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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]

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]

Key message: Here, we developed a reliable protocol for the fast and efficient gene-edited Anliu sweet orange plants production. The application of in vitro shoot grafting technology significantly reduced the growth cycle of transgenic seedlings, and the survival rate of cleft grafting was more than 90%. In addition, the mutation efficiency of the grafted geneedited sweet orange was significantly improved by short-term heat stress treatments. Thus, the combination strategy of grafting and heat stress treatments provided a reference for the fast and efficient multiplex gene editing of sweet orange. [ABSTRACT FROM AUTHOR]

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus industry worldwide. Most citrus cultivars such as sweet orange are susceptible to canker disease. Here, we utilized wild citrus to identify canker-resistant germplasms, and found that Atalantia buxifolia, a primitive (distant-wild) citrus, exhibited remarkable resistance to canker disease. Although the susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1) could also be induced in Atalantia after canker infection, the induction extent was far lower than that in sweet orange. In addition, three of amino acids encoded by transcription factor TFIIAγ in Atalantia (AbTFIIAγ) exhibited difference from those in sweet orange (CsTFIIAγ) which could stabilize the interaction between effector PthA4 and effector binding element (EBE) of LOB1 promoter. The mutation of AbTFIIAγ did not change its interaction with transcription factor binding motifs (TFBs). However, the AbTFIIAγ could hardly support the LOB1 expression induced by the PthA4. In addition, the activity of AbLOB1 promoter was significantly lower than that of CsLOB1 under the induction by PthA4. Our results demonstrate that natural variations of AbTFIIAγ and effector binding element (EBE) in the AbLOB1 promoter are crucial for the canker disease resistance of Atalantia. The natural mutations of AbTFIIAγ gene and AbLOB1 promoter in Atalantia provide candidate targets for improving the resistance to citrus canker disease. Author summary: It has been well documented that most citrus cultivars are susceptible to canker disease, while little is known about the resistance or susceptibility of primitive or wild citrus to canker disease. This study reveals that primitive citrus (Atalantia buxifolia) is highly resistant to citrus canker. Transcriptome data demonstrated that Atalantia had an active resistance response to the infection of Xcc, compared with susceptible sweet orange. Our results indicated that natural variations of AbTFIIAγ gene and AbLOB1 promoter contributed to the resistance. Hence, we propose that the natural mutations of AbTFIIAγ gene and AbLOB1 promoter could provide candidate targets for breeding canker resistant citrus. [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]

Summary: Long intergenic non‐coding RNAs (lincRNAs) play important roles in various biological processes in plants. However, little information is known about the evolutionary characteristics of lincRNAs among closely related plant species. Here, we present a large‐scale comparative study of lincRNA transcription patterns in nine citrus species. By strand‐specific RNA‐sequencing, we identified 18 075 lincRNAs (14 575 lincRNA loci) from 34 tissue samples. The results indicated that the evolution of lincRNA transcription is more rapid than that of mRNAs. In total, 82.8–97.6% of sweet orange (Citrus sinensis) lincRNA genes were shown to have homologous sequences in other citrus genomes. However, only 15.5–28.8% of these genes had transcribed homologous lincRNAs in these citrus species, presenting a strong contrast to the high conservation of mRNA transcription (81.6–84.7%). Moreover, primitive and modern citrus lincRNAs were preferentially expressed in reproductive and vegetative organs, respectively. Evolutionarily conserved lincRNAs showed higher expression levels and lower tissue specificity than species‐specific lincRNAs. Notably, we observed a similar tissue expression pattern of homologous lincRNAs in sweet orange and pummelo (Citrus grandis), suggesting that these lincRNAs may be functionally conserved and selectively maintained. We also identified and validated a lincRNA with the highest expression in fruit that acts as an endogenous target mimic (eTM) of csi‐miR166c, and two lincRNAs that act as a precursor and target of csi‐miR166c, respectively. These lincRNAs together with csi‐miR166c could form an eTM166‐miR166c‐targeted lincRNA regulatory network that possibly affects citrus fruit development. Significance Statement: Here, 18 075 lincRNAs have been identified from 34 tissue samples from nine citrus species. The data uncover the rapid turnover of lincRNA transcription among different citrus species and highlights the potential contribution of the eTM166‐miR166‐targeted lincRNA regulatory network to fruit development. [ABSTRACT FROM AUTHOR]

