis lines showed strong antioxidant activity and higher total contents of anthocyanins and flavonoids in leaves compared with the wild type. Conclusion: Our study concluded that the citrus Cs-UGT78D3 gene contributes to proanthocyanidins accumulation in seed coats and confers tolerance to high light stress by accumulating the total anthocyanin and flavonoid contents with better antioxidant potential (due to photoprotective activity of anthocyanin) in the transgenic Arabidopsis. [ABSTRACT FROM AUTHOR]

Neohesperidosides are disaccharides that are present in some flavonoids and impart a bitter taste, which can significantly affect the commercial value of citrus fruits. In this study, we identified three flavonoid-7- O - di -glucosyltransferase (dGlcT) genes closely related to 1,2-rhamnosyltransferase (1,2RhaT) in citrus genomes. However, only 1,2RhaT was directly linked to the accumulation of neohesperidoside, as demonstrated by association analysis of 50 accessions and co-segregation analysis of an F1 population derived from Citrus reticulata × Poncirus trifoliata. In transgenic tobacco BY2 cells, over-expression of CitdGlcT s resulted in flavonoid-7- O -glucosides being catalysed into bitterless flavonoid-7- O - di -glucosides, whereas over-expression of Cit1,2RhaT converted the same substrate into bitter-tasting flavonoid-7- O -neohesperidoside. Unlike 1,2RhaT, during citrus fruit development the dGlcT s showed an opposite expression pattern to CHS and CHI, two genes encoding rate-limiting enzymes of flavonoid biosynthesis. An uncoupled availability of dGlcTs and substrates might result in trace accumulation of flavonoid-7- O - di -glucosides in the fruit of C. maxima (pummelo). Past human selection of the deletion and functional mutation of 1,2RhaT has led step-by-step to the evolution of the flavor-related metabolic network in citrus. Our research provides the basis for potentially improving the taste in citrus fruit through manipulation of the network by knocking-out 1,2RhaT or by enhancing the expression of dGlcT using genetic transformation. [ABSTRACT FROM AUTHOR]

2,4-D retards senescence of postharvest citrus fruits by increasing exogenous auxin, endogenous ABA and SA contents, while decreasing ethylene production; and enhancing stress-defense capability through changing epicuticular wax morphology and lignin content in peelAuxin-like 2,4-dichlorophenoxyacetic acid (2,4-D), a high-efficiency anti-stalling agent for the post-harvest fresh fruit industry, has had its use restricted due to environmental concerns. However, no other substitutes for 2,4-D are available to the post-harvest industry. Insights into the molecular mechanism underlying the effects of 2,4-D on fruit quality preservation will provide a theoretical basis for exploring new safe and effective anti-stalling agents. This study comprehensively analysed changes in the peel of Olinda Valencia orange [Citrus sinensis (L.) Osbeck] induced by 500 ppm 2,4-D using ‘omic’-driven approaches. Transcriptional profiling revealed that transcriptional factor (mainly AP2/ERF, WRKY, and NAC family members), transport, and hormone metabolism genes were over-represented and up-regulated within 24h post-treatment (HPT). Stress defence genes were up-regulated, while cell wall metabolism genes were down-regulated after 48 HPT. However, secondary metabolism genes, especially phenylpropanoid and lignin biosynthesis-related genes, were over-represented at all the time points. Comparative proteomic analysis indicated that the expression of proteins implicated in stress responses (25%), hormone metabolism, and signal transduction (12%) significantly accumulated at the post-transcriptional level. Hormone levels detected by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) showed that abscisic acid, salicylic acid, and 2,4-D significantly increased, while ethylene production (detected by gas chromatography) decreased after 2,4-D treatment. In addition, lignin and water content in the fruit peel also increased and the epicuticle wax ultrastructure was modified. In conclusion, 2,4-D retarded fruit senescence by altering the levels of many endogenous hormones and by improving stress defence capabilities by up-regulating defence-related genes and proteins. [ABSTRACT FROM PUBLISHER]