In plant, a few 22-nt mi RNAs direct cleavages of their targets and trigger the biogenesis of phased small interfering RNAs (phasi RNAs) in plant. In this study, we characterized a mi RNA triggering phasi RNAs generation, miR3954, and explored its downstream target genes and potential function. Our results demonstrated that miR3954 showed specific expression in the flowers of citrus species, and it targeted a NAC transcription factor ( Cs7 g22460) and two non-coding RNA transcripts (lnc RNAs, Cs1 g09600 and Cs1 g09635). The production of phasi RNAs was detected from transcripts targeted by miR3954, and was further verified in both sequencing data and transient expression experiments. Phasi RNAs derived from the two lnc RNAs targeted not only miR3954-targeted NAC gene but also additional NAC homologous genes. No homologous genes of these two lnc RNAs were found in plants other than citrus species, implying that this miR3954-lnc RNAs-phasi RNAs- NAC pathway is likely citrus-specific. Transgenic analysis indicated that the miR3954-overexpressing lines showed decreased transcripts of lnc RNA, elevated abundance of phasi RNAs and reduced expression of NAC genes. Interestingly, the overexpression of miR3954 leads to early flowering in citrus plants. In summary, our results illustrated a model of the regulatory network of miR3954-lnc RNA-phasi RNAs- NAC, which may be functionally involved in flowering in citrus. [ABSTRACT FROM AUTHOR]

S-RNase-based self-incompatibility is found in Solanaceae, Rosaceae, and Scrophulariaceae, and is the most widespread mechanism that prevents self-fertilization in plants. Although 'Shatian' pummelo ( Citrus grandis), a traditional cultivated variety, possesses the self-incompatible trait, the role of S-RNases in the self-incompatibility of 'Shatian' pummelo is poorly understood. To identify genes associated with self-incompatibility in citrus, we identified 16 genes encoding homologs of ribonucleases in the genomes of sweet orange ( Citrus sinensis) and clementine mandarin ( Citrus clementine). We preliminarily distinguished S-RNases from S-like RNases with a phylogenetic analysis that classified these homologs into three groups, which is consistent with the previous reports. Expression analysis provided evidence that CsRNS1 and CsRNS6 are S-like RNase genes. The expression level of CsRNS1 was increased during fruit development. The expression of CsRNS6 was increased during the formation of embryogenic callus. In contrast, we found that CsRNS3 possessed several common characteristics of the pistil determinant of self-incompatibility: it has an alkaline isoelectric point (pI), harbors only one intron, and is specifically expressed in style. We obtained a cDNA encoding CgRNS3 from 'Shatian' pummelo and found that it is homolog to CsRNS3 and that CgRNS3 exhibited the same expression pattern as CsRNS3. In an in vitro culture system, the CgRNS3 protein significantly inhibited the growth of self-pollen tubes from 'Shatian' pummelo, but after a heat treatment, this protein did not significantly inhibit the elongation of self- or non-self-pollen tubes. In conclusion, an S-RNase gene, CgRNS3, was obtained by searching the genomes of sweet orange and clementine for genes exhibiting sequence similarity to ribonucleases followed by expression analyses. Using this approach, we identified a protein that significantly inhibited the growth of self-pollen tubes, which is the defining property of an S-RNase. [ABSTRACT FROM AUTHOR]

DNA methylation is a type of epigenetic modification involved in many biological processes. Recent studies have shown that DNA methylation plays important roles in regulating fruit development and ripening. In the present study, sweet oranges at six developmental stages were investigated to understand the possible roles of DNA methylation during fruit development and ripening. Analysis of genomic DNA methylation levels in different fruit tissues and developmental stages indicated that methylation levels were not static during fruit development and ripening. To explore the possible contributions of methylation-related genes in fruit development and ripening process, the full-length coding sequences corresponding to three types of DNA methyltransferase families ( CsMET1, CsCMT3, and CsDRM1), a chromatin-remodeling gene ( CsDDM1), and a demethylation gene ( CsDME1) were isolated. The expression profiles of these methylation-related genes were analyzed in vegetative tissues (seedlings and leaves) and reproductive tissues (three stages of flowering and six stages of fruit development). The results showed that CsDRM1 was highly expressed in seedlings, leaves, and flowers compared with other genes. During fruit development and ripening, CsMET1, CsCMT1, CsCMT2, CsDRM1, and CsDMEs showed higher expression in the peel than in the flesh. However, global DNA methylation level changes could not be explained by the expression of a single gene, suggesting that the methylation and demethylation system may cooperatively contribute to the final global DNA methylation pattern. [ABSTRACT FROM AUTHOR]

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community. [ABSTRACT FROM AUTHOR]

The direct analysis in real-time (DART) ion source and HPLC-electrospray mass spectrometry were applied in non-targeted metabolomic analyses of fruits of an orange bud mutant, 'Hong Anliu' along with its parental wild-type, 'Anliu'. Fruits of the two isogenic cultivars were sampled at three different ripening stages, i.e. 120, 170 and 220 days after flowering. More than 130 metabolites were tentatively identified, including acids, sugars, flavonoids, alkaloids, limonoids, coumarins, amino acids, and plant hormones. Metabolomic analyses revealed that, compared to its wild type, the bud mutant fruit is characterized by higher levels of monosaccharides and disaccharides and lower levels of organic acids such as citric acid, malic acid and quinic acid, which agrees well with the anticipated fruit quality benefits of the mutation. In addition, many secondary metabolites, such as flavonoids, showed significant differences between the two genotypes, indicating that the whole fruit metabolome is significantly changed due to the bud mutation. This study provided a comprehensive assessment of metabolites in orange fruits, and revealed metabolomic differences in fruits between two isogenic orange genotypes. The results are helpful for understanding how the bud mutation in 'Hong Anliu' impacts the physiological and biochemical processes of orange fruits. [ABSTRACT FROM AUTHOR]

ABA, ethylene, sucrose, and their related genes and pathways are involved in fruit ripening of citrus, and ABA may play a central role during the ripening process.Fruit ripening is a complex, genetically programmed process that occurs in conjunction with the differentiation of chloroplasts into chromoplasts and involves changes to the organoleptic properties of the fruit. In this study, an integrative analysis of the transcriptome and proteome was performed to identify important regulators and pathways involved in fruit ripening in a spontaneous late-ripening mutant (‘Fengwan’ orange, Citrus sinensis L. Osbeck) and its wild type (‘Fengjie 72-1’). At the transcript level, 628 genes showed a 2-fold or more expression difference between the mutant and wild type as detected by an RNA sequencing approach. At the protein level, 130 proteins differed by 1.5-fold or more in their relative abundance, as indicated by iTRAQ (isobaric tags for relative and absolute quantitation) analysis. A comparison of the transcriptome and proteome data revealed some aspects of the regulation of metabolism during orange fruit ripening. First, a large number of differential genes were found to belong to the plant hormone pathways and cell-wall-related metabolism. Secondly, we noted a correlation between ripening-associated transcripts and sugar metabolites, which suggests the importance of these metabolic pathways during fruit ripening. Thirdly, a number of genes showed inconsistency between the transcript and protein level, which is indicative of post-transcriptional events. These results reveal multiple ripening-associated events during citrus ripening and provide new insights into the molecular mechanisms underlying citrus ripening regulatory networks. [ABSTRACT FROM AUTHOR]