Citric acid plays an important role in fresh fruit flavor and its adaptability to post-harvest storage conditions. In order to explore organic acid regulatory mechanisms in post-harvest citrus fruit, systematic biological analyses were conducted on stored Hirado Buntan Pummelo (HBP; Citrus grandis) fruits. High-performance capillary electrophoresis, subcellular organelle expression microarray, real-time quantitative reverse transcription polymerase chain reaction, gas chromatography mass spectrometry (GC-MS), and conventional physiological and biochemical analyses were undertaken. The results showed that the concentration of organic acids in HBP underwent a regular fluctuation. GC-MS-based metabolic profiling indicated that succinic acid, γ-aminobutyric acid (GABA), and glutamine contents increased, but 2-oxoglutaric acid content declined, which further confirmed that the GABA shunt may have some regulatory roles in organic acid catabolism processes. In addition, the concentration of organic acids was significantly correlated with senescence-related physiological processes, such as hydrogen peroxide content as well as superoxide dismutase and peroxidase activities, which showed that organic acids could be regarded as important parameters for measuring citrus fruit post-harvest senescence processes. [ABSTRACT FROM AUTHOR]

Fruit quality is a very complex trait that is affected by both genetic and non-genetic factors. Generally, low temperature (LT) is used to delay fruit senescence and maintain fruit quality during post-harvest storage but the molecular mechanisms involved are poorly understood. Hirado Buntan Pummelo (HBP; Citrus grandis × C. paradis) fruit were chosen to explore the mechanisms that maintain citrus fruit quality during lengthy LT storage using transcriptome and proteome studies based on digital gene expression (DGE) profiling and two-dimensional gel electrophoresis (2-DE), respectively. Results showed that LT up-regulated stress-responsive genes, arrested signal transduction, and inhibited primary metabolism, secondary metabolism and the transportation of metabolites. Calcineurin B-like protein (CBL)–CBL-interacting protein kinase complexes might be involved in the signal transduction of LT stress, and fruit quality is likely to be regulated by sugar-mediated auxin and abscisic acid (ABA) signalling. Furthermore, ABA was specific to the regulation of citrus fruit senescence and was not involved in the LT stress response. In addition, the accumulation of limonin, nomilin, methanol, and aldehyde, together with the up-regulated heat shock proteins, COR15, and cold response-related genes, provided a comprehensive proteomics and transcriptomics view on the coordination of fruit LT stress responses. [ABSTRACT FROM PUBLISHER]

ABSTRACT Ethylene treatment promotes orange coloration in the flavedo of Satsuma mandarin (Citrus unshiu Marc.) fruit, but the corresponding regulatory mechanism is still largely unknown. In this study, we identified a C2H2‐type zinc‐finger transcription factor, CitZAT4, the expression of which was markedly induced by ethylene. CitZAT4 directly binds to the CitPSY promoter and activates its expression, thereby promoting carotenoid biosynthesis. Transient expression in Satsuma mandarin fruit and stable transformation of citrus calli showed that overexpressing of CitZAT4 inhibited CitLCYE expression, thus inhibiting α‐branch yellow carotenoid (lutein) biosynthesis. CitZAT4 overexpression also enhanced the transcript levels of CitLCYB, CitHYD, and CitNCED2, promoting β‐branch orange carotenoid accumulation. Molecular biochemical assays, including yeast one‐hybrid (Y1H), electrophoretic mobility shift (EMSA), chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP‐qPCR), and luciferase (LUC) assays, demonstrated that CitZAT4 directly binds to the promoters of its target genes and regulates their expression. An ethylene response factor, CitERF061, which is induced by ethylene signaling, was found to directly bound to the CitZAT4 promoter and induced its expression, thus positively regulating CitZAT4‐mediated orange coloration in citrus fruit. Together, our findings reveal that a CitZAT4‐mediated transcriptional cascade is driven by ethylene via CitERF061, linking ethylene signaling to carotenoid metabolism in promoting orange coloration in the flavedo of Satsuma mandarin fruit. The molecular regulatory mechanism revealed here represents a significant step toward developing strategies for improving the quality and economic efficiency of citrus crops. [ABSTRACT FROM AUTHOR]