The phytoene synthase ( PSY) gene product is involved in the carotenoid biosynthesis pathway. In this study, the promoter of the sweet orange gene CsPSY of sweet orange 'Cara Cara' ( Citrus sinensis Osbeck [ Cs]) was isolated and characterized. Its sequence included a number of regulatory elements predicted to be responsive to light and other environmental cues. The 926-bp region upstream of CsPSY was fused to the β-glucuronidase ( GUS) reporter gene, and introduced as a transgene into Arabidopsis thaliana. The pattern of GUS expression in the transgenic plants showed that the CsPSY promoter drove expression in the young seedlings, the leaves, the roots and in parts of the flower, in a developmentally regulated fashion. A promoter deletion analysis revealed that the region −479 to −306 nt positively regulated expression, the region −306 to −93 nt was associated with negative regulation of expression and the region −46 to +21 nt maintained basal promoter activity. In the presence of sucrose, gene expression was regulated via a cis-acting promoter element(s). Two highly repetitive sequences (Sp1 and A-box), located in the region from −479 to −306 nt, may act as light-responsive elements regulating CsPSY transcription during photomorphogenesis. [ABSTRACT FROM AUTHOR]

Distinguishing a bud mutant from its parental genotype using conventional molecular markers is extremely difficult because they are thought to be genetically near-identical owing to the nature of somatic mutations that give rise to the bud mutant phenotype. In this study, array-comparative genome hybridization (array-CGH) was used to investigate the genetic variation between a sweet orange bud mutant 'Hong Anliu' and its parental type 'Anliu'. Surprisingly, 25 candidate genes were shown to exhibit gene dosage variations, with 18 being underrepresented and 7 overrepresented in the bud mutant in comparison with the parental type. These gene dosage variations were validated by quantitative PCR. Interestingly, however, the transcription levels of some of the corresponding genes were not significantly different between the two genotypes. The detection of gene dosage variations in at least 25 candidate genes between 'Hong Anliu' and 'Anliu' may explain in part the genetic difference responsible for the phenotypic deviation of the bud mutant from its parental cultivar. Our findings also suggest that array-CGH is a powerful technology that can be used for detection of genome-wide variations among near-identical genotypes such as bud mutants and their parental cultivars. [ABSTRACT FROM AUTHOR]

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level. [ABSTRACT FROM PUBLISHER]

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence (CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. [ABSTRACT FROM AUTHOR]

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]

CYTOCHROME P450s genes are a large gene family in the plant kingdom. Our earlier transcriptome data revealed that a CYTOCHROME P450 gene of Citrus sinensis (CsCYT75B1) was associated with flavonoid metabolism and was highly induced after drought stress. Here, we characterized the function of CsCYT75B1 in drought tolerance by overexpressing it in Arabidopsis thaliana. Our results demonstrated that the overexpression of the CsCYT75B1 gene significantly enhanced the total flavonoid contents with increased antioxidant activity in transgenic Arabidopsis. The gene expression results showed that several genes that are responsible for the biosynthesis of antioxidant flavonoids were induced by 2–12 fold in transgenic Arabidopsis lines. After 14 days of drought stress, all transgenic lines displayed an enhanced tolerance to drought stress along with accumulating antioxidant flavonoids with lower superoxide radicals and reactive oxygen species (ROS) than wild type plants. In addition, drought-stressed transgenic lines possessed higher antioxidant enzymatic activities than wild type transgenic lines. Moreover, the stressed transgenic lines had significantly lower levels of electrolytic leakage than wild type transgenic lines. These results demonstrate that the CsCYT75B1 gene of sweet orange functions in the metabolism of antioxidant flavonoid and contributes to drought tolerance by elevating ROS scavenging activities. [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]

Interveinal chlorosis in old leaves is a common occurrence in citrus orchards in southern China. The present study investigates the 'Langfeng' navel orange (LF, Citrus sinensis) grafted onto a Trifoliate orange (TO, Poncirus trifoliata) rootstock, which exhibits healthy green leaves, and the 'Newhall' navel orange (NHE, C. sinensis) grafted onto TO, which has typical magnesium (Mg) deficiency-induced chlorosis. Chemical analysis of the rhizosphere soil revealed that the pH values were around 3.92 and that both Mg and calcium (Ca) were significantly deficient in the rhizosphere soil of both grafting combinations (LF/TO and NHE/TO). Furthermore, the chlorotic leaves of NHE/TO had significantly lower levels of Mg, Ca, and phosphorus (P), and the green leaves of NHE/TO had significantly lower levels of Mg and Ca compared to the green leaves of the LF/TO. This suggests that Mg deficiency may be the primary cause of chlorosis in NHE/TO. A greenhouse study using the same graft combinations showed that the LF/TO plants had better growth than the NHE/TO, possibly by promoting Mg uptake and/or improving Mg distribution to leaves, thereby increasing carbon dioxide (CO 2) assimilation and photosynthesis, optimizing carbohydrate distribution, and increasing plant biomass. This results in a phenotype that is tolerant to Mg deficiency. In conclusion, these findings suggest that the LF navel orange could be utilized in the development of new citrus varieties with improved Mg-use efficiency. • Interveinal chlorosis is common in southern China citrus orchards. • Study compares "Langfeng" navel orange (LF) on Trifoliate orange rootstock (TO) to "Newhall" navel orange (NHE) on TO. • Soil analysis showed Mg and Ca deficiencies and NHE/TO had lower levels of Mg, Ca, and P in chlorotic leaves. • Greenhouse study showed better growth in LF/TO plants, indicating improved Mg uptake and tolerance to Mg deficiency. • Findings suggest LF navel orange can be used in new citrus varieties with improved Mg-use efficiency. [ABSTRACT FROM AUTHOR]

[Display omitted] • Lane Late orange brown mutant emits a preferred fragrance. • Zong Cheng sesquiterpene content varied significantly from Lane Late orange. • Octanal was the characteristic aroma of Lane Late orange. • Valencene, β -myrcene and (E)- β -ocimene were the primary aromas of Zong Cheng. • Cs5g12900 had a crucial role in Zong Cheng biosynthesis of valencene. 'Zong Cheng' navel orange (ZC) is a brown mutant of Lane Late navel orange (LL) and emits a more pleasant odor than that of LL. However, the key volatile compound of this aroma and underlying mechanism remains unclear. In this study, sensory evaluations and volatile profiling were performed throughout fruit development to identify significant differences in sensory perception and metabolites between LL and ZC. It revealed that the sesquiterpene content varied significantly between ZC and LL. Based on aroma extract dilution and gas chromatography–olfactometry analyses, the volatile compound leading to the background aroma of LL and ZC is d -limonene, the orange note in LL was mainly attributed to octanal, whilst valencene, β -myrcene, and (E)- β -ocimene presented balsamic, sweet, and herb notes in ZC. Furthermore, Cs5g12900 and six potential transcription factors were identified as responsible for valencene accumulation in ZC, which is important for enhancing the aroma of ZC. [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]

• 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]

Fruit cuticle, which is composed of cutin and wax and biosynthesized during fruit development, plays important roles in the prevention of water loss and the resistance to pathogen infection during fruit development and postharvest storage. However, the key factors and mechanisms regarding the cuticle biosynthesis in citrus fruits are still unclear. Here, fruit cuticle of ‘Newhall’ navel orange ( Citrus sinensis Osbeck) was studied from the stage of fruit expansion to postharvest storage from the perspectives of morphology, transcription and metabolism. The results demonstrated that cutin accumulation is synchronous with fruit expansion, while wax synthesis is synchronous with fruit maturation. Metabolic profile of fruits peel revealed that transition of metabolism of fruit peel occurred from 120 to 150 DAF and ABA was predicted to regulate citrus wax synthesis during the development of Newhall fruits. RNA-seq analysis of the peel from the above two stages manifested that the genes involved in photosynthesis were repressed, while the genes involved in the biosynthesis of wax, cutin and lignin were significantly induced at later stages. Further real-time PCR predicted that MYB transcription factor GL1-like regulates citrus fruits wax synthesis. These results are valuable for improving the fruit quality during development and storage. [ABSTRACT FROM AUTHOR]