Carotenoids are natural pigments that influence the color of citrus fruit. The red-colored carotenoid β-citraurin is responsible for the peel color in "Newhall" orange (Citrus sinensis). Although jasmonates are known to regulate the biosynthesis and accumulation of carotenoids, their effects on β-citraurin biosynthesis in citrus fruit remain unclear. Here, we determined that treatment with methyl jasmonate (MeJA) significantly promotes fruit coloration and β-citraurin production in "Newhall" orange. A MeJA treatment induced the expression of CsMYC2 , which encodes a transcription factor that serves as a master regulator of jasmonate responses. CsMYC2 bound the promoter of the gene that encodes carotenoid cleavage dioxygenase 4b (CsCCD4b), the key gene for β-citraurin biosynthesis, and the promoters of genes that encode phytoene synthase (CsPSY), lycopene β-cyclase (CsLCYb), and β-carotene hydroxylase (CsBCH) and induced their expression. In addition, CsMYC2 promoted CsMPK6 expression. Notably, we found that CsMPK6 interacted with CsMYC2 and that this interaction decreased the stability and DNA-binding activity of CsMYC2. Thus, we conclude that negative feedback regulation attenuates JA signaling during the jasmonate-induced coloration of citrus fruit. Together, our findings indicate that jasmonates induce β-citraurin biosynthesis in citrus by activating a CsMPK6–CsMYC2 cascade, thereby affecting fruit coloration. [ABSTRACT FROM AUTHOR]

Uneven coloration is a common phenomenon in citrus fruit during the ripening stage, as affects the appearance and economic value of the fruit. The elevated expression of CsERF003 during the degreening process of both lemon and satsuma mandarin peels was reported. In this research, a similar performance of CsERF003 in the pericarp coloration process was also identified by transcriptome analysis of 'Fengjie 72–1' navel orange and Lane Late navel orange. However, the regulatory mechanism of CsERF003 is not clear yet. Overexpression of CsERF003 could deepen the color of citrus callus and promote peel degreening of Newhall navel orange, which was attributed to the upregulation of genes involved in chlorophyll degradation and carotenoid synthesis. Furthermore, CsERF003 acted as an activator to promote the expression of CsLCYE , but couldn't activate the expression of CsLCYB1 and CsLCYB2 ; CsERF003 could also bind to the promoter of CsSGR to activate its expression. Together, our findings shed light on the regulatory mechanism of CsERF003 in chlorophyll degradation and carotenoid accumulation, particularly in the α-branch of carotenoid metabolism. These insights offer valuable perspectives for the genetic enhancement of peel coloration in citrus. [Display omitted] • Overexpression of CsERF003 deepened the color of citrus callus and promoted peel degreening in Newhall navel orange. • CsERF003 activated the expression of CsLCYE and promoted the accumulation of lutein in the α-branch. • CsERF003 activated the expression of CsSGR and promoted the degradation of chlorophyll in the pericarp. [ABSTRACT FROM AUTHOR]