Four main flavanone glycosides (FGs) and four main polymethoxylated flavones (PMFs) were determined in fruits of ‘Cara Cara’ navel orange, ‘Seike’ navel orange, ‘Anliu’ and ‘Honganliu’ sweet orange ( Citrus sinensis ). No bitter neohesperidosides were detected in the FG profiles, indicating the functional inability of 1,2-rhamnosyltransferase, though relatively high transcription levels were detected in the fruit tissues of ‘Anliu’ and ‘Honganliu’ sweet oranges. Different to the FGs, the PMFs only exist abundantly in the peel and decreased gradually throughout fruit development of sweet oranges, suggesting the expression of methylation-related genes accounting for PMF biosynthesis have tissue-specificity. Significant changes in production of the eight flavonoids were found between red-flesh and blonde-flesh sweet oranges, indicating that lycopene accumulation might have direct or indirect effects on the modification of flavonoid biosynthesis in these citrus fruits. [ABSTRACT FROM AUTHOR]

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community. [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: Pummelo (Citrus grandis Osbeck) is one of the ancestors of sweet orange and economically important cultivated species of the family Rutaceae. The availability of the EST SSR markers for Pummelo represents a promising source to increase the number of markers available for the citrus species. In this study, we evaluated 212 Pummelo EST derived SSR markers (CgEMS) for their transferability across the genera, polymorphism, mapping ability and utility for genetic diversity analysis. Among these markers, 136 were amplified bands, 99 were transferable across the genera. Transferability of CgEMS to C. sinensis, C. reticulata, C. lemon, Fortunella sp and Poncirus sp was 76%, 76%, 75%, 74% and 73%, respectively. In contrast 52 (53%) markers were found to be polymorphic and segregating in a mapping population. Segregating markers can be categorized into four groups: full informative (8%), male informative (15%), female informative (19%) and partly informative (59%). The phylogenetic relationship between the citrus and its relatives obtained with CgEMS was in a good agreement with the established citrus taxonomy and phylogeny. CgEMS could potentially serve as perfect markers for determining variation in phenotype, fingerprinting, mapping and genetic diversity study in C. grandis. Their high level of cross genera transferability of the CgEMS markers has allowed phylogenetic inference within the Rutaceae. [Copyright &y& Elsevier]

Abstract: An integrative analysis of transcriptome and proteome was performed to identify differential genes/proteins of a red-flesh sweet orange Cara Cara in comparison with a common cultivar Newhall at ripening stages. At the transcript level, gene expression was measured with Massively Parallel Signature Sequencing (MPSS), and 629 genes of these two sweet orange cultivars differed by two fold or more (FDR<0.001). At the protein level, a combination of 2DE and MALDI-TOF-TOF MS identified 48 protein spots differed in relative abundance (P<0.05). The data obtained from comparing transcriptome with proteome showed a poor correlation, suggesting the necessity to integrate both transcriptomic and proteomic approaches in order to get a comprehensive molecular characterization. Function analysis of the differential genes/proteins revealed that a set of candidates was associated with carotenoid biosynthesis and the regulation. Overall, some intriguing genes/proteins were previously unrecognized related with the formation of red-flesh trait, which provided new insights into molecular processes regulating lycopene accumulation in a red-flesh sweet orange. In addition, some genes/proteins were found to be different in expression patterns between the Cara Cara and another red-flesh sweet orange Hong Anliu, and their potential roles were further discussed in the present study. [Copyright &y& Elsevier]