As is well known, postharvest abscisic acid (ABA) treatment of citrus fruits can significantly enhance fruit coloration by promoting the accumulation of carotenoids, however, the underlying molecular mechanism is still unclear. In this study, we further confirmed the effect of ABA treatment in promoting citrus fruit coloration by activating the expression of 4 key carotenoid metabolism genes (CsPSY , CsLCYB , CsHYD , and CsNCED2) during postharvest storage. A novel basic helix-loop-helix (bHLH) transcription factor, CsbHLH6, was identified using the promoters of 3 key ABA-induced carotenoid metabolism genes (CsLCYB , CsHYD , and CsNCED2) by yeast one-hybrid screening. Overexpression of CsbHLH6 enhanced carotenoid accumulation in citrus fruits and calli. Interference with CsbHLH6 expression in citrus calli and fruits indicated that CsbHLH6 was essential for ABA-induced carotenoid biosynthesis and fruit coloration. As a nucleus-localized transcriptional activator, CsbHLH6 directly bound to the promoters of 6 key carotenoid metabolism genes (CsPSY , CsLCYB , CsLCYE , CsHYD , CsZEP , and CsNCED2), subsequently activating their expression in vitro and in vivo. Furthermore, ABA signaling significantly raised CsbHLH6 promoter activity, thus enhancing the regulation of carotenoid accumulation by CsbHLH6. In summary, our study reveals the molecular mechanism by which CsbHLH6 regulates ABA-induced citrus fruit coloration during postharvest storage, which is crucial for improving the postharvest appearance quality of citrus fruits. • Abscisic acid (ABA) treatment promotes fruit coloration during postharvest storage. • CsbHLH6 plays an essential role in postharvest ABA-mediated fruit coloration. • CsbHLH6 activates the expression of 6 key ABA-induced carotenoid biosynthesis genes. • ABA signaling greatly enhances the regulation of carotenoid accumulation by CsbHLH6. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is one of the essential macro-elements for plants. Sugar and organic acid are important factors affecting sensory characteristics of citrus fruit quality. The aim of this study was to investigate how P fertilizer affects quality improvement particularly sucrose (Suc), fructose (Fru), glucose (Glu) and citric acid (CA) accumulations in Cara Cara navel. P fertilizer improved fruit quality of Cara Cara navel, as supported by decreasing titratable acid (TA), CA and increasing soluble solid (TSS), sugars and the ratio of TSS and TA. At the early stage of fruit development, P fertilizer had greater roles in degrading Suc into Fru and Glu due to the increased activities of Suc-degrading enzymes including acid invertase, neutral invertase and Suc synthase-cleavage activity. Coversely, at the mid and late stages of fruit development, P fertilizer had greater roles in re-synthesizing Suc due to the increased activities of Suc-synthesizing enzymes including Suc phosphate synthase and Suc synthase-synthetic activity. These results indicated that application of P fertilizer increased soluble sugars concentrations by improving Suc metabolism and sink strength in fruit conferred by the upregulations of the activities of Suc-degrading and Suc-synthesizing enzymes. P fertilizer decreased CA accumulations at least partially by inhibiting synthesis of CA due to the decreased activities of CA-synthesizing enzymes including citrate synthetase and phosphoenolpyruvate carboxylase. This study suggested that P fertilizer, particularly fertilized with 0.40 kg/plant, increased soluble sugars but decreased CA accumulations in citrus fruit. • Phosphorus (P) fertilizer improved fruit quality of Cara Cara navel. • P enhanced sugar accumulations by improving sucrose metabolism and sink strength. • P fertilizer reduced citric acid accumulations partially by inhibiting its synthesis. [ABSTRACT FROM AUTHOR]

• A navel orange mutant 'Gannan No.1' has higher biomembrane stability and fruit quality during storage. • Higher unsaturation degree of fatty acids contributes to the membrane stability of citrus fruit. • The ascorbic acid-glutathione-tocopherol triad contributes to the anti-peroxidation of membrane lipids in citrus fruit. Little is known about the variations of fresh fruit biomembrane and its physiological and biochemical characteristics during storage. A navel orange mutant 'Gannan No.1' (Citrus sinensis Osbeck) showed higher membrane stability and titratable acid while lower calyx senescence compared with wild-type 'Newhall'. The membrane damage was significantly reduced in 'Gannan No.1' under 10% polyethylene-glycol (41.16% vs. 8.77%) and 30% polyethylene-glycol (52.59% vs. 16.11%) treatments on day 45 after harvest. Consistently, membrane electrolyte leakage and malondialdehyde were significantly decreased in 'Gannan No.1', and superoxide dismutase and glutathione reductase were activated. A metabolic analysis was performed to evaluate membrane fatty acid unsaturation and peroxidation. Linolenic acid and hexadecylenic acid contributed to the higher degree of unsaturated fatty acids in 'Gannan No.1'. Furthermore, 'Gannan No.1' accumulated stress-resistant metabolites such as proline, α-tocopherol and glutathione. Correlation analysis of membrane homeostasis indexes with quality parameters showed the importance of biomembrane stability in maintaining citrus fruit quality. [ABSTRACT FROM AUTHOR]

The loss of organic acids during postharvest storage is one of the major factors that reduces the fruit quality and economic value of citrus. Citrate is the most important organic acid in citrus fruits. Molecular evidence has proved that γ-aminobutyric acid (GABA) shunt plays a key role in citrate metabolism. Here, we investigated the effects of exogenous GABA treatment on citrate metabolism and storage quality of postharvest citrus fruit. The content of citrate was significantly increased, which was primarily attributed to the inhibition of the expression of glutamate decarboxylase (GAD). Amino acids, including glutamate, alanine, serine, aspartate and proline, were also increased. Moreover, GABA treatment decreased the fruit rot rate. The activities of antioxidant enzymes and the content of energy source ATP were affected by the treatment. Our results indicate that GABA treatment is a very effective approach for postharvest quality maintenance and improvement of storage performance in citrus production. [ABSTRACT FROM AUTHOR]

• Gene co-expression analysis showed that blue module is related to fruit wax synthesis. • CsERF003, CsMYB7 and CsMYB102 in the blue module can activate CsKCS2 expression. • CsERF003, CsMYB7 and CsMYB102 promote cuticular wax synthesis in Arabidopsis leaves. Cuticular wax is a hydrophobic protective layer covering the surface of citrus fruit, which plays an important role in preventing fruit water loss and improving fruit appearance quality. Our previous study has demonstrated that CsKCS2 is highly expressed at fruit ripening and involved in fruit cuticular wax synthesis in Citrus sinensis. However, the upstream regulators of CsKCS2 and the transcription factors regulating fruit cuticular wax synthesis at ripening remain unclear. Here, we performed a gene co-expression analysis with the published transcriptomic data of five developmental stages of Citrus sinensis fruit. The results showed that the gene expression trend of the blue module was synchronized with the accumulation pattern of fruit cuticular wax during fruit development and ripening. Further dual luciferase assay suggested that the promoter of CsKCS2 could be activated by CsERF003, CsMYB7 and CsMYB102 in the blue module. Moreover, ectopic expression in Arabidopsis revealed that CsERF003, CsMYB7 and CsMYB102 could increase cuticular wax content in leaves by upregulating the expression of wax-related genes (such as KCS, CER1, CER3 and CER4). Our results provide new insights into the transcriptional regulation of fruit cuticular wax synthesis, and important information for the mining of regulatory factors involved in cuticular wax synthesis in Citrus sinensis fruit. [ABSTRACT FROM AUTHOR]

To elucidate the effects of exogenous brassinosteroids (BRs) treatment on the postharvest behavior of citrus fruit, Satsuma mandarin ( Citrus unshiu ) fruit were treated with 5 mg L −1 24-epibrassinolide (EBR) and stored at 12–16 °C with a relative humidity of 90–95%. After 50 d treatment, the disease incidence of the control and EBR-treated fruit was 18.7% and 4%, respectively, suggesting that EBR treatment can significantly enhance the postharvest biotic stress tolerance of Satsuma mandarins. Moreover, the fruit quality analysis revealed that EBR treatment induced a rapid weight loss during the first 6 d after treatment, which was similar to the effect of a conditioning treatment, while it exhibited no negative effects on inner quality. Further, we measured the contents of hydrogen peroxide (H 2 O 2 ) and the primary metabolites as well as the expression of the stress-related genes in the fruit pericarp. The results indicated that H 2 O 2 content was significantly increased by EBR treatment. In addition, analyses on the primary metabolites showed that some stress-related metabolites, such as ornithine, proline, GABA, d -xylose and d -galactose, were increased in the EBR-treated fruit. Expression of the stress-related genes was markedly up-regulated in EBR-treated fruit. These results suggest that the effects of EBR on reducing the postharvest disease incidence of citrus fruit may be associated with the accumulation of H 2 O 2 and stress-related metabolites and the induction of stress-related genes. [ABSTRACT FROM AUTHOR]

Fruit shape is an important trait for fruit appearance and commercial value. Diversity of fruit-shape has been utilized in the breeding of pummelo (Citrus maxima), a basic species in Citrus. However, little is known about genetic basis of fruit shape in citrus. In this study, we identified 16 OVATE family protein (OFP) genes in the pummelo genome. Phylogenetically, they were classified into three subfamilies, which was consistent with the classification of their Arabidopsis orthologs. Synteny analysis suggested that segment and tandem duplications were responsible for their expansion in pummelo. Expression pattern analysis of Citrus OFPs (CitOFP s) showed that CitOFP19 had significantly higher expression level in the ovaries of round pummelo than in those of pear-shaped pummelo. Heterologous overexpression of CitOFP19 in tomato resulted in pear-shaped ovary and fruit shape. Taken together, this study characterized OVATE gene family in Citrus genome and assessed the function of CitOFP19. • We summarized the distribution pattern of the fruit shape index in the natural pummelo populations. • We identified the OVATE gene family in Citrus and segment and tandem duplications responsible for their expansion. • Heterologous overexpression of CitOFP19 in tomato resulted in pear-shaped ovary and fruit shape, which provided the basis for investigating its function in Citrus. [ABSTRACT FROM AUTHOR]