Abstract: The effects of low boron (B) on plant growth, photosynthesis, B concentration and distribution of ‘Newhall’ orange (Citrus sinensis Osb.) plants grafted on either Trifoliate orange (Poncirus trifoliata (L.) Raf.) or Carrizo citrange [C. sinensis (L.) Osb.× Poncirus trifoliata (L.) Raf.] rootstocks were investigated. One-year-old plants of the two scion-rootstock combinations were grown for 183 days in sand:perlite (1:1, v/v) medium under greenhouse conditions. The plants were irrigated with half-strength Hoagland''s nutrient solutions containing four B concentrations (0.01, 0.05, 0.10 and 0.25mgl−1). The growth of root, stem of scion and leaves was less affected by low B treatments when ‘Newhall’ scion was grafted on Carrizo citrange than on Trifoliate orange. Thus, the growth of scions under low B conditions was mainly depended on the rootstock used, i.e., Carrizo citrange-grafted plants were more tolerant to low B compared to the plants grafted on Trifoliate orange. Boron concentrations in all plant parts decreased significantly by decreasing the B supply in the nutrient solution. Leaves were the dominant sites of B accumulation and showed the greatest reduction in B concentration compared to the other plant parts, as B concentration in the nutrient solution decreased. Irrespective of the rootstock, B levels in the upper–younger leaves were substantially higher than in basal-older leaves when plants were exposed to low B concentrations (≤0.05mgl−1), suggesting that under such conditions B was preferentially translocated to upper–younger leaves to support their growth. Furthermore, B distribution in different plant parts indicated that there was a restriction in translocation of B from root to scion tissues (stems and leaves of scion) under conditions of limited B availability. In addition, low B treatments decreased leaf photosynthetic rate, stomatal conductance and transpiration rate but increased intercellular CO2 concentration in the leaves of ‘Newhall’ plants, irrespective of the rootstock used. [Copyright &y& Elsevier]

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]

• 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]

• Potassium (K) fertilizer improved fruit quality in Cara Cara navel fruit. • K enhanced sugar accumulations by improving sucrose metabolism and sink strength. • K fertilizer increased citrate accumulations partially by promoting its synthesis. Sugars and organic acid are key factors for sensory properties of fruit quality. Potassium (K) is an essential macroelement and is also known as quality element. This study aims to investigate how K affects quality improvement particularly sucrose, fructose, glucose and citrate accumulations in Cara Cara navel orange. K fertilizer increased fruit sucrose, fructose, glucose and citrate concentrations and the ratios of total soluble sugars and citrate, particularly the ratios increased more in the treatment of 0.50 kg/plant K fertilizer. These results showed that suitable amount of K fertilizer could improve fruit quality of Cara Cara navel orange. Further study showed that, at early stage of fruit development, K fertilizer played more important roles in degrading sucrose due to the increased activities of sucrose-degrading enzymes including invertase and sucrose synthase-cleavage activity. Conversely, at late stage of fruit development, K fertilizer played more important roles in re-synthesizing sucrose due to the higher activities of sucrose-synthesizing enzymes including sucrose synthase-synthetic activity and sucrose phosphate synthase. The above results suggested that the upregulations of sucrose-degrading and sucrose-synthesizing enzyme activities caused by K fertilizer were conducive to improving sucrose metabolism and sink strength, and then increased the concentrations of sucrose, fructose and glucose in fruit. K fertilizer increased the activities of citrate-synthesizing enzymes including citrate synthetase and phosphoenolpyruvate carboxylase, but had no significant impacts on the activities of citrate-degrading enzyme, indicating that K fertilizer increased citrate accumulations at least partially by promoting its synthesis. This study finally suggested that K fertilizer improved the accumulations of soluble sugar and citrate in Cara Cara navel orange. [ABSTRACT FROM AUTHOR]