Highlights: [•] More aldehydes and larger wax platelets were found in mature fruits of Newhall than Satsuma. [•] Lupeol and lupenone were identified for the first time in citrus fruit wax. [•] Epicuticular wax constitutes about 80% of total wax in Newhall. [•] CER3, CER6, CER1–3, MYB96 are key genes associated with the formation of wax in citrus fruit. [ABSTRACT FROM AUTHOR]

• The hypoxia-induced alterations in mitochondrial morphology and ultrastructure were characterized. • The mitochondrial proteomes of wax-treated and the control were compared. • Key hypoxia-responsive proteins were identified from mitochondrial proteome via WGCNA. Hypoxia frequently occurs in postharvest logistics, which greatly influences fruit storability. Here, we for the first time studied the dynamic variations of mitochondrial morphology in living citrus fruit cells, and revealed that waxing treatment-induced hypoxia strongly triggered mitochondrial fission and fragmentation. Correspondingly, hypoxia caused a decline in mitochondrial membrane potential and mobility. Besides, impairment of energetic and redox status was also found in waxed fruit. The proteomic changes of mitochondria after waxing treatment were also characterized. Using weighted gene co-expression network analysis (WGCNA), we identified 167 key hypoxia-responsive proteins, which were mainly involved in fatty acid, amino acid and organic acid metabolism. Metabolite analysis verified that waxing treatment promoted the accumulation of several hypoxic metabolites, such as ethanol, acetaldehyde, succinic acid and γ-aminobutyric acid (GABA). Taken together, our findings provide new insights into the cytological and proteomic responses of mitochondria to hypoxia during fruit storage. [ABSTRACT FROM AUTHOR]

• QTL3 was detected for fruit cuticular wax and postharvest water loss. • CER1-1 and CER1-3 were candidate genes in QTL3. • CER1-1 and CER1-3 were involved in alkane synthesis to reduce plant water loss. • Coating experiment verified the potential role of alkanes in reducing fruit water loss. Postharvest water loss causes fruit softening, wilting, and a decline in quality and commodity value. Cuticular wax is a key barrier against non-stomatal water loss and plays a crucial role in fruit quality maintenance. However, there has been limited research on the genetic basis of fruit wax and postharvest water loss. Here, we found that HJ (Citrus reticulata) and ZK (Poncirus trifoliata) fruit had significant differences in postharvest water loss and cuticular wax. HJ fruit had a lower wax content and a faster water loss, and the main aliphatic wax components were alkanes and aldehydes. By contrast, ZK fruit had a higher wax content and a slower water loss, and the main aliphatic wax components were only alkanes. Correlation analysis revealed that fruit water loss seemed to be correlated with cuticular wax alkane in the F 1 pseudo-testcross population of HJ and ZK. Furthermore, through high-density genetic map and bulk segregant analysis, alkanes and fruit water loss were co-localized to QTL3, resulting in the identification of CER1-1 and CER1-3 as the candidate genes. Heterologous overexpression in Arabidopsis revealed that they were involved in alkane synthesis and reduction of leaf water loss. In addition, coating with C28 alkane could reduce postharvest fruit water loss of six citrus varieties. Collectively, we speculated that cuticular wax alkanes might play an important role in limiting fruit water loss. The study provides new insights into the development of coating agents and breeding of citrus varieties with better storage performance. [ABSTRACT FROM AUTHOR]

Abstract: The precocious trifoliate orange (Poncirus trifoliata [L.] Raf.), an early flowering mutant of P. trifoliata, has a short juvenile phase of about 14 months, significantly shorter than other citrus. In this report, using the stems of precocious trifoliate orange seedlings as explants, an improved protocol mediated by Agrobacterium tumefaciens was developed to evaluate regeneration and transformation frequency. The transformed plants continued to reveal the precocious trait of non-transgenic mature precocious trifoliate orange, and as such could prove to be an efficient system for the verification of gene function in the fruits/flower via genetic transformation in woody plants. [Copyright &y& Elsevier]

Abstract: Pink or red-fleshed fruit mutations are commonly found in grapefruit, sweet orange, and occasionally in lemon, which combine novel appearance with fine eating quality. In order to identify the major coloured pigments, high performance liquid chromatography (HPLC) with ultraviolet detection was applied for the separation and characterization of carotenoids from five pink or red-fleshed citrus cultivars. As a result, both lycopene and β-carotene with similar HPLC profiles were detected in the five citrus cultivars, ‘Fengdu’ red-fleshed pomelo (Citrus grandis (L.) Osbeck), ‘Guanxi’ sweet pomelo red mutant, ‘Hirado Buntan’ pomelo, ‘Cara Cara Navel’ orange (C. sinensis (L.) Osbeck) and ‘Star Ruby’ grapefruit (C. paradisi Macf.). ‘Star Ruby’ contained the highest lycopene and β-carotene content in its flesh. However, although the significant correlation between the concentrations of lycopene and β-carotene was detected with a coefficient of 0.9692 (P <0.01), ratios of lycopene/β-carotene were different among the cultivars. Post-harvest biosynthesis verified that the flesh itself synthesized carotenoids rather than acquiring them via transport from other tissues. In addition, a product feedback regulation mechanism might be involved in the process of carotenoid biosynthesis. [Copyright &y& Elsevier]

• A novel biological postharvest treatment by staying with apple (SWA) is proposed. • SWA treatment promotes peel coloration and maintain fruit freshness. • SWA treatment has similar effects to ethylene treatment on citrus peel coloration. • SWA treatment significantly alleviates calyx senescence caused by ethylene treatment. A novel biological treatment of staying with commercially mature apple fruits was proposed to promote the peel coloration of 'Newhall' navel orange fruit (Citrus sinensis Osbeck), which could conduce to the formation of uniform orange red peel color. At 15 days after treatment (DAT), the staying with apple (SWA) treatment led to a nearly 4-fold increase in the total carotenoid content compared with the control. Specifically, the red color apocarotenoid β-citraurin showed the highest degree of enhancement (~6 fold) under SWA treatment. Simultaneously, the expression level of β-citraurin biosynthetic gene carotenoid cleavage dioxygenase 4b (CCD4b) was dramatically induced (about 35 folds). Furthermore, SWA had the same effect of promoting coloration and obviously alleviate calyx senescence of citrus fruit compared with ethylene treatment. In conclusion, SWA treatment is a potential environment-friendly and recyclable postharvest technology to improve peel color and maintain fruit freshness for citrus. [ABSTRACT FROM AUTHOR]

• Oleocellosisdecreased the primary metabolitesandincreased the secondary metabolites. • Oleocellosis accelerated flavedo lignification. • Oleocellosissignificantly up-regulated the genes involved in flavedolignin accumulation. • Oleocellosis disorder improved citrus fruitpathogen resistance. Oleocellosis, a physiological disorder that affects the appearance quality of citrus fruit, is usually accompanied by the lignification of fruit flavedo. However, the mechanism of lignin accumulation in citrus flavedo in response to oleocellosis remains largely unknown. Here, the molecular mechanism of flavedo lignification caused by oleocellosis in citrus was dissected by metabolome, histochemistry and transcriptome analysis of healthy and oleocellosis disorder green lemon (Citrus limon Burm. f. Eureka) fruit. The metabolome analysis showed a remarkable decrease in primary metabolite content while an increase in secondary metabolite content in oleocellosis-damaged flavedo. The histochemical analysis revealed that oleocellosis disorder accelerated flavedo lignification and resulted in lignin accumulation. The transcriptomic analysis showed that in oleocellosis-damaged flavedo, phenylpropanoid biosynthesis was significantly activated, and 79 differentially expressed genes (DEGs) related to lignin accumulation were identified, among which 65 (82.3 % of the total DEGs) were significantly up-regulated, particularly the PAL , C4H, CCR , F5H, POD and UGT72E genes. The results of penicillium inoculation showed that oleocellosis disorder could enhance fruit pathogen resistance. In conclusion, the occurrence of oleocellosis may enhance the pathogen resistance of citrus fruit by lignin accumulation. [ABSTRACT FROM AUTHOR]